JP2010540444A - Composition comprising resveratrol for regulating the enrichment or activity of a gene product - Google Patents

Abstract

The present invention relates to compositions comprising resveratrol that can regulate gene expression to a degree greater than that seen with resveratrol alone or with caloric restriction. The present invention specifically includes resveratrol, a chelating agent, hyaluronic acid, and / or vitamin D, which increases the concentration or activity of a survival / longevity gene product and / or induces cytotoxicity or when administered to a recipient. It relates to a composition comprising resveratrol that reduces the concentration or activity of the gene product it causes. Most preferably, the resveratrol stabilizing composition comprises the chelating agents phytic acid (inositol hexaphosphate; IP6), hyaluronic acid, and vitamin D. The invention further relates to the use of such compositions in the treatment or prevention of cancer, cardiovascular disease, diseases associated with aging, and other conditions and diseases.

Description

(Cross-reference to related applications)
No. 10 / 971,017 (filed and pending on Oct. 25, 2004), which is hereby incorporated by reference. U.S. Provisional Patent Application No. 60 / 513,225 (Oct. 23, 2003) US Provisional Patent Application No. 60 / 973,817 (filed September 20, 2007; pending), 61 / 023,227 (2008) Filed January 24, 2008; pending), 61 / 023,230 (filed January 24, 2008; pending), 61 / 023,234 (filed January 24, 2008); Pending), 61 / 048,756 (filed April 29, 2008; pending) and 61 / 048,769 (filed April 29, 2008; pending) All of these patent applications are Overall Luo, herein incorporated by reference.

The present invention relates to compositions comprising resveratrol that can regulate gene expression to a degree greater than that seen with resveratrol alone or with caloric restriction. The present invention specifically includes resveratrol, chelating agents, hyaluronic acid, and / or vitamin D, which increases the concentration or activity of the survival / longevity gene product and / or induces cytotoxicity when administered to a recipient or It relates to a composition comprising resveratrol that reduces the concentration or activity of the gene product it causes. The present invention particularly relates to the use of the above compositions in the treatment or prevention of cancer, cardiovascular disease, diseases associated with aging, and other conditions and diseases.

BACKGROUND OF THE INVENTION Despite high levels of risk factors such as cholesterol, diabetes, hypertension and high intakes of saturated fats, French men are the lowest mortality from ischemic heart and cardiovascular diseases in western industrialized countries. (36% lower than the US and 39% lower than the UK). The so-called “French paradox” (especially low mortality due to cardiovascular disease) can be mainly due to habitual consumption of wine (Non-patent Document 1).

  Resveratrol (3,4 ', 5-trihydroxy-trans-stilbene) has been demonstrated to have beneficial properties related to human health, such as found naturally in grape skins It is a phenolic compound (Das, S. et al., (2007), “Resveratrol: A Therapeutic Promise For Cardiovascular Diseases”, Regent Patents Cardiovasc. Drug Disc. 2 pages 3 to 38 (1); (2007), “Natural antioxidants in Alzheimer's disease”, Expert Opin. Investig. Drugs. 2): 1921-1931; Baumann L., (2007), “Botanical Ingredients In Cosmetics” J. Drugs Dermatol. 6 (11): 1084-1088; Meeran, SM et al. 2008), “Cell Cycle Control As A Basis For Cancer Chemopration Through Dietary Agents” Front. Biosci. 13: 2191-202; de la Lastra, CA. -Oxidant Agent: Machinery Andd "Clinical Implications", Biochem. Soc. Trans. 35 (Part 5): 1156-1160; Das, S. et al. (2007), "Anti-Inflammatory Responses Of Rivers", Inflamm. (No. 3), pp. 168-173; Cacciola, V. et al., (Epub July 31, 2007), “Resvertrol: From Basic Science To The Clinic”, Cell Cycle 6 (20): 2495. ˜2510; Opie, L .; H. (Epub, June 7, 2007), "The Red Wine Hypothesis: From Concepts To Protective Signaling Modules", Eur. Heart J.H. 28 (14), 1683-1693; Chen, Y .; (2007), “Review. Pro-And Anti-Angiogenesis Effects Of Research”, In Vivo 21 (2): 365-370 [In Vivo 21 (6): 1172 and 21 (5). No.): Errata on page 955]; Holme, A.M. L. (2007), “Reversetrol In Cell Fate Decisions”, J. MoI. Bioenerg. Biomembr. 39 (1): 59-63; (Epub, Jan. 3, 2007), “Resevertrol: A Review Of Preclinical Studies For Human Cancer Prevention”, Toxicol. Appl. Pharmacol. 224 (3): 274-283). In particular, resveratrol is thought to be beneficial for the work of the heart and in extending the life of human cells. Resveratrol is generally produced as an alcoholic extract from plant sources when used in dietary supplements.

  A calorie restricted diet has been shown to extend survival and longevity by upregulating survival / longevity genes or downregulating genes whose expression increases cytotoxicity (Edwards, MG, et al., (2007), “Gene Expression Profiling Of Aging Reveals Activation Of A P53-Mediated Transactional Program”, BMC Genomics 8: 80,; Anderson, R.M., et al. -2005-Mid-2006 ", Exp. Gerontol. 41 (12): 1247-1249; NDRUCH, R. et al., (2001), “Microarray Profiling of Gene Expression in Aging and Its Alteration by Callic Restriction in Mice”, J. Nutrition 131, C.3. (Transcribable Profiles Associated With Ageing And Middle Age-Onset Coloric Restriction In Mouse Hearts, Proc. Natl. Acad. Sci. , R. et al. 2002), "Effects of Caloric Restriction On Gene Expression", Nestle Nutr. Workshop Ser. Clin. Perm. Program. Volume 6: 17-28, 28-32; November 1), "Downregulation of Plasma Insulin Levels And Hepatic Pargamma Expression Durst The First Of Caloric Restriction In Mic.," Gerontol. 43 (3): 146-153; Rodgers, J. et al. T.A. (Epub, November 26, 2007), "Metabolic Adaptations Through The PGC-1 Alpha And SIR Pathways", FEBS Lett. 582 (1): 46-53; Windell, W .; R. (2007), "Gene Expression Profiling Of Long-Lived Dwarf Mice: Longevity-Associated Genes And Relations Diet Diet, Gender And MC." 8: 353; Zhu, M .; (Epub, June 6, 2007), “Adipogenic Signaling In Rat White Adipose Tissue: Modulation By Aggregation And Calibration Restriction”, Exp. Gerontol. 42 (8): 733-744; Chiarpotto, E .; (2006), “Molecular Mechanisms of Carolina Restriction's Protection Against Age-Related Sclerosis”, IUBMB Life. 58 (12): 695-702; Lu, J. et al. (2007), “Differential Gene Expression Of Skin Tissues Between Mice With Weighed Controlled By The Equal Employment Exclusion”. Biol. Med. (Maywood). 232 (4): 473-480; Masternak, M .; M.M. (2007), “pPARS In Calorially Restricted And Genetically Long-Lived Mice”, pPAR Res. 2007: 28436; Fu, C .; (2006) “Tissue Specific And Non-Specific Changes In Gene Expression By Aging And By Early Stage CR”, Mech. Ageing Dev. 127 (12): 905-916). As shown by the references listed above, mice are widely used as a model for comparing human gene expression. Without limitation, the validity of the mouse model for human gene expression reflects the fact that 98% of the human and mouse genes are homologous and that the mouse and human have approximately the same number of genes (eg, about 30,000). is doing.

  Despite the proven benefits of a calorie-restricted diet, the strictness of the necessary dietary management limits the use of this approach to prolong life. Therefore, it would be desirable to provide an alternative means of avoiding the need for dietary restrictions and gaining the benefits of caloric restriction that is acceptable for widespread use. The present invention addresses this and other needs.

Renaud, S.M. (1998), “The French Paradox And Wine Drinking”, Novartis Found. Symp. 216: 208-222, 152-158

SUMMARY OF THE INVENTION The present invention relates to a composition comprising resveratrol capable of regulating gene expression to a degree greater than that seen with resveratrol alone or with caloric restriction. The present invention specifically includes resveratrol, chelating agents, hyaluronic acid, and / or vitamin D, which increases the concentration or activity of the survival / longevity gene product and / or induces cytotoxicity when administered to a recipient or It relates to a composition comprising resveratrol that reduces the concentration or activity of the gene product it causes. Most preferably, the resveratrol stabilizing composition comprises the chelating agents phytic acid (inositol hexaphosphate; IP6), hyaluronic acid, and vitamin D. The present invention further relates to the use of compositions that up-regulate a survival / longevity gene or down-regulate a gene whose expression increases cytotoxicity when administered to a recipient. The present invention particularly relates to the use of the above compositions in the treatment or prevention of cancer, cardiovascular disease, diseases associated with aging, and other conditions and diseases.

  Specifically, the present invention modulates the concentration or activity of a product of a survival / longevity gene or gene whose expression increases cytotoxicity when administered to a recipient as compared to resveratrol alone or caloric restriction Compositions comprising resveratrol are provided. Administration is preferably by oral ingestion.

In the present invention, the adjustment is
(A) oxidative phosphorylation,
(B) actin filament length or polymerization,
(C) intracellular transport,
(D) Organelle biosynthesis,
(E) Insulin signaling,
(F) glycolysis,
Further provided are embodiments of the composition that alter (G) gluconeogenesis, or (H) fatty acid metabolism.

  The invention further provides an embodiment of the composition wherein the gene product is a survival / longevity gene product, and in particular the gene product is sSurtuin 1 or forkhead Foxo1 transcription factor.

  The present invention provides an embodiment of a composition wherein the gene product is a gene product that increases cytotoxicity, and in particular the gene product is encoded by the uncoupling protein 3, Pgc-1, or pyruvate dehydrogenase kinase 4 gene. Provide further.

The present invention provides a composition comprising:
A gene comprising (a) trans-resveratrol, and (b) a metal chelator, wherein the trans-resveratrol is encapsulated, whereby the product of the survival / longevity gene or its expression increases cytotoxicity Further provided are embodiments of the composition that substantially protect the ability of the composition to modulate the concentration or activity of the product from loss due to exposure of trans-resveratrol to light or oxygen.

  The present invention further provides a method for ameliorating symptoms associated with a disease in an existing individual or for preventing symptoms from developing in an individual before the disease occurs in the individual, which comprises resveratrol alone Or administering to the individual a composition comprising resveratrol that modulates the concentration or activity of the product of the survival / longevity gene or gene product whose expression increases cytotoxicity as compared to caloric restriction, Veratrol is provided in an effective amount that results in modulation of the concentration or activity of a gene that improves disease symptoms, and the disease is selected from the group consisting of cardiovascular disease, cancer, macular degeneration, diseases associated with aging, and inflammation Is done.

The present invention provides the adjustment
(A) oxidative phosphorylation,
(B) Actin filament length or polymerization,
(C) intracellular transport,
(D) Organelle biosynthesis,
(E) Insulin signaling,
Further provided are embodiments of the method such as (F) glycolysis (G) gluconeogenesis, or (H) altering fatty acid metabolism.

  The present invention further provides such method embodiments wherein the survival / longevity gene product is a sirtuin 1 or forkhead Foxo1 transcription factor. The present invention further provides an embodiment of the method wherein the gene whose expression increases cytotoxicity encodes uncoupling protein 3 or pyruvate dehydrogenase kinase 4.

In the present invention, the composition comprises
A gene comprising (a) trans-resveratrol, and (b) a metal chelator, wherein the trans-resveratrol is encapsulated, whereby the product of the survival / longevity gene or its expression increases cytotoxicity Further provided is an embodiment of such a method that substantially protects the ability of the composition to modulate the product concentration or activity from loss due to exposure of trans-resveratrol to light or oxygen.

  The present invention further provides an embodiment of a method wherein the disease is cancer or a disease associated with aging (particularly a neurodegenerative disease).

  The present invention further provides an embodiment of the method wherein the composition further comprises quercetin, hyaluronic acid and / or vitamin D.

  The present invention further provides such method embodiments where the modulation is compared to resveratrol alone or the modulation is compared to caloric restriction.

  The invention further provides an embodiment of the method wherein the gene product is a survival / longevity gene product, and in particular, the gene product is a sirtuin 1 or forkhead Foxo1 transcription factor.

  The present invention provides embodiments of the method wherein the gene product is a gene product that increases cytotoxicity, and in particular the gene product is encoded by the uncoupling protein 3, Pgc-1, or pyruvate dehydrogenase kinase 4 gene. Provide further.

FIG. 6 shows changes in body weight of mice administered resveratrol or a composition of the present invention (Longevinex®) compared to control animals and animals maintained on a calorie restricted diet. FIG. 6 shows serum insulin concentrations of mice administered resveratrol or a composition of the invention (Longevinex®) compared to control animals and animals maintained on a calorie restricted diet. Mice that received resveratrol (P = 0.97) or a composition of the present invention (Longevinex®) (P = 0.07), animals that maintained a caloric restricted diet (P = 0) Fig. 10 shows the serum glucose concentration compared to 10). FIG. 2 shows a schematic diagram of the mechanism of action in harmony with the observed biological activity of the composition of the invention.

  The present invention relates to a composition comprising resveratrol that can regulate gene expression to a degree greater than that seen with resveratrol alone or with caloric restriction (especially a food composition comprising resveratrol (ie, a recipe). Composition)) suitable for oral ingestion by the ent. The present invention specifically includes resveratrol, a chelating agent, hyaluronic acid, and / or vitamin D, and when administered to a recipient, a gene that upregulates a survival / longevity gene or whose expression increases cytotoxicity Relates to a composition comprising resveratrol that down-regulates. Most preferably, the composition that stabilizes resveratrol comprises the chelating agents phytic acid (inositol hexaphosphate; IP6), hyaluronic acid, and vitamin D. The invention further relates to the use of such compositions that up-regulate survival / longevity genes or down-regulate genes whose expression increases cytotoxicity when administered to a recipient. The mineral chelating agent of the present invention provides an anti-aging effect as evidence in genome differentiation.

A. Resveratrol As used herein, the term “resveratrol” has the structure:

を有するフィトアレキシンの３，４’，５−トリヒドロキシ−トランス−スチルベンを指す。

Refers to the phytoalexin 3,4 ', 5-trihydroxy-trans-stilbene.

  Resveratrol is used in the prevention or treatment of cardiovascular disease (see, eg, Das, S. et al. (2007), “Resveratrol: A Therapeutic Promise For Cardiovascular Diseases”, Regent Patents Cardiovasc. No.): 133-138; Opie, L. H., et al., (Epub, June 7, 2007), “The Red Wine Hypothesis: From Concepts To Protective Signaling Modules”, Eur. ): 1683-1693; Bertelli, AA, (Epub, May 24, 2007), “Wi e, Research And Cardiovascular Disease: Instructions For Use ", Atherosclerosis 195 Volume (No. 2):. 242-247 pages; Providencia, R, (2006 years)," Cardiovascular Protection From Alcoholic Drinks: Scientific Basis Of The French Paradox ", Rev. Port.Cardiol.25 (11): 1043-1058; Maulik, N., (2006), "Reactive Oxygen Specialties Drivers?" Antioxid. 8 (11-12): 2161-2168; Olas, B. et al. (2005), "Resveratrol, A Phenolic Antioxidant With Effects On Plate Platelets Functions," Vol. ˜260; Bradamante, S. et al. (2004), “Cardiovascular Protective Effects of Research”, Cardiovas Drug Rev. 22 (3): 169-188; Hao, H .; D. (2004), “Mechanisms of Cardiovascular Protection By Reversion”, J. et al. Med. Food 7 (3): 290-298), prevention or treatment of cancer (Jang, M. et al., (1997), “Cancer Chemoventive Activity of Research, A Natural Product Derived Volume 75”. 218-220; Das, S. et al. (2007), “Anti-Inflammatory Responses of Research”, Inflamm. Allergy Drug Targets 6 (3): 168-173; de Ia Lara et al. , (2007), “Resveratrol As An Antioxidant And Pro-Oxidant. Agent: Mechanical And Clinical Implications, Biochem. Soc. Trans. 35 (Part 5): 1156-1160; Atar, M., et al. (Epub Pub. 3 January 2007), “Resvertrol: A Ore Rev: A Ore Rev. "Studies For Human Cancer Prevention", Toxicol. Appl. Pharmacol. 224 (3): 274-283; Meeran, SM et al. (2008), "Cell Cycle Control As Af. Agents ", Front. B osci., 13: 2191-2202; (2006), “Reversetrol As A Chemopreventive Agent: A Promising Molecular For Fighting Cancer”, Curr. Drug Targets 7 (4): 423-442; Signorelli, P .; (2005), “Resveratrol As An Anticancer Nutrient: Molecular Basis, Open Questions And Promises”, J. Am. Nutr. Biochem. 16 (8): 449-466), prevention or treatment of macular degeneration (US Patent Application No. 61/023, 234; King, RE, et al. (2005) "Reseverol reductions oxidation and promotion of." human retinal pigment epithelial cells via extracellular signal-regulated kinase inhibition, Chem. Biol. Interact. 151 (2): 143-149; DNA in retinigine pigment epithelial cells. Vit min E and other antioxidants inhibit A2E-epoxide formation ", J. Biol. Chem. 278 (20): 18207-18213), diseases associated with aging, and the incidence of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease Or attenuation or prevention of other conditions and illnesses including severity (Baxter, RA, (2008) "Anti-Aging Properties Of Research: Review And Report Of A New Antioxidant Skirt." Cosmet. Dermatol.7 (1): 2-7; Engel, N .; (2008), “Aging And Anti-Aging: Unexpected Side Effects of Every Day Meditation Through Sirtuinl Modulation,” Int. J. MoI. Med. (Epub, Dec. 21, 2007), "Resveratrol Is An Effective Inducer Of Carg-Driven Tnf-Alpha Gene Therpy", Cancer Gene Ther. 15 (3): 133-139; Putics, A.M. (2008) "Resvertrol Industries The Heat-Shock Response And Protects Human Cells From Seven Heat Stress", Antioxid. Redox Signal. 10 (1): 65-75: Bass, T .; M.M. (Epub, Aug. 14, 2007), "Effects of Reversal on Lifepan In Drosophila melanogano And Caenorhabditis elegans", Mech. Ageing Dev. 128 (10): 546-552; Stefani, M .; (Epub, September 5, 2007), "The Effect Of Reversal On A Cell Model Of Human Aging", Ann. N. Y. Acad. Sci. 1114: 407-418; Heiss, E .; H. (Epub, July 11, 2007), “Chronic Treatment With Resveratrol Industries Redox Stress- And Axia Telangectasiasia-Mutated Pent. 53 (Dependent Sensence Pent. Biol. Chem. 282 (37): 26759-26766; Mayo Clinic (2007), “A Compound In Red Wine Makes Mice Live Longer, Healthier”, Mayo Clin. Health Lett. 25 (5): 4; Kim, D.M. (Epub, June 21, 2007), “Sirtl Deacetylase Protects Against Neurogeneration in Models for Alzheimer ’s Disease And Amytropic Lateral ScL”. 26 (13): 3169-3179), and anti-inflammatory activity (Das, S. et al. (2007), "Anti-Inflammatory Responses of Research", Inflamm. Allergy Drug Targets 6 (3): US Pat. No. 7,345,178, which is considered the cause of a variety of beneficial biological effects including US Pat. No. 7,345,178, which is incorporated herein by reference. reference).

  Resveratrol can be synthesized chemically (Farina, A. et al., (2006), “An Improved Synthesis Of Research”, Nat. Prod. Res. 20 (3): 247-252. ) Or more preferably from plant sources. Resveratrol is found in at least 72 species of plants distributed within 31 genera and 12 families (see Counett, C. et al. (2006), “Chocolate And Cocoa: New Sources Of. Trans-Resvertrol And Trans-Picid ”, Food Chem. 98: 649-657; Jang, M. et al., (1997),“ Cancer Chemoventive Activity 2 Revelation of Revel. 218-220; Wang, Y. et al., (2002), “An LC-MS Method For Analyz. ... Ng Total Resveratrol In Grape Juice, Cranberry Juice, And In Wine ", J Agricult Food Chem 50 Volume (No. 3): 431 to 435 pages). All families that have been found to contain resveratrol are the gates of seed plants: Grapeaceae, Myrtaceae, Dipterocarpaceae, Cyperaceae, Gnetsum, Leguminous, Pine, Mulberry, Beech (Langcake, P., et al., (1976) "The Production Of Reversion By Visiti Vinifera And Other Jr. of the Pit et al. Yoshiaki, T. et al. (2002), “Biogenetic Reactions On Stilbenetramers From Vitace. aeous Plants ", Tetrahedron 58: 9265-9271). Resveratrol is most frequently reported to be in non-edible plants: vines, Eucalyptus, spruce, and the tropical deciduous tree Bauhinia racemosa, Pterobium Hexepetalum (Cassady, A. et al. (2000)) “Isoflavones, Lignans, And Stilbenes-Origins, Metabolism And Potential Potential Human Health,” J. Science Food Agric. 80: 104: 104. Gol. Molecule What Time Time Has Come? And Gone? " . Clin Biochem 30 Volume:. 91-113 pages). Resveratrol is especially found in grape skins and giant redbirds (see, Burns, J. et al. (2002), “Plant Foods and Herbal Sources of Research”, J. Agric. Food Chem. 50 (11): 3337-3340), in cocoa and chocolate (Counet, C. et al., (2006), “Chocolate And Trans Cocoa: New Sources of Trans-Android Control Trans-Picid”, Food Chem. Vol. Especially found in Peanut buds are also a rich source of resveratrol.

B. Modulation of Gene Product Concentration or Activity The present invention increases the concentration or activity of a survival / longevity gene product and / or decreases the concentration or activity of a gene product that induces or causes cytotoxicity when administered to a recipient. Related to the composition to be made. The increase (or decrease) in concentration or activity as used herein can be achieved by any mechanism. For example, such an increase (or decrease) can reflect the regulation of gene expression, resulting in an increase (or decrease) in gene expression encoding a survival / longevity gene product, or such expression or activity A gene that regulates (eg, induces or suppresses) or a product thereof results in either gene that regulates such expression or activity. Alternatively, or jointly, such an increase (or decrease) in concentration or activity can reflect modulation of the recipient's ability to degrade or stabilize any of the above gene products. Alternatively or jointly, such an increase (or decrease) in concentration or activity may modulate the recipient's ability to increase, accelerate, suppress or slow down the activity of any of the above gene products. Can be reflected.

  The modulation of concentration or activity discussed above can be the modulation of intracellular, intercellular and / or tissue concentration or activity of such a gene product that induces or causes such survival / longevity gene products or cytotoxicity. . Such modulation is confirmed by DNA expression assays, gene product activity assays, gene product level assays, gene product turnover rate assays, etc. performed on one or more types of cells, tissues, etc. be able to.

  An increase in the concentration of the survival / longevity gene product may be, for example, increased transcription of the gene encoding the survival / longevity gene product, increased transcription of the gene that induces expression of the gene encoding the survival / longevity gene product, survival / longevity. Caused by decreased transcription of genes that suppress the expression of genes encoding gene products, decreased degradation or increased stability of expressed molecules of survival / longevity gene products (leads to increased accumulation of survival / longevity gene products) Can be done. Similarly, a decrease in the concentration of a survival / longevity gene product can include, for example, a decrease in transcription of a gene encoding a survival / longevity gene product, a decrease in transcription of a gene that induces expression of a gene encoding a survival / longevity gene product, Increased transcription of genes that suppress the expression of the gene encoding the survival / longevity gene product, increased degradation or decreased stabilization of the molecule expressing the survival / longevity gene product (leads to increased loss of survival / longevity gene product) Can be brought about by.

  One aspect of the present invention thus relates to the use of resveratrol and compositions comprising resveratrol that regulate gene expression, and in particular regulate the expression of “survival / longevity” genes and / or “injury-inducing” genes. . A compound as used herein is said to “modulate” gene expression if its administration results in a change in the expression of such gene (as compared to a control) of at least 10%. Modulation can include an increase in expression (“up-regulation”) or it can include a decrease in expression (“down-regulation”). The term upregulation thus means an increase in expression of at least 10%, at least 20%, at least 50%, at least 2-fold, at least 5-fold, or most preferably at least 10-fold (compared to the control). To do. The term downregulation conversely means a decrease in expression of at least 10%, at least 20%, at least 50%, at least 2-fold, at least 5-fold, or most preferably at least 10-fold (compared to the control). .

  The second aspect of the invention thus relates to the use of resveratrol and compositions comprising resveratrol that modulate the concentration or activity of the expression product of the “survival / longevity” gene and / or the “injury-inducing” gene. A compound as used herein is an agent of such expressed product when its administration results in a change in intracellular, intercellular or tissue concentration or activity (as compared to a control) of at least 10% of such gene product. Say "modulate" concentration or activity. Modulation can include, for example, “increased accumulation” or “enhanced activity” or, for example, it can include “reduced accumulation” or “decreased activity”. The term “increased accumulation” (or “enhanced activity”) is at least 10%, at least 20%, at least 50%, at least 2-fold, at least 5-fold, or most preferably at least 10-fold (compared to a control). ) Concentration (or activity). The term “reduction in accumulation” or “reduction in activity” conversely means at least 10%, at least 20%, at least 50%, at least 2-fold, at least 5-fold, or most preferably at least 10-fold (compared to the control). )) Concentration (or activity).

  As used herein, a “survival / longevity” gene is a gene whose expression contributes to the survival or longevity of a subject (eg, a mammal, particularly a human) that expresses such a gene. Conversely, a “damage-inducing” gene is a gene whose expression is involved in DNA, cell, or tissue damage in such a subject. Such genes are responders to biological stressors, which regulate radiation (eg, sunlight, gamma rays, UV light, etc.), radiation mimetic agents (eg, Initiates action in response to stress factors such as vitamin D), heat, and half-starvation (calorie restriction or mimetics thereof, resveratrol).

  Examples of survival / longevity genes are provided in Table 1. Examples of genes whose expression increases cell damage are provided in Table 2. These tables provide NCBI's “ENTREZGENE” accession numbers for genes. Most preferably such gene is a human gene. The sirtuin 1 gene is known to control the rate of aging in the body and mimic the beneficial effects of calorie restriction, thanks to its ability to produce DNA repair enzymes. Resveratrol trans form (not cis-resveratrol) activates the sirtuin 1 gene (Alcendor, RR (2007), “Sirl Regulates Aging And Resistive Stress In The Heart,” Circulation Research 100 (No. 10): 1512-1521; Howitz, KT, et al. (2003), “Small Molecule Activators of Sirtuins Extended Saccharomies 6: N1”, 19: N4. The present invention particularly relates to compositions that increase the concentration of a sirtuin 1 survival / longevity gene product. The present invention further relates to compositions that increase the concentration of forkhead Foxo1 (daf-16, dFoxO) transcription factor survival / longevity gene products, among others.

Ｃ．本発明の好ましい組成物
本発明は、特に、レスベラトロールの比活性が安定化されているかまたは増大しているレスベラトロールを含む組成物に関する。本明細書で使用される用語の「比活性」とは、投与されたレスベラトロールの量（質量）当たりの遺伝子調節の程度（対照と比較して）の比率を指す。

C. Preferred compositions of the present invention The present invention relates in particular to compositions comprising resveratrol in which the specific activity of resveratrol is stabilized or increased. As used herein, the term “specific activity” refers to the ratio of the degree of gene regulation (compared to a control) per amount (mass) of resveratrol administered.

  Preferably, the composition comprises a chelating agent, hyaluronic acid, and / or vitamin D. The present invention particularly comprises the above composition (preferably the composition of the present invention) comprising resveratrol and at least one compound selected from the group consisting of an antioxidant (chelating agent), hyaluronic acid, and vitamin D. Product provides a composition dosage of about 10 mg to about 2 g, more preferably about 100 mg to about 500 mg). Preferably, the composition of the present invention contains resveratrol, an antioxidant, hyaluronic acid, and vitamin D.

  The term “chelating agent” as used herein refers to an organic compound that binds and removes free metal ions from a solution. Examples of suitable chelating agents include ethylenediaminetetraacetic acid (EDTA), histidine, tetracycline family antibiotics, pyridoxal 2-chlorobenzoylhydrazone, desferrioxamine, dexrazoxane, deferasirox, pioversin, pseudodans, citrate NDGA (nordihydroguaiaretic acid: l, 4-bis [3,4-dihydroxyphenyl] 2,3-dimethylbutane), ferulic acid and phytic acid. Preferably, the composition of the present invention provides a composition dosage of chelating agent of from about 1 g to about 15 g, more preferably from about 2 g to about 12 g.

  Phytic acid is a particularly preferred chelating agent for the purposes of the present invention. As used herein, the term “phytic acid” refers to inositol hexaphosphate ((2,3,4,5,6-pentaphosphonooxycyclohexyl) dihydrogen phosphate, also known as “IP6”) ( See, Thorne Research, Inc., (2002), “Inositol Hexaphosphate. Monograph”, Altern. Med. Rev. 7 (3): 244-248; Vucenik, I. et al., (2006), “ Protection Against Cancer By Dietary IP6 And Inisotol, ”Nutr. Cancer. 55 (2): 109-125; Lopez, MA et al., (2004),“ Iron Avalibi. it: An Updated Review ", Int. J. Food Sci. Nutr., 55 (8): 597-606; Singh, RP, et al. "Mechanisms", Anticancer Res. 25 (4): 2891-2903; Vucenik, I. et al. 133 (11th volume 1): 3778S-3784S; V., (2003), “Myo-Inositol-1, 2,3,4,5,6-Hexakisphosphate”, Phytochemistry. 64 (6): 1033-19043; Vohra, A. et al., (2003). Physics: Microbiological Sources, Production, Purification, And Potential Biotechnical Applications, Crit. Rev. Biotechnol. Vol. 23 (No. 1): 29-60 C; H. (2002), “Physic Acid (IP6), Novell Broad Spectrum Anti-Neoplastic Agent: A Systemic Review”, Complement Ther. Med. 10 (4): 229-234; Grases, F.M. (1999), “Phytate (IP6) Is A Powerful Agent For Presenting Certifications In Biological Fluids: Usefulness In Relative Resistance Treatment”, Anticancer Res. 19 (5a): 3717-3722; Jariwalla, R .; J. et al. (1999), “Inositol Hexaphosphate (IP6) As An Anti-Neoplastic And Lipid-Lowering Agent”, Anticancer Res. 19 (5a): 3699-3702; Katayama T .; (1999), "Hypolipidic Action Of Physic Acid (IP6): Prevention Of Fatty River", Anticancer Res. 19 (5a): 3695-3698). The structure of phytic acid is provided below:

フィチン酸は、全粒穀類（ｗｈｏｌｅ ｇｒａｉｎ）、穀類（ｃｅｒｅａｌ）、マメ科植物、木の実、および種子中にかなりの量が見出され、発芽する植物のための主要なエネルギー源である（Ｇｒａｆ， Ｅ．、（１９８３年）、「Ａｐｐｌｉｃａｔｉｏｎｓ ｏｆ Ｐｈｙｔｉｃ Ａｃｉｄ」、Ｊ． Ａｍ． Ｏｉｌ． Ｃｈｅｍ． Ｓｏｃ ６０巻：１８６１〜１８６７頁）。フィチン酸およびその低リン酸化型（ＩＰ３など）は、ほとんどの哺乳類細胞中にも見出され、そこでそれらはさまざまな重要な細胞機能の調節を助けている（Ｓｚｗｅｒｇｏｌｄ， Ｂ． Ｓ．ら、（１９８７年）、「Ｏｂｓｅｒｖａｔｉｏｎ Ｏｆ Ｉｎｏｓｉｔｏｌ Ｐｅｎｔａｋｉｓ− Ａｎｄ Ｈｅｘａｋｉｓｐｈｏｓｐｈａｔｅｓ Ｉｎ Ｍａｍｍａｌｉａｎ Ｔｉｓｓｕｅｓ Ｂｙ ^３１Ｐ ＮＭＲ」、Ｂｉｏｃｈｅｍ． Ｂｉｏｐｈｙｓ． Ｒｅｓ． Ｃｏｍｍｕｎ． ２６４巻：８７４〜８８１頁）。フィチン酸は、好ましくは米ぬかの形で提供される（Ｓｒｉｎｉｖａｓａｎ， Ｍ．、（２００７年）、「Ｆｅｒｕｌｉｃ Ａｃｉｄ’ Ｔｈｅｒａｐｅｕｔｉｃ Ｐｏｔｅｎｔｉａｌ Ｔｈｒｏｕｇｈ Ｉｔｓ Ａｎｔｉｏｘｉｄａｎｔ Ｐｒｏｐｅｒｔｙ」、Ｊ． Ｃｌｉｎ． Ｂｉｏｃｈｅｍ． Ｎｕｔｒ． （２００７年）４０巻（２号）：９２〜１００頁；Ｋｉｍ， Ｍ．Ｊ．ら、（２００７年）、「Ｆｅｒｕｌｉｃ Ａｃｉｄ Ｓｕｐｐｌｅｍｅｎｔａｔｉｏｎ Ｐｒｅｖｅｎｔｓ Ｔｒｉｍｅｔｈｙｌｔｉｎ−Ｉｎｄｕｃｅｄ Ｃｏｇｎｉｔｉｖｅ Ｄｅｆｉｃｉｔｓ ｉｎ Ｍｉｃｅ」、Ｂｉｏｓｃｉ． Ｂｉｏｔｅｃｈｎｏｌ． Ｂｉｏｃｈｅｍ． （２００７年）７１巻（４号）：１０６３〜１０６８頁）。フィチン酸は、銅および鉄等の二価のカチオンをキレート化することによる抗酸化剤として機能し、それによって細胞傷害および発癌に関与する反応性の酸素種の発生を防止すると報告されている（Ｈａｒｌａｎｄ， Ｂ．Ｆ．ら、（１９８７年）、「Ｐｈｙｔａｔｅ Ｉｎ Ｆｏｏｄｓ」、Ｗｏｒｌｄ Ｒｅｖ． Ｎｕｔｒ． Ｄｉｅｔ ５２巻：２３５〜２５９頁）。フィチン酸（例えば、米ぬかとして）の好ましい組成物薬用量は、２０００〜１２，０００ｍｇの範囲である。

Phytic acid is found in significant amounts in whole grains, cereals, legumes, nuts, and seeds and is the primary energy source for germinating plants (Graf, E., (1983), "Applications of Physic Acid", J. Am. Oil. Chem. Soc 60: 1861-1867). Phytic acid and its hypophosphorylated forms (such as IP3) are also found in most mammalian cells where they help regulate various important cellular functions (Szwergold, BS et al., ( 1987), “Observation Of Inositol Pentakis-And Hexakisphosphates In Mammalian Tissues By ³¹ P NMR”, Biochem. Biophys. Res. Commun. 264: 874-881). Phytic acid is preferably provided in the form of rice bran (Srinivasan, M., (2007), “Ferric Acid 'Therapeutic Throughput It's Antioxidant Property”, J. Clin. Bio40. (2): 92-100; Kim, MJ, et al. (2007), "Ferric Acid Supplementation Presents Trimethylin-Initiated Cognitive Defects in Mice", Biosci. Biotech. 4): 1063-1068). Phytic acid is reported to function as an antioxidant by chelating divalent cations such as copper and iron, thereby preventing the generation of reactive oxygen species involved in cell injury and carcinogenesis ( Harland, BF et al. (1987), “Phytate In Foods”, World Rev. Nutr. Diet 52: 235-259). A preferred composition dosage of phytic acid (eg, as rice bran) is in the range of 2000 to 12,000 mg.

As used herein, the term “hyaluronic acid” (also known as hyaluronan) means that D-glucuronic acid and DN-acetylglucosamine are alternating β-1,4 glycosidic bonds and β-1 , 3 refers to a linear polymer composed of repeating disaccharides ([-β (1,4) -GlcUA-β (1,3) -GlcNAc-] _n ) linked together through glycosidic bonds. Hyaluronic acid can be 25,000 disaccharide repeats (n) long:

ヒアルロン酸は、人体中で天然に生成されるが、体が老化すると量が減少する水保有分子である。ヒアルロン酸は、細胞の細胞周囲マトリクスの基底を形成する多機能のグリコサミノグリカンである。ヒアルロン酸は、異なるけれども関連する３つの酵素によって合成される（ヒアルロナンシンターゼ：ＨＡＳｌ、ＨＡＳ２およびＨＡＳ３）（Ｗｅｉｇｅｌ， Ｐ．Ｈ．ら、（１９９７年）、「Ｈｙａｌｕｒｏｎａｎ Ｓｙｎｔｈａｓｅｓ」、Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７２巻：１３９９７〜１４０００頁；Ｔａｍｍｉ， Ｍ．Ｉ．ら、（２００２年）、「Ｈｙａｌｕｒｏｎａｎ Ａｎｄ Ｈｏｍｅｏｓｔａｓｉｓ： Ａ Ｂａｌａｎｃｉｎｇ Ａｃｔ」、Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７７巻：４５８１〜４５８４頁；Ｋａｋｅｈｉ， Ｋ．ら、（２００３年）、「Ｈｙａｌｕｒｏｎｉｃ Ａｃｉｄ： Ｓｅｐａｒａｔｉｏｎ Ａｎｄ Ｂｉｏｌｏｇｉｃａｌ Ｉｍｐｌｉｃａｔｉｏｎｓ」、Ｊ． Ｃｈｒｏｍａｔｏｇｒ． Ｂ Ａｎａｌｙｔ． Ｔｅｃｈｎｏｌ． Ｂｉｏｍｅｄ． Ｌｉｆｅ Ｓｃｉ． ７９７巻（ｌ〜２号）：３４７〜３５５頁；Ｒａｄａｅｖａ， Ｉ．Ｆ．ら、（１９９７年）、「Ｈｙａｌｕｒｏｎｉｃ Ａｃｉｄ： Ｂｉｏｌｏｇｉｃａｌ Ｒｏｌｅ， Ｓｔｒｕｃｔｕｒｅ， Ｓｙｎｔｈｅｓｉｓ， Ｉｓｏｌａｔｉｏｎ， Ｐｕｒｉｆｉｃａｔｉｏｎ， Ａｎｄ Ａｐｐｌｉｃａｔｉｏｎ （Ｒｅｖｉｅｗ）」、Ｐｒｉｋｌ． Ｂｉｏｋｈｉｍ． Ｍｉｋｒｏｂｉｏｌ． ３３巻（２号）：１３３〜１３７頁；Ｓｔｏｏｌｍｉｌｌｅｒ， Ａ．Ｃ．ら、（１９７０年）、「Ｔｈｅ Ｂｉｏｓｙｎｔｈｅｓｉｓ Ｏｆ Ｈｙａｌｕｒｏｎｉｃ Ａｃｉｄ Ｂｙ Ｇｒｏｕｐ Ａ Ｓｔｒｅｐｔｏｃｏｃｃｕｓ」、Ｅｘｐｏｓ． Ａｎｎｕ． Ｂｉｏｃｈｉｍ． Ｍｅｄ． ３０巻：６５〜７８頁）。米国特許出願公開第２００４／０２３４４９７号は、抗がん剤送達のためのヒアルロン酸の使用を開示している。その公開物の全体の開示を、参照により本明細書に組み込む。

Hyaluronic acid is a water-bearing molecule that is naturally produced in the human body but decreases in amount as the body ages. Hyaluronic acid is a multifunctional glycosaminoglycan that forms the basis of the pericellular matrix of cells. Hyaluronic acid is synthesized by three different but related enzymes (hyaluronan synthase: HAS1, HAS2 and HAS3) (Weigel, PH et al. (1997), “Hyalulan Syntheses”, J. Biol. Chem. 272: 13997-14000; Tammi, MI et al., (2002), "Hyaluran And Homeostasis: A Balancing Act", J. Biol. Chem. 277: 4584-1458; Kakehi, K. et al. (2003), “Hyaluronic Acid: Separation And Biological Implications”, J. Chromatogr. B Analyte. Techno. Biomed. Life Sci. 797 (1-2): 347-355; Radaeva, IF et al., (1997), "Hyuralonic Acid: Biological Role, Construction, Ill., Science, Ill. (Review) ", Prikl. Biokhim. Mikrobiol. 33 (2): 133-137; Stoolmiller, A.C. et al. (1970)," The Biosynthesis Of Hyaluronic Acid. Annu. Biochim. Med., 30: 65-78). US Patent Application Publication No. 2004/0234497 discloses the use of hyaluronic acid for anticancer drug delivery. The entire disclosure of that publication is incorporated herein by reference.

  Hyaluronic acid has traditionally been extracted from rooster trout, bovine or fish vitreous humor, microbial products or other sources (Rangwaswamy, V. et al. (Epub 24 Oct. 2007), " Ann. Efficient Process For Production And Purification Of Purification Of Hyaluronic Acid From Streptococcus Equi Subsub.Zopidemicus, p.3, Biotechnol. Hyaluronan Oligosaccharides For Angogen cis Study ", J. Biomed. Mater. Res. B Appl. Biometer. 78 (2): 385-392; Blank, LM, et al., (2005)," Stable Production Of Hyaluronic Inc. “Zoeidemicus Chemostats Operated At High Dilution Rate”, Biotechnol. Bioeng. 90 (6): 685-693; Kakehi, K. et al. (C), “H. B Analyt. Biol Life Sci. 797 (1-2): 347-355; Volpi, N., et al. 6): 619-625; Tawada, A. et al., (2002), “Large-Scale Preparation, Purification, And Charac- terization of Hyaluronan Oligosaccharides From 4-Morsco Togol, 52-Mers”. . July 2002, 12 (7): 421-426; Mahoney, D. et al. J. et al. (2001) “Novel Methods For The Preparation And Charactarization Of Hyaluronan Oligosaccharides Of Defined Length”, Glycobiology. 11 (12): 1025-1033; Mcdonald, J. et al. Et al. (2002), “Hyalulanan Miniview Series”, J. Am. Biol. Chem. 277 (7): 4575-4579; Radaeva, I .; F. (1997), “Hyaluronic Acid: Biological Role, Structure, Synthesis, Isolation, Purification, And Application (Review)”, Prikl. Biokhim. Mikubiol. 33 (2): 133-137). Most preferably, the hyaluronic acid of the present invention is obtained from the top of a rooster. Hyaluronic acid is widely available commercially and such formulations may be suitable for the purposes of the present invention. The compositions of the present invention preferably provide a composition dosage of hyaluronic acid from about 1 mg to about 400 mg, more preferably from about 50 mg to about 200 mg.

As used herein, the term “vitamin D” refers to a prohormone that dissolves in fats and oils. The two main forms of vitamin D are vitamin D ₂ (ergocalciferol) and vitamin D ₃ (cholecalciferol) (DeLuca, HF et al. (1998), “Mechanisms And Functions Of Vitamin D Nutr. Rev. 56: S4-S10):

ビタミンＤは、多くの生物学的作用を示す。ビタミンＤは、骨疾患（成長中の子供のくる病、高齢者における骨粗しょう症）を回避するその能力について広く公知である一方で、それはがんとの闘いにおける主役となりつつある。免疫力およびがんにおけるビタミンＤの役割に関して、ビタミンＤは、走化性の（親和性の）好中球の動員および移動を向上させる。ビタミンＤ欠乏によるくる病の患者は、適切に移動することができない不活発な好中球を有することが観察される。ビタミンＤは、単球のマクロファージへの成熟化を促進する。これにより腫瘍に向かって戦闘を開始する免疫戦闘細胞の大きくなった軍隊が生じる。ビタミンＤは、幅広く市販されており、そのような製剤は、本発明の目的に適合する。

Vitamin D exhibits many biological effects. While vitamin D is widely known for its ability to avoid bone disease (rickets in growing children, osteoporosis in the elderly), it is becoming a major player in the fight against cancer. With regard to the role of vitamin D in immunity and cancer, vitamin D improves the recruitment and migration of chemotactic (affinity) neutrophils. It is observed that patients with rickets due to vitamin D deficiency have inactive neutrophils that cannot migrate properly. Vitamin D promotes maturation of monocytes into macrophages. This creates a large army of immune combat cells that begin fighting towards the tumor. Vitamin D is widely available commercially and such formulations are suitable for the purposes of the present invention.

  Vitamin D is essential for optimal muscle, bone, brain, immunity and cardiovascular health and has been rediscovered by aging researchers around the world. Vitamin D supplementation up to 2000 IU has been shown to significantly reduce mortality, thus adding vitamin D to the molecular lineup currently considered to be a true longevity factor (Autier, P. et al. (2007), “Vitamin D Supplementation Total Totality: A Meta-Analysis Of Randomized Controlled Trials”, Arch Intern Med. 167 (17): 173. Its anti-calcification properties (Zittermann, A. et al. (2007), “Vitamin D And Vascular Calibration”, Curr. Opin. Lipidology 18 (1): 41-46) It is described as another powerful agent that inhibits gradual overmineralization with age and is comparable to that of other mineral chelators in the compositions of the present invention. The 1200 IU dose is three times higher than the recommended daily allowance, but it is well within the safe upper limit established by the National Academy of Sciences (2000 IU) and is beneficial in human clinical trials This is consistent with additional dosages recently found (Lappe, JM et al., (2007), “Vitamin D and calcium supplementation cancer risk: results of abandoned tri. Nutr., 85 (6): 1586-1591). A dosage of 2,000 IU is roughly equivalent to the natural vitamin D3 produced by 15-30 minutes of full-day midday summer sun exposure in the southern zone where no side effects have been reported. The compositions of the present invention preferably provide a composition dosage of vitamin D from about 100 IU to about 100,000 IU, more preferably from about 1,000 IU to about 50,000 IU.

  The compositions of the present invention comprise additional active ingredients and inert compounds that act to increase the biological activity of resveratrol (eg, flavors, sweeteners, dyes, vitamins, amino acids (eg, lysine, proline, etc.) ), Minerals, nutrients, etc.) can be included.

  In particular, quercetin (3,3 ', 4', 5,7-pentahydroxy-2-phenylchromen-4-one),

ブテイン、フィセチン、ミリセチン、ケンフェロール、シス−レスベラトロールまたはピセタノールをその組成物に加えることができる。

Butein, fisetin, myricetin, kaempferol, cis-resveratrol or picetanol can be added to the composition.

かかる化合物は、例えば、Ｓｔｅｃｈｅｒ， Ｇ．ら、（２００１年）（「Ｄｅｔｅｒｍｉｎａｔｉｏｎ Ｏｆ Ｆｌａｖｏｎｏｉｄｓ Ａｎｄ Ｓｔｉｌｂｅｎｅｓ Ｉｎ Ｒｅｄ Ｗｉｎｅ Ａｎｄ Ｒｅｌａｔｅｄ Ｂｉｏｌｏｇｉｃａｌ Ｐｒｏｄｕｃｔｓ Ｂｙ ＨＰＬＣ Ａｎｄ ＨＰＬＣ−ＥＳＩ−ＭＳ−ＭＳ」、Ｆｒｅｓｅｎｉｕｓ Ｊ． Ａｎａｌ Ｃｈｅｍ． ２００１年９月：３７１巻（１号）：７３〜８０頁）により記述されている。

Such compounds are described, for example, in Stecher, G. et al. (2001) ("Determination Of Flavoroids And Stilbenes In Red Wine And Related Biological Products By HPLC And HPLC-ESI-MS-MS", Fresenius J. e. 73 to 80).

  Resveratrol is glucuronidated in the human liver, which may reduce bioavailability. Flavonoids such as quercetin can act to block the glucuronic acid binding of resveratrol and thus improve the bioavailability of resveratrol (see de Santi, C. et al. (2000). ), “Glucuronidation Of Research, A Natural Product Present In Grape And Wine, In The Human Liver”, Xenobiotica 30 (11): 1047-1054 ti; (2000), "Sulphation Of Research, A Natural Compound Present In Wine, And It Inhibition By Natural Flavonoids", Xenobiotica 30 (866), 8: S66; (2000), "Sulphation Of Research, A Natural Product Present In Grapes And Wine, In The Human Liver And Dudenum", Xenobiotica 30: 6 (No. 6). Quercetin can also act synergistically with or independently of resveratrol to provide a beneficial function (Kampkoetter, A. et al. (Epub 16 October 2007), “Increase. Of Stress Resistance And Lifespan Of Caenorhabditis Elegans By Quercetin ", Comp. Biochem. Physiol. B Biochem. Mol. Biol. 149 (2): 314-32. (See also, Kaindl, U., et al., (2008), “The Dietary Antioxidants Research And Quercetin Protect Cells From Exogenous Pro-Oxidative C., No. 46, x. Melzig, M.F, et al. (2002), “Induction of Neutral Endopeptidase And Antidetensin-Converting Enzyme Activity of Sk-N-Sh Cells.” 56-558; Hsu, CL, et al. (2006), “Induction Of Cell Apoptosis In 3T3-L1 Pre-Adiposites By Flavoroids Is Associated with T. Att. 11): 1072-1079; Chan, MM, et al. (2000), “Synergy Between Ethanol And Polythen Phenylphenol, Quercetin, And The River Inhibit, In The Inhibit Inhibit. Biochem. Pharmacol. 60 (10): 1539-1548; Nicholson S. K. et al., (2008), “Effects of Dietary Polyphenols On Gene Expression in Human Vascal Endo. 67 (l): 42-47; Lemos, C., et al. (2007), "Modulation Of Folate Uptake In Cultured Human Colon Adenocarcinoma Caco-2 Cells By Dietary Compounds. Eur." J. et al. Nutr 46 (6): 329-336).

  Emulsifiers, fillers, binders and the like can also be included in the compositions of the present invention.

  In one embodiment, the composition of the present invention comprises a novel combination, which comprises small molecules such as quercetin and resveratrol with a wide range of prophylactic and therapeutic health properties; and / or collagen construct nutrients ( Eg, vitamin C-ascorbate, lysine, proline, etc.); and shortened (low molecular weight) chain hyaluronic acid (HA) or its specific components (glucosamine, glucuronate) or chondroitin sulfate (they are A linear disaccharide (a molecule resembling sugar) that contributes as a constituent, but in this combination it contributes as a synergistic co-therapeutic agent in non-cellular (connective) tissues surrounding living cells) A combination of saminoglycans. A combination of glycosaminoglycans (hyaluronan, glucuronic acid or chondroitin) and / or ascorbic acid, lysine or proline, and small molecules working at the intracellular base to promote collagen production is therapeutic or preventive covering the entire tissue matrix Including action. The combination of the present invention is intended to be taken orally by a human or animal as a dietary supplement. In its sub-embodiments, such compositions can include a combination of resveratrol and hyaluronan in a dietary supplement, which is useful for treating a variety of diseases, including several cancers. Resveratrol is an anticancer molecule and is known to have other curative and life-prolonging properties. Hyaluronan (hyaluronic acid, HA) can be taken as an oral supplement or administered intravenously targeting cancer cells. When combined with or conjugated to other molecules, hyaluronan delivers other anticancer and healing agents such as resveratrol to the tumor site. The combination may or may not include chelating agents, antioxidants and / or emulsifiers as described in the above-invented copending patent application. Resveratrol and HA have potent healing properties for animals and humans when encapsulated or otherwise applied together with or without their additives.

  Most preferably, the composition of the present invention has a specific activity of resveratrol, wherein the resveratrol of the composition is maintained in the presence of oxygen gas, or no chelating agent, hyaluronic acid, or vitamin D To have a specific activity that is greater than the specific activity of resveratrol maintained in Preferably, the amount of components other than resveratrol in the composition is such that resveratrol in the composition is maintained in the presence of oxygen gas or without chelating agent, hyaluronic acid, or vitamin D. More than at least 10% activity, at least more than 20% activity, at least more than 50% activity, at least 2 times activity, at least 5 times activity, or at least 10 times the activity of resveratrol being maintained , And it has such specific activity over time (eg, 1, 2, 4, 6, 10) at ambient temperature and humidity (ie, without the need for special precautions for temperature or humidity). , 12, 18, 24, or 36 months or longer).

D. Packaging of Preferred Compositions of the Invention Resveratrol is generally unstable to light and oxidation (Shaanxi University of Science & Technology, Xianyang China, (2007), "Study On The Stable Instibilities". Rhizoma Polygoni cuspidate ", Zhong Yao Cai. 30 (7): 805-80). The resveratrol of the present invention is preferably prepared, packaged and / or stored in a manner that maximizes its specific activity. Prepare, package and / or store resveratrol in low light (or in the dark) and / or in low oxygen to minimize photo-induced degradation (eg photoisomerization) or oxygen-induced degradation Is preferred. Preferred compositions of the present invention are formulated as dietary supplements for oral consumption in the form of pills, lozenges, capsules, elixirs, syrups and the like. Other modes of administration (eg, intranasal, parenteral, intravenous, intraarterial, topical, etc.) can be used instead.

  In the first example of such a preferred package, the composition of the present invention is formulated as an airtight capsule where the encapsulation is done to prevent or minimize exposure to oxygen. In one embodiment, such encapsulation is performed in an oxygen free environment. For example, the components of the composition of the present invention can be encapsulated in an inert gas (eg, nitrogen, argon, etc.) environment. Preferably, a nitrogen bubble (5-20% of the capsule volume) can be introduced into the capsule to further stabilize and protect the component against oxygen (see herein by reference). PCT Publication No. WO 01/088631) incorporated. The international application has a corresponding US patent application. Suitable capsules useful in the encapsulation of resveratrol and other oxidizable ingredients in dietary supplements include Licaps® (Capsugel), hermetic gelatin capsules. The presence of phytic acid, which has the ability to protect the component from metal-induced oxidation, increases such antioxidant prevention measures. A particularly preferred example of a composition comprising such resveratrol is Longevinex® (Resveratrol Partners, LLC, San Dimas, Calif.) Comprising resveratrol and phytic acid. Longevinex® is taken from the active ingredient (per capsule), 5 mg vitamin E (as mixed tocopherol), 215 mg total resveratrol (French red wine and giant knotwood) (Polygonum cuspidatum), 100 mg Of trans-resveratrol), 25 mg quercetin dihydrate, 75 mg phytic acid (rice bran extract), 380 mg rice bran oil, 55 mg sunflower lecithin.

  Once the composition is sealed in an airtight capsule, maintaining the hypoxic or anoxic environment in the packaging surrounding the capsule should prevent the composition from being destroyed or leaked in the sealed capsule. Important to protect against oxidation. Therefore, an oxygen-absorbing packet is preferably used to reduce the presence of free oxygen. Desirable vacuum or nitrogen-filled packaging (bottles, pill cases, etc.) of hermetic materials.

  In other embodiments, the components and compositions of the present invention can be prepared like a microencapsulation process (see, generally, Rubiana, M. et al. (2004), “Drug Delivery Systems: Past , Present, and Future ", Current Drug Targets, Volume 5 (5): 449-455). Microencapsulation is the process of coating microparticles or droplets (sizes ranging from a few nanometers to 1 micron) with a protective layer to produce small capsules with controlled properties. Suitable micron-sized encapsulated formulations are available from Maxx Performance Inc. (Chester, NY), Blue California (Rancho Santa Margarita, CA), Southwest Research Institute (San Antonio, TX), Coating Place, Inc. (Verona, WI), Microtek Laboratories (Dayton, OH), Particle Sciences, Inc. It can be obtained using a microencapsulation process such as (Bethlehem, PA). A third generation Longevinex® (“Longevinex-3®”) (Resevertrol Partners, LLC) containing vitamin D3, vitamin E, resveratrol, quercetin, and phytic acid is a composition of the present invention. A particularly preferred microencapsulated form of the product.

  The present invention further includes a practical method of stabilizing quercetin and other readily oxidized dietary supplement ingredients that can be contacted with an oxidizing metal.

E. Usefulness of the Composition of the Invention The composition of the invention increases the specific activity of resveratrol. The compositions of the present invention may therefore be used for cardiovascular disease, cancer, macular degeneration, aging, neurodegenerative diseases (eg, Alzheimer's disease) where modulation of “survival / longevity” and / or “injury-inducing” gene expression is desirable , Parkinson's disease, etc.), and treatment of diseases such as inflammation (or improvement of disease symptoms). Over time, when minerals such as calcium and iron accumulate in the human body, genes react in a detrimental form. Liu, Y .; (2005) “Global Genomic Approaches To The Iron-Regulated Proteome”, Ann. Clin. Lab. Sci. 35 (3): 230-239; Templeton, D.M. M.M. (2003), “Genetic Regulation Of Cell Function In Response To Iron Overload Or Chelation”, Biochim. Biophys. Acta. 1619 (2): 113-124; Ikeda, H .; (1992), “Evidence That An Iron Chelator Regulates Collagen Synthesis By Degrading The Stability of Proliferation mRNA”, Hepatology 15: 28 (2). The present invention has unique utility in the treatment of macular degeneration, cancer and aging conditions.

1. Macular degeneration In the United States, the extension of human life span over the past 20-30 years has caused the spread of macular degeneration, an eye disease associated with aging. Although it does not result in complete blindness, the disease deprives them of their central vision used for reading as well as color vision. Macular degeneration affects the visual center of the eye called the macula. The plaque is a part of the retina in which a color-vision cell (retinal cone) is located.

Macular degeneration is a disease with progressive aging that can be classified into the following four stages.
1. Due to the inability to swallow and remove cell debris from the fundus of the “dirt cleansed” cells, called retinal pigment epithelium (RPE), which begins around the 30s of life, ly-poh-fus-kin This results in the formation of a small microscopic deposit called. Lipofuscin is caused by iron and copper-induced cellular debris oxidation, the accumulation of which is associated with premature aging and shortened lifespan of the organism. The prevalence of macular degeneration is more common in whites than people with dark skin, and whites have more lipofuscin deposits in their retina. Some of this cellular debris in the retina consists of worn-out vitamin A that detaches from night-vision (rod) cells every morning in the human eye. The debilitating function of RPE cells is caused by the accumulation of iron and calcium within the RPE.
2. Late in the 50s of life, there is progressive calcification of the underlying retinal layer of cellophane called Bruch's membrane that exists between the RPE and the blood supply layer (choroid) To do. The drusen that occurs in the retina is partly composed of cholesterol, but this lipid is not originated from the blood circulation or the liver where most cholesterol is produced. Calcification within the Bruch's membrane further reduces leakage of lipids (fats), proteins, and cell debris from the photoreceptor layer, resulting in the formation of yellow spots called drusen on the retina. Drusen can be observed in an eye examination using an ophthalmoscope. There is currently no way to remove drusen.
3. RPE cell death is the third stage of this progressive disease. This is sometimes called RPE dropout. When RPE cells weaken or die and the Bruch membrane is clogged with calcium, the photoreceptors cannot then be fed with nutrients and begin to die one after another. There is currently no cure for stages 1-3 of macular degeneration. Stages 1-3 are referred to as the “dry” form of macular degeneration because it does not result in bleeding or edema or neovascularization. About 85% of patients with macular degeneration have a “dry” form of the disease.
4). When the Bruch membrane breaks or the Bruch membrane is fully calcified, the photoreceptor layer is deprived of oxygen and forms new blood vessels that can penetrate into the photoreceptor layer in plaques There can be vision loss (called neovascularization) or blood serum leakage or apparent red blood cell release, which leads to edema or bleeding. This is a more advanced and threatening form of macular degeneration, often referred to as “wet” type macular degeneration due to the presence of blood serum or red blood cell leakage into the photoreceptor layer. If this stage of the disease is caught early, it can be treated with a laser beam, thereby occluding the leaking blood vessels. However, this treatment is only effective in slowing the progression of the disease and does not cure it.

  The cell cleansing process facilitated by lysosomes cannot keep up with the accumulation of metabolic waste throughout life. The parafoveal ring, which has the highest rod cell density and therefore more discoid, worn-out vitamin A, is where macular degeneration begins and the highest concentration of lipofuscin is found in the retina. As a result, RPE cells die one after the other with increasing age, thereby increasing the burden of the remaining RPE cells maintaining a healthy retina.

  In the past, lipofuscin has been considered a harmless and exhausted byproduct of cellular metabolism. One aspect of the present invention is that lipofuscin, which is formed by iron and copper-induced oxidation and hardens in lysosomal bodies in retinal pigment epithelial cells, sensitizes and damages the retina by a mild amount of irradiation and oxidation Concerning the perception of things. The retina becomes increasingly sensitive to blue light damage with age. The formation of drusen in the retina is associated with the inability of RPE cells to produce superoxide dismutase, an endogenous antioxidant enzyme. Mice deficient in superoxide dismutase develop the characteristics of macular degeneration in humans associated with typical aging. Superoxide dismutase protects retinal cells against unbound (free) iron. High iron diet and cellular environment have been shown to reduce superoxide dismutase activity.

  Retinal photoreceptors and retinal pigment epithelial cells are thought to be particularly vulnerable to damage by low molecular weight iron complexes. Since antioxidants in the blood circulation are not always able to cross the blood retinal barrier, the retina produces antioxidants that bind iron that protects itself. Iron chelators prevent the negative effects of unbound (free) iron (not bound to protein). Heme oxygenase also acts in a manner similar to iron chelators to prevent retinal damage induced by free iron.

  A number of drugs have been used experimentally to clean lipofuscin and drusen. Statin drugs commonly used to lower blood serum levels of cholesterol are also being tested to prevent lipofuscin deposition in animals. Statins have reduced lipofuscin formation, but are toxic to the liver, resulting in early death of these animals. Piracetam, a derivative of the neurotransmitter GABA, currently available as a dietary supplement, has succeeded in reducing lipofuscin formation in brain tissue by using it. Solvinyl is an enzyme inhibitor (akrose reductase inhibitor) that has undergone unsuccessful human trials in the 1990s to prevent retinal problems associated with diabetes. Solvinyl has been shown to partially reduce lipofuscin deposition in rodent retinal pigment epithelial cells. Hydergine is a drug used to treat senile dementia. In rodent studies, it was reported that Hydergine reduced brain lipofuscin levels but also caused early animal death. East Indian spice turmeric contains an antioxidant molecule called curcumin. Curcumin was used in an experimental mouse study to reduce lipofuscin in the brain. The purslane is a flowering plant rich in magnesium, β-carotene and ω-3 oils. The supply of purslane to mice has been shown to reduce the deposition of lipofuscin in the mouse brain.

  In a lab dish study, an antioxidant molecule, sulforaphane, found in 1992 in cabbage and broccoli was successfully used to reduce lipofuscin deposition in RPE cells exposed to blue light.

  Intraperitoneal administration of lipoic acid to aged rats results in decreases and increases in lipofuscin and enzyme activity in the brain, cortex, cerebellum, striatum, hippocampus, and hypothalamus, respectively. These results suggest that the natural metabolic antioxidant lipoic acid should serve as a therapeutic tool to prevent neurological dysfunction in the elderly. The natural antioxidant product lipoic acid, which is produced in living tissue and can also be used as a dietary supplement, has been shown in Labdish studies to protect RPE cells from oxidative damage.

  Lipofuscin formation increases dramatically in tissues following consumption of alcohol. Supplementation with high doses of grape seeds with flavonols prevents increased lipofuscin formation. Lipofuscin is the end product of a lipid peroxidation that increases dramatically following ethanol consumption.

  Oolong and green tea drinks reverse cognitive impairment and lipofuscin formation in mice. Epigallocatechin-3-gallate (EGCG), a major component of green tea, upregulates the activity of heme oxygenase in the Labdish study. Heme oxygenase is a protective enzyme against iron-induced oxidation that occurs in the retina.

  Supplemental estrogen supply has been shown to reduce lipofuscin deposition in brain tissue. In a Labdish study, feeding lutein and zeaxanthin to RPE cells reduced lipofuscin formation. In rodents fed supplemental acetyl-L-carnitine, reduced lipofuscin deposition has been measured in brain cells.

  US Pat. No. 5,747,536 discloses L-carnitine, lower alkanoyl L-carnitine or drugs to produce a medicament for the prevention and treatment of cardiovascular disorders, peripheral vascular disease and peripheral diabetic neuropathy. Physiologically acceptable salts thereof are described for use in therapy in combination with resveratrol, resveratrol derivatives or resveratrol-containing natural products.

  Melanin is an antioxidant that binds to iron in the retina. As aging progresses and the level of melanin in the retina decreases, lipofuscin accumulates more.

In one embodiment, the present invention provides:
(A) chelating agents for metal (s) such as iron, copper, heavy metals, such as inositol hexaphosphate (IP6), transresveratrol, quercetin, or any polyphenol or bioflavonoid,
(B) a calcium chelator, such as inositol hexaphosphate (IP6),
(C) a heme oxygenase activator, for example transresveratrol, picetanol, or any natural analog of resveratrol, or similar small molecules such as fisetin, myricetin, quercetin or other bioflavonoids,
(D) agents that reduce the affinity of oxygen for red blood cells, such as inositol hexaphosphate (IP6), and optionally
(E) Other antioxidants such as vitamin E, lutein / zeaxanthin, alpha lipoic acid,
To a composition comprising a combination of

The formulation is
(1) limiting oxidation in retinal tissue (photoreceptors, retinal pigment epithelial cells (RPE), choroid, especially mitochondria and lysosomes in RPE cells),
(2) block the accumulation of lipofuscin deposits,
(3) prevents drusen formation, and (4) functions to limit calcification to retinal tissue, particularly Bruch's membrane.

2. Cancer The main challenge in cancer treatment is to selectively direct cytotoxic agents to tumor cells (Luo, Y. et al. (2000), “A Hyaluronic Acid-Taxol Antibiotic Biojugated Targeted Toe. Cancer Cells ", Biomacromolecules 1 (2): 208-218). Many targeting approaches have been tried to reduce the undesirable side effects of small molecule anticancer drugs. One of the most promising methods involves the combination or covalent binding of cytotoxins with macromolecular carriers, particularly hyaluronic acid (Luo, Y. et al. (1999), “Synthesis And Selective Cytotoxicity Of A Hyaluronic Acid-Antitumor”. Bioconjugate ", Bioconjug.Chem. 10 (5): 755-763; Luo, Y. et al. (2000)," A Hyaluronic Acid-Taxol Anticancer Biotargeted To Cancer Bio, " ): 208-218; Luo, Y. et al., (1999), "Hyaluronic Acid-N-Hydr." xysuccinimide: A Useful Intermediate for Bioconjugation ”, Bioconjug. Chem. 12 (6): 1085-1088; Luo, Y. et al. Res., 19 (4): 396-402).

  In one embodiment, the present invention relates to a resveratrol and hyaluronic acid-containing composition comprising resveratrol, hyaluronan, and optionally vitamin D and / or IP6 for the treatment of cancer. These components are synergistic with each other to mediate the effect of curing and / or preventing cancer in humans and / or improving the immunity (eg, immune system response) of a patient threatened by a tumor. It is thought to work. This aspect of the invention recognizes that in part, natural molecules can, in some cases, increase cancer immunity in a manner similar to that observed in mice with proven cancer. Is based.

  When supplied with such a composition, dendritic cells, who are guardians of the innate immune system, can be alerted and can significantly increase the activity of neutrophils, macrophages and natural killer cells. Enhancement of vitamin D receptors by resveratrol is yet another major advantage of a combined approach to treating or preventing cancer. This approach appears to be more appropriate for the elderly in the highest risk group for cancer, often immune compromised due to poor nutrition or lack of nutrient absorption. The fact that this treatment can now be measured immediately by non-invasive cancer cell counting techniques may be costly and unclear animal testing may not be necessary to prove efficacy It means that.

  Vitamin D exhibits many biological effects. While vitamin D is widely known for its ability to avoid bone diseases (growth children's rickets, elderly osteoporosis), it is becoming a major player in the fight against cancer. Most recently, it is also gaining attention as an antibiotic. Mice deficient in vitamin D show an incomplete response from phagocytic cells in the face of infection or inflammation. Vitamin D deficiency is often associated with recurrent infections. Compared to animals with adequate vitamin D levels, only about half of the macrophage cells accumulate at the site of inflammation in animals deficient in vitamin D.

  When delving deeper into the role of vitamin D in immunity and cancer, vitamin D improves chemotaxis (affinity) to neutrophils and mobilizes them to move. It is observed that patients with rickets due to vitamin D deficiency have slow neutrophils that cannot migrate properly. Vitamin D stimulates the maturation of monocytes into macrophages. This results in an expanded army of immune combat cells that confront the tumor. Greater attention is now being given to vitamin D as an anti-cancer weapon, with studies showing that supplemental vitamin D significantly reduces the risk for all types of cancer. Studies using 1100 IU vitamin D3 produced a 60-77% reduction in cancer risk among women in Nebraska in just 4 years.

  Despite the lowest risk of cancer in the sun's equatorial region, where vitamin D levels are higher in the sun-exposed population, the protective effects of vitamin D against cancer are repeatedly rejected. Has been neglected. Oral consumption of vitamin D eliminates skin cancer concerns that result from overexposure to unfiltered sunlight. One recent analysis shows that colon cancer risk can be halved by taking 2000 IU of vitamin D per day, and breast cancer risk can be halved by taking 3500 IU of vitamin D per day. ing. The median dietary intake of vitamin D is only about 230 IU per day, so the potential for food fortification or supplementation to prevent or treat cancer is now a reality.

  In order for tissues to take advantage of and benefit from vitamin D, the tissue must have proteins in their outer layer (cell membrane) that are designed to accept and bind to vitamin D. For example, although vitamin D receptor gene expression (production) is at a low level, about 80% of human breast tumors produce vitamin D cell receptors.

  The ability of vitamin D to block cancer can be increased when it is aided by faint estrogen-like molecules in food. Resveratrol, an estrogen-like molecule normally found in red wine, upregulates vitamin D receptors in breast cancer cells without increasing cancer growth. Resveratrol can substantially increase the sensitivity of breast cancer cells to the anti-cancer properties of vitamin D.

  Laboratory experiments show that low doses of vitamin D3 do not reduce breast tumor cell growth, but when combined with resveratrol, the number of tumor cells is reduced by as much as 40%. At higher concentrations, vitamin D3 reduces the number of breast cancer cells in a lab dish by about 25%, and this reduction improves to 50% when combined with resveratrol. Estrogen increases vitamin D receptor gene expression, but it also stimulates the growth of breast tumors. Resveratrol does not have this drawback. Resveratrol enhances or “weaponizes” the cancer-suppressing effects of vitamin D.

  Moreover, resveratrol alone has been shown to calm phagocytic responses to foreign invaders such as phagocytic bacteria and tumor cells. Resveratrol attenuates the production of reactive oxygen species (free radicals) and normalizes particle uptake in macrophage cells. Therefore, resveratrol blocks the immune cell's excessive response that can give rise to autoimmunity.

  Resveratrol interferes with cancer in so many ways that it is difficult for cancer to find a route that is not obstructed by resveratrol. Resveratrol produces in the tumor cells a cellular energy compartment called mitochondria that releases an enzyme called cytochrome C oxidase that normally leads to a cascade of other enzymes that induce programmed cell death called apoptosis. However, recent experiments have shown that resveratrol releases cytochrome C from ovarian tumor cells, which is actually digested by the enzymes produced inside the tumor cells (a kind of intracellular cannibalization form). ) It shows that cell death is caused by a process called autophagy. This is a form of cell suicide in which resveratrol is activated in tumor cells rather than healthy cells.

  The contribution of innate immunity in tumor research is relatively ignored in cancer biology. Phagocytosis, or “cell eating”, is the cornerstone of the innate immune response. The focus has been on dendritic cells that appear to be guardians of innate immune responses. The number of immunopotentiators investigated has been limited.

  Skepticism surrounds the benefits of the innate immunity approach to cancer treatment. For example, patients using immunosuppressants do not necessarily develop cancer more frequently. However, this may be misunderstood. An overreactive immune system can lead to more tissue and organ damage that can be fatal to cancer patients. Most drugs used for breast cancer treatment induce immune suppression.

  The most powerful iron chelator in nature is inositol hexaphosphate (IP6) found in seeds and bran parts of whole grains. A low dose of IP6 has been found to reduce the growth of rhabdomyosarcoma cells by 50%. Removal of IP6 allows these tumor cells to recover and grow again. IP6-treated mice with injected tumors show 50 times smaller tumors than untreated mice. IP6 has also been shown to reduce the growth of injected fibrosarcoma cells in mice and prolong the survival of mice.

  In the test of IP6 immunity enhancing properties, it has been shown to increase free radical (superoxide) production and neutrophil cell digestion in the presence of bacteria. IP6 increases the release of interleukin-8.

  The action of natural killer cells involved in tumor cell destruction is enhanced by IP6.

  In one embodiment, the hyaluronic acid of the composition is conjugated to a chemotherapeutic agent. The invention particularly relates to such a composition wherein the chemotherapeutic agent is taxol. The present invention is particularly and preferably related to such compositions comprising a chelating agent and / or vitamin D. Most malignant solid tumors contain elevated levels of hyaluronic acid (Rooney, P. et al. (1995), “The Role Of Hyaluronan In Tumour Neovascularization (Review)”, Int. J. Cancer 60 ( 5): 632-636), and these high levels of HA production provide a matrix that promotes invasion (Hua, Q. et al., (1993), “Internalization Of Hyaluronan By Chronicles Occurrers Via Receptor- "Mediated Endocytosis", J. Cell. Sci. 106 (Part 1): 365-375; Luo, Y. et al. (2000), A Hyaluronic Acid-Taxol Antitumor Bioconjugate Targeted To Cancer Cells ", Biomacromolecules l wound (No. 2): 208 - 218 pages). Thus, chemotherapeutic agents conjugated to hyaluronic acid can target tumor cells and provide effective antitumor dosages at lower overall concentrations.

Briefly, a preferred method of conjugation involves the formation of the chemotherapeutic agent NHS (N-hydroxysuccinimide derivative. Such a derivative can be obtained by combining a molar excess of dry pyridine with taxol and succinic anhydride in CH ₂ Cl ₂ at room temperature. The reaction mixture is then stirred for several days at room temperature and then concentrated in vacuo The residue is dissolved in 5 ml of CH ₂ Cl ₂ and the product is Taxol-2′-hemisuccinate can be purified on silica gel (washed with hexane and eluted with ethyl acetate) to give the desired product (Luo, Y. et al. (1999), “ Synthesis And Selective Cytotoxicity Of A Hyaluronic Acid-Antitumo .. Bioconjugate ", Bioconjug Chem 10 Volume (No. 5): 755-763 pages).

  The N-hydroxy-succinimide derivative of the chemotherapeutic agent is then coupled to adipic acid dihydrazide functionalized hyaluronic acid. The adipic acid dihydrazide functionalized hyaluronic acid is preferably prepared according to Pouani, T .; (1994) ("Functionalized Derivatives Of Hyaluronic Acid Oligosaccharides-Drug Carriers And Novel Biomaterials" Bioconjugate Chem. 5: 339Pu. (1994) ("Novel Hydrology Of Hyaluronic Acid: Synthesis, Surface Morphology, And Solid-State NMR", J. Am. Chem. Soc. 116: 7515 er sr; P. (1997) (“Synthesis And In Vitro Degradation Of New Polyhydride Cross-Linked Hydrogels Of Hyaluronic Acid”, pages 6 to 68: Bioconjugate Chem. 8: 68). Accordingly, hyaluronic acid is preferably dissolved in water and excess adipic dihydrazide (ADH). The pH of the reaction mixture is adjusted to 4.75 by adding acid. Next, 1 equivalent of 1-ethyl-3- [3- (dimethylamino) -propyl] carbodiimide (EDCI) is added as a solid. The pH of the reaction mixture is maintained at 4.75 by addition of acid. The reaction is quenched by adding 0.1 N NaOH to adjust the pH of the reaction mixture to 7.0. The reaction mixture is then transferred to a pretreated dialysis tube (Mw cutoff 3,500) and dialyzed exhaustively against 100 mM NaCl, then dialyzed against 25% EtOH / H 2 O and finally water. The solution is then filtered through a 0.2 m cellulose acetate membrane, snap frozen and lyophilized (Luo, Y. et al. (1999), “Synthesis And Selective Cytotoxicity of Hyaluronic Acid-Anti-Tumor Bioconjugate”, Bioconjugate. Chem., 10 (5): 755-763).

3. Aging Calcification and rusting are the main promoters of aging. The human body consists of sticky substances called collagen or connective tissue that fill between cells that must be constantly replaced or renewed from the inside and cells that must also be constantly regenerated. As the human body ages at the cellular level, cellular debris called lipofuscin accumulates gradually. The formation of lipofuscin is facilitated by the progressive accumulation of iron and calcium inside the cell body called lysosomes and mitochondria. A cell cleaning and regeneration process called autophagy prevents the accumulation of lipofuscin. The progressive inability of cell debris removal results in diminished cell function and subsequent premature cell death. Young cells efficiently remove debris from the interior. Old cells cannot efficiently remove debris and accumulate lipofuscin.

  Cell calcification and rusting impairs the cleaning of cellular debris (lipofuscin) from cells by enzymes produced by lysosomes, resulting in a deficiency in cellular energy (ATP) produced by mitochondria in the cells. The composition of the present invention prevents and / or reverses cellular aging and / or connective tissue aging, in particular cell aging and / or caused by the accumulation of major minerals (eg iron, calcium, etc.). Prevent and / or reverse connective tissue aging. As a result, recipients of the compositions of the present invention exhibit increased lifespan and enhanced health and structure of cells and connective tissue.

  The human body ages at the cellular level by the gradual accumulation of cell debris called lipofuscin, which is facilitated by the progressive accumulation of iron and calcium inside the cell body called lysosomes and mitochondria. Cell cleansing and regeneration processes called autophagy prevent the accumulation of lipofuscin during the age of young growth, but this lysosomal mechanism is attenuated by intracellular iron and calcium accumulation once full growth is reached . The progressive inability to remove cell debris results in diminished cell function and subsequent premature cell death. Young cells efficiently remove debris from the interior. Old cells cannot efficiently remove debris and accumulate lipofuscin. The mitochondria that provide cell energy for the lysosomal bodies to perform their cell-cleansing activities are also gradually calcified and have a high iron content once the childhood growth is over. By the age of 80, only about 5% of mitochondria will function. Iron and calcium chelators have been proposed to improve mitochondrial aging that affects cellular functions such as lysosomal enzyme activity.

  The human body ages in connective tissue due to a disorder of cells called fibroblasts that regenerate collagen and hyaluronic acid (the latter is a space-filled, water-retaining molecule). Collagen formation is promoted by vitamins and amino acids (vitamin C, lysine, proline) in food. Fibroblasts are stimulated to produce hyaluronic acid by estrogen that is naturally produced in the body, as well as by estrogen-like molecules found in plants called phytoestrogens that are provided in the diet for hyaluronic acid itself be able to. Young women exhibit darker hair, smoother skin, and more flexible joints due to abundant hyaluronic acid, thanks to their ability to produce estrogen. These are all youthful qualities.

  The inability to regenerate hyaluronic acid results in the tissue losing its physical integrity due to the loss of hyaluronic acid's space-filling properties. If there is not enough hyaluronic acid, a dehydrated state will occur, and the tissue will shrink and shrink completely. For example, skin deficient in hyaluronic acid appears wrinkled and dry. The joint cavity will lack the necessary relaxation and space fillers to prevent the bone from rubbing against the bone. The eye begins to shrink in size. Hair is thinned by inadequate hydration. These are the most prominent visible or cosmetic signs of aging.

  In one embodiment, the present invention eliminates both cellular senescence and extracellular (connective tissue) aging, eg, (a) young functions of living cells, excess minerals that are mostly calcium and iron. And thereby promoting autophagy (cleaning of cell debris such as lipofuscin by lysosomal enzymes), and (b) metal chelators such as phytic acid, ferrate, quercetin, resveratrol, etc. In order to prevent the degradation of HA due to supply, the production of hyaluronan is actively protected by the stimulation of fibroblasts by HA and phytoestrogens (resveratrol, quercetin, gemistine, etc.).

In one embodiment, the present invention is a dietary supplement that addresses both cellular and non-cellular aging by the following capabilities:
(A) A dietary supplement that stimulates the regeneration of living cells from the inside through enzymatic degradation of cell debris by intracellular lysosomal bodies. This is facilitated by including metal (iron, copper, heavy metal) chelating molecules and calcium chelating molecules in the formulation. Lysosomes lose their ability to enzymatically digest cell debris by progressive accumulation of iron, copper and other metals, and calcium crystallization.

  (B) A dietary supplement that stimulates fibroblasts to produce hyaluronic acid again at the level of young people. This is accomplished by providing an orally consumed molecule that stimulates fibroblasts to produce hyaluronic acid.

  (C) Metal chelating molecules that help maintain youthful lysosomal function include vitamin E or vitamin C, lipoic acid, metal chelators (IP6 phytate, quercetin, biobravonoids or polyphenols, resveratrol Dietary supplements that have been identified as antioxidants. Resveratrol works by its ability to stimulate the production of the enzyme heme oxygenase, which helps control iron.

  (D) A dietary supplement in which the molecules that prevent calcium crystallization are magnesium and IP6 phytate.

  (E) Orally consumed molecules that stimulate fibroblasts to produce hyaluronic acid are hyaluronic acid, glucosamine, chondroitin, or estrogen-like molecules such as genistein, lignan, hydroxytyrosol, or estrogen Nutritional supplements such as other composed molecules. Orally consumed HA stimulates HA and chondroitin synthesis more greatly. Similarly, glucosamine stimulates fibroblasts to produce HA. Alternatively, or in addition, glucosamine promotes the production of hyaluronic acid synovial fluid, which is primarily involved in synovial fluid lubrication and buffering (McCarty, MF, (1998), “Enhanced Synthetic Production Of Hyaluronic Acid”. May Explore Rapid Clinical Response To High-Dose Glucosamine In Osteoarthritis ", Medical Hypotheses 50: 507-510, 1998).

  (F) A dietary supplement wherein the molecules consumed orally that stimulate collagen production are vitamin C, proline and lysine.

In such embodiments, the present invention relates to a dietary supplement containing resveratrol and hyaluronic acid that restores youthful function and appearance to human cells and tissues. The present invention particularly relates to such a composition further comprising a chelating agent and / or vitamin D. Most preferably, the composition comprises the chelating agent phytic acid (inositol hexaphosphate; IP6). The composition of the present invention synergistically increases the specific activity of resveratrol and / or hyaluronic acid, and therefore the composition of the present invention provides an increase in activity over that obtained by administering the ingredients individually. provide. In such an embodiment, the present invention relates to a method for restoring human cells and tissues to restore youthful function and appearance, and includes the following steps:
(A) stimulating the regeneration of living cells from the interior through enzymatic degradation of cell debris by intracellular lysosomal bodies, preferably providing a metal chelating molecule that helps maintain youthful lysosomal function Such molecules may be antioxidants, such as vitamin E or vitamin C, lipoic acid, metal chelators (such as IP6 phytate, quercetin, bioflavonoids or polyphenols, and / or resveratrol). And (b) stimulating fibroblasts to produce hyaluronic acid (providing an orally consumed molecule that stimulates fibroblasts to produce hyaluronic acid, and Such orally consumed molecules include, for example, hyaluronic acid, glucosamine, chondroitin, and / or Or an estrogen-like molecule such as genistein, lignan, hydroxytyrosol, or other molecules configured like estrogen).
Preferably, such stimulation is achieved by food administration of a composition comprising a specified compound in combination with an orally consumable molecule that more preferably stimulates the production of collagen, such as vitamin C, proline, etc. And / or lysine.

While not intending to be limited by any mechanism, the four preferred components are believed to act in the following manner when administered together.
(A) Vitamin D: Vitamin D3 acts like an agent that mimics the response to solar radiation, a biological stress factor. In particular, vitamin D3 up-regulates the number and motility of protective genes involved in the activation of the immune system, particularly neutrophils, and reduces the sun / skin production of vitamin D. Help overcome the decrease in vitamin D3 production. Moreover, vitamin D3 acts synergistically to break down IP6 to IP3, which appears to be the main active molecule. Resveratrol also works synergistically to increase cell sensitivity to vitamin D3 (sensitize cell surface vitamin D receptors). Vitamin D serves to break down IP6 to its main active form, IP3. Vitamin D is also thought to act as an immune system enhancer that increases innate immunity in humans. With regard to this ability, vitamin D has been experimentally shown to have important cancer prevention and cancer healing properties.
(B) Resveratrol: Resveratrol increases the sensitivity of the vitamin D receptor on the cell surface and is therefore believed to act as an enhancer and an anticancer agent against vitamin D. Resveratrol upregulates the vitamin D receptor on the surface of cancer cells, increasing the sensitivity of cancer cells to vitamin D (Wietzke, JA et al. (2003), “Phytoestrogen Regulation Of A Vitamin”. D3 Receptor Promoter And 1, 25-Dihydroxyvitamin D3 Actions In Human Breast Cancer Cells ", J. Steroid Biochem. Molec. Biol. 84 (2-3); 2005), “Regulation Of The Human Vitamin D3 Receptor Promoter In Breast Cancer Cel. ... S Is Mediated Through Sp1Sites ", Molec Cell Endocrinol 230 Volume (Nos. 1 and 2): 59 to 68 pages). Resveratrol is also considered to be a monoamine oxidase inhibitor (MAO inhibitor).
(C) Hyaluronic acid: Hyaluronic acid is a molecule that gels water which contributes as its scaffolding agent and wettable powder in the human body. As aging progresses, less hyaluronic acid is produced, resulting in wrinkled skin, thinning hair, and unlubricated joints. The chelating agent of the composition also serves to protect hyaluronic acid in the body. Hyaluronic acid components and mineral chelating components (eg, resveratrol, quercetin, phytic acid IP6, ferrate) serve as an overall anti-aging strategy to maintain youthful function within cells and connective tissue . Hyaluronic acid is considered to have affinity for cancer cells. It is believed to serve as a molecule for delivery and targeting in the blood circulation (drug delivery agent) and to address connective tissue aging. The disruption and loss of connective tissue integrity between cells gives signs of aging (eg, skin wrinkles, thinning hair, joint stiffness, height loss, etc.). The addition of hyaluronic acid to the composition of the present invention is believed to contribute to the active production of further hyaluronic acid by fibroblasts in the human body, thus preserving connective tissue (collagen) in a youthful state. (Yadav, AK et al., (2008), “An Insight On Hyaluronic Acid In Drug Targeting And Drug Delivery”, J. Drug Target. 16 (2): o 107; H. et al., (2005), “Hyaluranan: Pharmaceutical Characterization And Drug Delivery”, Drug Deliv. 12 (6): 327-342; Joddar, B. et al., (2006). Elastogenic Effects Of Exogenous Hyaluronan Oligosaccharides On Vessel Smooth Muscular Cells, Biomaterials 27 (33): 5699-5707; Giris, K. H. e. Biological Overview ", Life Sci. 80 (21): 1921-1943).
(D) Phytic acid: Phytic acid, preferably in the form of rice bran, is believed to act as a chelating agent for iron and copper and as an inhibitor of calcium crystallization. Phytic acid is also thought to reduce the availability of metal minerals that contribute as growth factors in tumor cells. It is also thought to act as a neutrophil priming and motility agent. In addition, phytic acid is neuroprotective and thus has been found to attenuate the severity of conditions associated with neurodegenerative diseases, particularly Parkinson's disease, proflexion, and Alzheimer's disease (Xu, Q. et al. (Epub, Dec. 27, 2007), “Neuroprotective Effect Of The Natural Iron Chelator, Phytic Acid In A Cell Culture Of Parkinson's Disage” 1 to 108, Tox. . The components of the composition of the present invention are believed to enhance such neuroprotective action.

  The iron chelator quercetin, when present, works to increase the immediate bioavailability of resveratrol by allowing it to pass more through the liver before it is metabolized. Conceivable.

  The individual components of the composition are believed to act synergistically, for example, to increase the effects of resveratrol. Without intending to be limited thereto, it is proposed that body management or iron and calcium chelation controls the rate of aging after full growth is achieved. During childhood growth, all iron and calcium are devoted to the production of new bone and new red blood cells (hemoglobin). The cessation of childhood growth results in excess iron, copper and calcium, which then gradually (a) calcifies and (b) rusts the tissue. Lysosomes begin to accumulate iron and calcium, thereby causing their dysfunction. Mitochondria begin to malfunction and at the same time they gradually rust and calcify. It is believed that the composition of the present invention can limit or slow its progressive rusting and calcification of cells and organelles, thereby facilitating the slowing or reversal of the aging process. Chelation is what regulates genes. The gene is then smoothly up-regulated or down-regulated. Resveratrol and copper chelators are (1) a controller of calcium concentration by upregulation of osteocalcin, a hormone that helps retain calcium in bone, and (2) iron concentration by heme oxygenase, an antioxidant enzyme. It is thought to act as a controller.

  MAO inhibitors and iron chelators have been proposed as therapeutics for Parkinson's disease (Youdim, MB, et al., (2004), “Novel Biological Drugs Targeting Monooxide Inhibition Anti-Antion of Anti-Non-Antial Anti-Antion of Anti-Non-Arpide) Parkinson's Disease And Other Neurodegenerative Diseases ", J. Neurol. Transm. 111 (10-11): 1455-1471; Yanez, M. et al., (2006)," (-)-Trans-ps-Trans-ps Viniferin, A Polyphen I Present In Wines, Is An Inhibitor Of Noradrenaline And 5-Hydroxytryptamine Up Take And Mono Oxidase Activity, p. 54. Eur. (Reveratrol And Quercetin, Two Natural Polyphenols, Reduced Optical Neuro Cell Induced By Neuroinflammation, pp. 403, J. Neurosci. 3 years), “Quercetin Potentates L-Dopa Reversal Of Drug-Induced Catalepsy In Rats: Possible COMMT / MAO Inhibition, Pharmacol. 68 (2): 81-X8; “Prospective Study Of Dietary Pattern And Risk Of Parkinson Disease”, Am. Clin. Nutr. 86 (5): 1486-1494; Johnson, S .; (2001), “Is Parkinson's Disease The Heterozygote Form Of Wilson's Disease: PD = 1/2 WD?”, Med. Hypotheses 56 (2): 171-173). Compositions of the present invention containing MAO inhibitors and copper chelators, resveratrol, iron chelators and MAO inhibitors, quercetin, and a broad metal chelant phytic acid are useful in neurodegenerative diseases (particularly Parkinson). Disease, anteflexia, and Alzheimer's disease) or for improving the symptoms of such diseases.

  Now that the present invention has been described in general terms, the same is provided by way of example and is not intended to limit the invention unless explicitly stated by reference to the following examples. It will be easier to understand.

Example 1
Comparison of the effect of resveratrol and the composition of the present invention To determine whether the composition of the present invention is more effective than resveratrol alone in mediating the biological activity of resveratrol In addition, gene expression analysis was performed to compare the regulation of gene expression achieved by calorie restriction with the regulation of gene expression obtained by the composition of the present invention.

  Accordingly, the ability to upregulate the survival / longevity genes of resveratrol alone and compositions comprising resveratrol of the present invention or down-regulate genes whose expression increases cytotoxicity as a positive control. Comparison was made using the expression profile of calorie restricted ("CR") animals and the expression profile of normally fed animals as a negative control. Male B6CHF1 mice (2 months of age), thus a 40% calorie restricted diet, a commercially available supply of trans-resveratrol (Sigma Chemical; 1.25 mg / kg per day), Supply of composition comprising resveratrol (Longevinex®; Reversatrol Associates, LLC; 100 mg trans-resveratrol capsules per day in 80 kg humans (ie 2.5 mg / kg resveratrol per day) Trol (1.25 mg / kg trans-resveratrol per day), 0.31 mg / kg quercetin dihydrate per day, 0.94 mg / kg rice bran extract per day, 4.75 mg / kg per day Rice bran oil and per day 0.70 mg / kg sunflower lecithin)). The mice were observed until they reached 5 months of age.

  Body weight, serum glucose level, serum insulin level and lipid peroxidation reactants in brain and muscle tissue were measured. The results indicated that Longevinex® did not cause weight gain and was distinguishable from control animals (FIG. 1). Serum insulin levels were found to be approximately the same as that seen in calorie restricted animals (FIG. 2). Serum glucose levels were found to be lower than those seen in calorie restricted animals (FIG. 3).

(Example 2)
Comparison of the effects of resveratrol and the composition of the invention on gene expression in heart tissue of the gene expressed in the heart tissue of mice receiving resveratrol or the composition of the invention (Longevinex®) Profiles were compared to those of mice on a calorie restricted diet and control mice. Gene expression was measured using an Affymetrix MG430 2.0 array containing 45,101 probe sets per array. When the array represented the same gene by multiple probes, the probe set with the highest signal intensity was used. Unknown genes, including uncharacterized EST and cDNA sequences, were not analyzed. The array therefore provided a means to analyze 20,341 genes with a single Entrez Gene ID. The analysis is substantially as described in Lee, C.-K. (2002), “Transcribable Profiles Associated With Ageing And Middle Age-Onset Coloric Restriction In Mouse Hearts”, Proc. Natl. Acad. Sci. (U.S.A.) 99: 14988-14993. The average value of all arrays in a group was calculated. The mean value of the treated group was compared with the mean value of the control group, and any statistical significance was determined using a two-tailed t-test (P <0.01). The results of the analysis are shown in Table 3 (CO, control; CR, calorie restriction; RES, resveratrol; LGX, Longevinex®; FC, fold change. FC is the mean value of the treated group as the control group This value is then logarithmically transformed (bottom 2) for statistical purposes, for example, 100 genes in the control and 200 expressed genes in the treated group have 2 Fc Will have (ie, a 2-fold increase in expression), and a gene that expresses 100 in the control and 50 in the treated group will have a Fc of −2 (ie, a 2-fold decrease in expression).

  Treatment of human umbilical vein epithelial cells with ferulic acid, quercetin or resveratrol resulted in over-regulation of over 363 genes out of 10,000 investigated and up-regulation of over 233 genes (Nicholson, S. K. et al., (2008), "Effects of Dietary Polyphenols On Gene Expression in Human Vascular Endoceral Cells", Proc. (I): 42-47). On the other hand, Table 3 shows that 2,829 genes were found to show statistically significant changes in expression in treated versus control mice. For these genes, 7% were found to show altered expression in mice that received only caloric restriction, and 8% were found to show altered expression in mice that only received resveratrol. It was issued. The combination of calorie restriction and resveratrol administration failed to alter the expression of some additional genes. In contrast, administration of Longevinex® was found to change 61% expression of 2,829 genes. Administration of Longevinex® to calorie restricted mice was found to alter the expression of an additional 2% of genes. Administration of Longevinex® to mice receiving resveratrol was found to alter the expression of an additional 21% of genes. Therefore, Longevinex® alone or in combination with other regimens was found to affect 85% (2,406) of the total genes exhibiting altered expression. .

特に興味深いいくつかの遺伝子は、本発明の配合物（Ｌｏｎｇｅｖｉｎｅｘ（登録商標））が、レスベラトロールより大きい程度まで、生存／長寿遺伝子を上方制御するかまたはその発現が細胞傷害を増大させる遺伝子を下方制御することを示す発現パターンを示した：
（Ａ）遺伝子のサーチュインファミリー、特にサーチュイン１は、寿命の延長に決定的に重要な媒介物であると考えられている（Ｂｏｉｌｙ， Ｇ．ら、（２００８年）、「ＳｉｒＴｌ Ｒｅｇｕｌａｔｅｓ Ｅｎｅｒｇｙ Ｍｅｔａｂｏｌｉｓｍ Ａｎｄ Ｒｅｓｐｏｎｓｅ Ｔｏ Ｃａｌｏｒｉｃ Ｒｅｓｔｒｉｃｔｉｏｎ Ｉｎ Ｍｉｃｅ」、ＰＬｏＳ ＯＮＥ ３巻（３号）：ｅｌ７５９；Ｈｕａｎｇ， Ｊ．ら、（２００８年）、「ＳＩＲＴｌ Ｏｖｅｒｅｘｐｒｅｓｓｉｏｎ Ａｎｔａｇｏｎｉｚｅｓ Ｃｅｌｌｕｌａｒ Ｓｅｎｅｓｃｅｎｃｅ ｗｉｔｈ Ａｃｔｉｖａｔｅｄ ＥＲＫ／Ｓ６ｋ１ Ｓｉｇｎａｌｉｎｇ ｉｎ Ｈｕｍａｎ Ｄｉｐｌｏｉｄ Ｆｉｂｒｏｂｌａｓｔｓ」、ＰＬｏＳ ＯＮＥ ３巻（３号）：ｅｌ７１０）。レスベラトロールを受けているマウスは１．２２倍の発現における減少を示すのみであり、カロリー制限食を受けるマウスはサーチュイン１発現における１．１２倍の低下を示すのみであるが、サーチュイン１の発現が、Ｌｏｎｇｅｖｉｎｅｘ（登録商標）を受けているマウスにおいては１．７１倍減少されることが見出された。
（Ｂ）Ｐｇｃ−１α（ペルオキシソーム増殖活性化受容体γコアクチベーター１α；ｐｐａｒｇｃ１ａ）は、エネルギー代謝およびミトコンドリアの生物発生を制御する転写補助因子であり、その発現は長期間のカロリー制限によって骨格筋組織中で増加する（Ｃｏｎｌｅｙ， Ｋ．Ｅ．ら、（２００７年）、「Ｍｉｔｏｃｈｏｎｄｒｉａｌ Ｄｙｓｆｕｎｃｔｉｏｎ ａｎｄ Ａｇｅ」、Ｃｕｒｒ． Ｏｐｉｎ． Ｃｌｉｎ． Ｎｕｔｒ． Ｍｅｔａｂ． Ｃａｒｅ． １０巻（６号）：６８８〜６９２頁；Ｗｕ， Ｚ．ら、（２００７年）、「Ｔａｒｇｅｔｉｎｇ ＰＧＣ−１ Ａｌｐｈａ Ｔｏ Ｃｏｎｔｒｏｌ Ｅｎｅｒｇｙ Ｈｏｍｅｏｓｔａｓｉｓ」、Ｅｘｐｅｒｔ Ｏｐｉｎ． Ｔｈｅｒ． Ｔａｒｇｅｔｓ １１巻（１０号）：１３２９〜１３３８頁）。Ｐｇｃ−１αの発現を、レスベラトロールを受けているマウスはわずかに１．６倍の発現の増加を示したのみであり、カロリー制限食を受けるマウスは増加を示さなかったが、Ｌｏｎｇｅｖｉｎｅｘ（登録商標）を受けているマウスは、１．９４倍のＰｇｃ−１αの発現の増加を示した。
（Ｃ）脱共役タンパク質−３は、Ｐｇｃ−１αの標的であって脂肪酸代謝における役割を果たすと考えられ、その発現は、長期間カロリー制限すると心臓組織中で増加する（Ｂｅｚａｉｒｅ， Ｖ．ら、（イーパブ２００７年１月３日）、「Ｕｎｃｏｕｐｌｉｎｇ Ｐｒｏｔｅｉｎ−３： Ｃｌｕｅｓ Ｉｎ Ａｎ Ｏｎｇｏｉｎｇ Ｍｉｔｏｃｈｏｎｄｒｉａｌ Ｍｙｓｔｅｒｙ」、ＦＡＳＥＢ Ｊ． ２１巻（２号）：３１２〜３２４頁；Ｃｈａｎ， Ｃ．Ｂ．ら、（２００６年）、「Ｕｎｃｏｕｐｌｉｎｇ Ｐｒｏｔｅｉｎｓ： Ｒｏｌｅ Ｉｎ Ｉｎｓｕｌｉｎ Ｒｅｓｉｓｔａｎｃｅ Ａｎｄ Ｉｎｓｕｌｉｎ Ｉｎｓｕｆｆｉｃｉｅｎｃｙ」、Ｃｕｒｒ． Ｄｉａｂｅｔｅｓ Ｒｅｖ． ２巻（３号）：２７１〜２８３頁）。脱共役タンパク質−３発現において、レスベラトロールを受けているマウスはわずかに２．０２倍の発現の増加を示したのみであり、カロリー制限食を受けるマウスはわずかに１．８倍の増加を示したのみであったが、Ｌｏｎｇｅｖｉｎｅｘ（登録商標）を受けているマウスは、２．７９倍の脱共役タンパク質−３の発現の増加を示した。
（Ｄ）ピルビン酸デヒドロゲナーゼキナーゼ４は、絶食中脂肪酸代謝を促進するために燃料の選択を調整する（Ｓｕｇｄｅｎ， Ｍ．Ｃ．ら、（２００６年）、「Ｍｅｃｈａｎｉｓｍｓ Ｕｎｄｅｒｌｙｉｎｇ Ｒｅｇｕｌａｔｉｏｎ Ｏｆ Ｔｈｅ Ｅｘｐｒｅｓｓｉｏｎ Ａｎｄ Ａｃｔｉｖｉｔｉｅｓ Ｏｆ Ｔｈｅ Ｍａｍｍａｌｉａｎ Ｐｙｒｕｖａｔｅ Ｄｅｈｙｄｒｏｇｅｎａｓｅ Ｋｉｎａｓｅｓ」、Ａｒｃｈ． Ｐｈｙｓｉｏｌ． Ｂｉｏｃｈｅｍ． １１２巻（３号）：１３９〜１４９頁；Ｐｉｌｅｇａａｒｄ， Ｈ．ら、（２００４年）、「Ｔｒａｎｓｃｒｉｐｔｉｏｎａｌ Ｒｅｇｕｌａｔｉｏｎ Ｏｆ Ｐｙｒｕｖａｔｅ Ｄｅｈｙｄｒｏｇｅｎａｓｅ Ｋｉｎａｓｅ ４ Ｉｎ Ｓｋｅｌｅｔａｌ Ｍｕｓｃｌｅ Ｄｕｒｉｎｇ Ａｎｄ Ａｆｔｅｒ Ｅｘｅｒｃｉｓｅ」、Ｐｒｏｃ． Ｎｕｔｒ． Ｓｏｃ． ６３巻（２号）：２２１〜２２６頁； Ｓｕｇｄｅｎ， Ｍ．Ｃ．、（２００３年）、「ＰＤＫ４： Ａ ｆａｃｔｏｒ ｉｎ ｆａｔｎｅｓｓ？」、Ｏｂｅｓ． Ｒｅｓ． １１巻（２号）：１６７〜１６９頁）。それは、Ｐｇｃ−１αの標的であり、長期間のカロリー制限によって多くの組織中に誘導される。ピルビン酸デヒドロゲナーゼキナーゼ４の発現において、レスベラトロールを受けているマウスはわずかに２．７８倍の発現の増加を示したのみであり、カロリー制限食を受けるマウスはわずかに１．４８倍の増加を示したのみであったが、Ｌｏｎｇｅｖｉｎｅｘ（登録商標）を受けているマウスは、３．２５倍のピルビン酸デヒドロゲナーゼキナーゼ４の発現の増加を示した。

Some genes of particular interest include genes whose formulation (Longevinex®) up-regulates the survival / longevity gene to an extent greater than resveratrol or whose expression increases cytotoxicity. An expression pattern showing down-regulation was shown:
(A) The sirtuin family of genes, in particular sirtuin 1, is considered to be a critical mediator for life extension (Boilly, G. et al. (2008), “SirTl Regulates Energy Metabolism And Responses”. To Caloric Restriction In Mice ", PLoS ONE Volume 3 (No. 3): el759; Huang, J. et al. Volume (3): el710). Mice receiving resveratrol only show a 1.22 fold decrease in expression and mice receiving a calorie restricted diet show only a 1.12 fold decrease in sirtuin 1 expression, while sirtuin 1 Expression was found to be reduced 1.71 times in mice receiving Longevinex®.
(B) Pgc-1α (peroxisome proliferator activated receptor γ coactivator 1α; ppargc1a) is a transcriptional cofactor that regulates energy metabolism and mitochondrial biogenesis, and its expression is skeletal muscle by long-term calorie restriction. Increased in tissues (Conley, KE et al. (2007), “Mitochondral Dysfunction and Age”, Curr. Opin. Clin. Nutr. Metab. Care. 10 (6): 688-692; Wu, Z. et al. (2007), “Targeting PGC-1 Alpha To Control Energy Homeostasis”, Expert Opin. Ther. Targets 11 (10): 1329-1338). Mice receiving resveratrol showed only a 1.6-fold increase in expression of Pgc-1α, while mice receiving a calorie restricted diet showed no increase, but Longevinex® Mice receiving the trademark) showed a 1.94-fold increase in expression of Pgc-1α.
(C) Uncoupling protein-3 is thought to be a target of Pgc-1α and play a role in fatty acid metabolism, and its expression increases in heart tissue upon long-term calorie restriction (Bezaire, V. et al., (Epub, Jan. 3, 2007), “Uncoupling Protein-3: Blues In An Ongoing Mitochondrial Mystery”, FASEB J. 21 (2): 312-324; Chan, CB et al., (2006). ), “Uncoupling Proteins: Role In Insulin Resistance And Insulin Insufficiency”, Curr. Diabetes Rev. 2 (No. 3): 271-283). In uncoupling protein-3 expression, mice receiving resveratrol showed only a 2.02-fold increase in expression, while mice receiving a calorie-restricted diet showed a 1.8-fold increase. Although only shown, mice receiving Longevinex® showed a 2.79-fold increase in uncoupled protein-3 expression.
(D) Pyruvate dehydrogenase kinase 4 regulates fuel selection to promote fasting fatty acid metabolism (Sugden, MC, et al. (2006), “Mechanisms Underlying Regulation of the Expressions of Activities”. Mammalian Pyruvate Dehydrogenase Kinases ", Arch. Physiol. Biochem. 112 (No. 3): 139-149; Pilegaard, H. et al. ercise ", Proc. Nutr. Soc. 63 (2): 221-226; Sugden, MC, (2003)," PDK4: A factor in fatness? ", Obes.Res. 2): 167-169). It is a target for Pgc-1α and is induced in many tissues by prolonged caloric restriction. Mice receiving resveratrol showed only a 2.78-fold increase in expression of pyruvate dehydrogenase kinase 4, while mice receiving a calorie-restricted diet only increased 1.48-fold. The mice receiving Longevinex® showed a 3.25-fold increase in pyruvate dehydrogenase kinase 4 expression.

  Analysis of genes that are up- or down-regulated by the formulation of the present invention (Longevinex®) revealed that oxidative phosphorylated genes involved in mitochondrial ATP production are markedly up-regulated. (Table 4).

（実施例４）
本発明の組成物により影響を受ける生化学経路
最近の研究は、複雑な形質が、複雑な遺伝子の座位および環境要因によって調節される分子ネットワークの創発（ｅｍｅｒｇｅｎｔ）特性であることを示唆している。Ｃｈｅｎ， Ｙ．ら、（イーパブ２００８年３月１６日）、「Ｖａｒｉａｔｉｏｎｓ Ｉｎ ＤＮＡ Ｅｌｕｃｉｄａｔｅ Ｍｏｌｅｃｕｌａｒ Ｎｅｔｗｏｒｋｓ Ｔｈａｔ Ｃａｕｓｅ Ｄｉｓｅａｓｅ」、Ｎａｔｕｒｅ ４５２巻（７１８６号）：４２９〜４３５頁）。

Example 4
Biochemical pathways affected by the composition of the present invention Recent studies suggest that complex traits are an emerging feature of molecular networks regulated by complex gene loci and environmental factors . Chen, Y. et al. (Epub, Mar. 16, 2008), “Variations In DNA Elucidate Molecular Networks That Cause Disease”, Nature 452 (7186): 429-435).

  In fact, research over the last decade has shown that most chronic diseases, such as cancer, cardiovascular and respiratory diseases, nervous system diseases, diabetes, and autoimmune diseases, are multiple cells. It has been shown to show dysregulation of signal transduction pathways (Harikumar, KB et al., (Epub Pub. 15 Feb. 2008), “Resvertrol: A Multiagent Aged-Associated Chronic Diseases”, Cell Cell. 2008: Volume 7 (No. 8)). The formulations of the present invention therefore evaluated their effect on the expression of biochemical pathways and found that (Table 5) affects the expression of genes involved in 220 biological processes (P <0). .05).

カロリー制限は、これらの過程の５％と関係する遺伝子に影響を及ぼし、レスベラトロールの投与はこれらの過程の１０％と関係する遺伝子に影響を及ぼした。本発明の配合物（例えば、Ｌｏｎｇｅｖｉｎｅｘ（登録商標））は、これらの過程の８５％に影響を及ぼすことが見出された。カロリー制限したマウスへのレスベラトロールの投与は、これらの過程のいずれにおいてもどの遺伝子にも影響を及ぼすことに失敗した。カロリー制限したマウスへのＬｏｎｇｅｖｉｎｅｘ（登録商標）の投与は、これらの過程の８％と関係する遺伝子に影響を及ぼすことが見出された。レスベラトロールとＬｏｎｇｅｖｉｎｅｘ（登録商標）の両方の投与は、これらの過程の１２％と関係する遺伝子に影響を及ぼすことが見出された。表６は、カロリー制限（ＣＲ）、レスベラトロール単独（Ｒｅｓ）、または本発明の組成物（ＬＧＸ）により引き起こされる酸化的リン酸化経路（ＧＯ：０００６１１９）の遺伝子の調節を示す。

Caloric restriction affected genes associated with 5% of these processes, and administration of resveratrol affected genes associated with 10% of these processes. Formulations of the present invention (eg, Longevinex®) have been found to affect 85% of these processes. Administration of resveratrol to calorie restricted mice failed to affect any gene in any of these processes. Administration of Longevinex® to calorie restricted mice was found to affect genes associated with 8% of these processes. Administration of both resveratrol and Longevinex® was found to affect genes associated with 12% of these processes. Table 6 shows gene regulation of the oxidative phosphorylation pathway (GO: 0006119) caused by calorie restriction (CR), resveratrol alone (Res), or the composition of the present invention (LGX).

表７は、カロリー制限（ＣＲ）、レスベラトロール単独（Ｒｅｓ）、または本発明の組成物（ＬＧＸ）により引き起こされるグルコース代謝経路（ＧＯ：０００６００６）の遺伝子の調節を示す。

Table 7 shows gene regulation of the glucose metabolic pathway (GO: 006006) caused by calorie restriction (CR), resveratrol alone (Res), or the composition of the invention (LGX).

表８は、カロリー制限（ＣＲ）、レスベラトロール単独（Ｒｅｓ）、または本発明の組成物（ＬＧＸ）により引き起こされるトリカルボン酸代謝経路（ＧＯ：０００６０９９）の遺伝子の調節を示す。

Table 8 shows the regulation of genes of the tricarboxylic acid metabolic pathway (GO: 00000099) caused by calorie restriction (CR), resveratrol alone (Res), or the composition of the present invention (LGX).

表９は、カロリー制限（ＣＲ）、レスベラトロール単独（Ｒｅｓ）、または本発明の組成物（ＬＧＸ）により引き起こされる脂肪酸代謝経路（ＧＯ：０００６６３１）の遺伝子の調節を示す。

Table 9 shows the regulation of genes of the fatty acid metabolic pathway (GO: 0006631) caused by calorie restriction (CR), resveratrol alone (Res), or the composition of the present invention (LGX).

心臓組織中の２０，３４１の遺伝子の発現の調査により、２，８２９の遺伝子が、発現において統計的有意差（Ｐ＜０．０１）を示すことが明らかとなった。これらの７％（ほぼ１８７の遺伝子）は、カロリー制限食のみを受けた動物で発現の変化を示し、８％（ほぼ２２６の遺伝子）は、レスベラトロールのみを受けた動物で発現の変化を示し、レスベラトロールを受けかつカロリー制限食を受けた動物では発現の変化を示す追加の遺伝子は無かった。対照的に、２０，３４１の遺伝子の６１％（ほぼ１，７２９の遺伝子）が、本発明の配合物（例えば、Ｌｏｎｇｅｖｉｎｅｘ（登録商標））のみを受けている動物で発現の変化を示し、追加の２％の遺伝子（ほぼ５６の遺伝子）が、本発明の配合物（例えば、Ｌｏｎｇｅｖｉｎｅｘ（登録商標））を受け、かつカロリー制限食を受けていた動物で発現の変化を示し；追加の２１％の遺伝子（ほぼ５９４の遺伝子）が、本発明の配合物（例えば、Ｌｏｎｇｅｖｉｎｅｘ（登録商標））とレスベラトロールを受けた動物で発現の変化を示し；追加の１％の遺伝子（ほぼ２８の遺伝子）が、本発明の配合物（例えば、Ｌｏｎｇｅｖｉｎｅｘ（登録商標））とレスベラトロールを受け、かつカロリー制限食を受けていた動物で発現の変化を示した。

Investigation of the expression of 20,341 genes in heart tissue revealed that 2,829 genes showed statistically significant differences (P <0.01) in expression. Of these, 7% (approximately 187 genes) showed altered expression in animals that received only a calorie-restricted diet, and 8% (approximately 226 genes) exhibited altered expression in animals that received only resveratrol. There were no additional genes showing altered expression in animals that received resveratrol and received a calorie-restricted diet. In contrast, 61% of the 20,341 genes (approximately 1,729 genes) showed altered expression in animals receiving only the formulations of the invention (eg, Longevinex®) and added 2% of genes (approximately 56 genes) show altered expression in animals that received a formulation of the invention (eg, Longevinex®) and were on a calorie restricted diet; an additional 21% Of genes (approximately 594 genes) show altered expression in animals that received a formulation of the invention (eg, Longevinex®) and resveratrol; an additional 1% gene (approximately 28 genes) ) Showed altered expression in animals that received a formulation of the present invention (eg, Longevinex®) and resveratrol and were on a calorie restricted diet.

  The above data demonstrates that the formulations of the present invention (eg, Longevinex®) were effective in modulating gene expression in heart tissue to a degree that even exceeded that of caloric restriction. is doing. Similar effects have been observed in tissues other than the heart. Investigation of the expression of 20,341 genes in brain tissue revealed that 3,572 genes showed statistically significant differences in expression (P <0.01). Of these, 124 genes showed altered expression in animals receiving only a calorie restricted diet, 424 genes showed altered expression in animals receiving only resveratrol, and 10 genes were less The animals that received veratrol and received a low calorie diet showed altered expression. In contrast, 2560 genes show altered expression in animals receiving only the formulations of the present invention (eg, Longevinex®), and 19 additional genes are present in the formulations of the present invention (eg, , Longevinex®) and showing a change in expression in animals that were on a calorie restricted diet; 430 additional genes were added to the formulations of the present invention (eg, Longevinex®) and Resvera Shows changes in expression in animals that received trawl; 5 additional genes in animals that received a formulation of the invention (eg, Longevinex®) and resveratrol and received a calorie-restricted diet Change in expression was shown.

Example 4
Model mechanism of action of the composition of the present invention The formulations of the present invention thus far exceed the regulation of gene expression seen with respect to caloric restriction, lipid metabolism, glucose metabolism, oxidative phosphorylation, Krebs cycle, ATP synthesis and fatty acids. It has been found to alter gene expression in the main pathway of β-oxidation. In summary, it has been found that the formulations of the present invention have greater specific activity than resveratrol alone, both in terms of the number of affected genes and the number of different biochemical pathways. This result is important because caloric restriction (CR) is considered an obvious way to extend lifespan in all forms of organisms. In general, a 50% reduction in caloric intake doubles the life of any organism. The above experiments have demonstrated that the composition of the present invention exerts a stronger impact on genome expression than resveratrol or CR, indicating that some techniques exceed the effects of CR. First time. Moreover, the compositions of the present invention have been found to affect genomic expression at an earlier stage of life than CR (the CR diet must be followed for life to differentiate genes).

  While not intending to be bound by any mechanism of action, the above results indicate that the formulations of the present invention act by increasing the activity of the forkhead Foxo1 (daf-16, dFoxO) transcription factor. This suggests (Fig. 5). Investigations in model organisms indicate that Foxo1 mediates longevity expression by enhancing gene expression. Insulin / IGF-1 signaling phosphorylates Foxo1, thereby leading to its elimination from the nucleus and down-regulating its action. The formulations of the invention reduce insulin and IGF-1 signaling, thereby reducing Foxo1 phosphorylation. Consistent with this model is the observation that the insulin receptor signaling pathway (eg, GO: 008286; genes Ide, Igfbp4, and Igfbp6) is affected by the formulations of the present invention. Foxo1 expression is increased 1.75 times. The formulations of the present invention result in decreased glycolysis and increased gluconeogenesis (eg, GO: 006006), enhanced Pgc-1α expression, thereby stimulating Pdk4 expression (eg, 1.94-fold increase in Pparge1α) And 3.25-fold increase in Pdk4), increased expression of lipid metabolism genes (eg, 2.79-fold increase in Ucp3, 1.49-fold increase in Cpt1a, and 1.45-fold increase in Cpt1b) Lipid and fatty acid metabolism genes GO: 0006629 and GO: 0006635 are affected in a unique way by the formulations of the invention, which are thus affected by caloric restriction and resveratrol. Major processes (eg chromatin remodeling, RNA polymerase II promotion The genes GO: 0006333 and GO: 0006367 are affected in a unique way by the formulations of the present invention, and the gene GO: 0006512 is less susceptible to transcription from the protein and the ubiquitin cycle. Thus, in summary, the mechanism of action presented is that the composition of the present invention delivers resveratrol to cells where it is cell wall. And enter the cytoplasm and promote the transfer of the Foxo1 gene into the cell nucleus, thereby producing a longevity effect.

  All publications and patents mentioned in this specification are referenced to the same extent as if each individual publication or patent application was shown to be expressly and individually incorporated by reference in its entirety. Is incorporated herein by reference. Although the present invention has been described in connection with specific embodiments thereof, it will be appreciated that it can be further modified, and that this application generally follows the principles of the present invention and Any variation, application, or adaptation of the present invention, including those that depart from the present disclosure as falling within the scope of known or customary practice of the technology and that may apply to the essential features shown above It is intended to cover.

Claims (20)

  A composition comprising resveratrol, wherein when administered to a recipient, the concentration of a product of a survival / longevity gene or a gene product whose expression increases cytotoxicity relative to resveratrol alone or caloric restriction or A composition comprising resveratrol that modulates activity. The adjustment in the recipient is
(A) oxidative phosphorylation,
(B) actin filament length or polymerization,
(C) intracellular transport,
(D) Organelle biosynthesis,
(E) Insulin signaling,
(F) glycolysis,
The composition comprising resveratrol according to claim 1, which alters (G) gluconeogenesis or (H) fatty acid metabolism.   A composition comprising resveratrol according to any of claims 1 to 2, wherein the product of the survival / longevity gene is sirtuin 1 or forkhead Foxo1 transcription factor.   4. A composition comprising resveratrol according to any of claims 1 to 3, wherein the gene whose expression increases cytotoxicity encodes uncoupling protein 3 or pyruvate dehydrogenase kinase 4. The composition is
Comprising (a) trans-resveratrol, and (b) a metal chelator, wherein said trans-resveratrol is encapsulated, whereby the product of the survival / longevity gene or expression thereof is cytotoxic. 5. The ability of the composition to modulate the product concentration or activity of the gene to be increased substantially protects against loss of the trans-resveratrol from exposure to light or oxygen. A composition comprising resveratrol according to claim 1.   6. A composition comprising resveratrol according to claim 5, wherein the metal chelator is nordihydroguaiaretic acid.   6. A composition comprising resveratrol according to claim 5, wherein the metal chelator is phytic acid.   The composition comprising resveratrol according to claim 5, wherein the composition further comprises quercetin.   The composition comprising resveratrol according to any of claims 5 to 8, wherein the composition further comprises hyaluronic acid.   The composition comprising resveratrol according to any of claims 5 to 9, wherein the composition further comprises vitamin D.   11. A composition comprising resveratrol according to any of claims 5 to 10, wherein the resveratrol encapsulation is microencapsulation.   Use of a composition comprising resveratrol for improving a symptom associated with a disease of an existing individual or for producing a medicament for preventing the onset of the symptom in the individual before the disease occurs in the individual A composition comprising said resveratrol modulates the concentration or activity of a product of a survival / longevity gene or a gene whose expression increases cytotoxicity compared to resveratrol alone or caloric restriction And the use of a composition comprising resveratrol selected from the group consisting of cardiovascular disease, cancer, macular degeneration, diseases associated with aging, and inflammation. In the individual, the adjustment is
(A) oxidative phosphorylation,
(B) actin filament length or polymerization,
(C) intracellular transport,
(D) Organelle biosynthesis,
(E) Insulin signaling,
(F) glycolysis,
Use of a composition comprising resveratrol according to claim 12, which alters (G) gluconeogenesis or (H) fatty acid metabolism.   Use of a composition comprising resveratrol according to any of claims 12 to 13, wherein the product of the survival / longevity gene is sirtuin 1 or forkhead Foxo1 transcription factor.   Use of a composition comprising resveratrol according to any of claims 12 to 14, wherein the gene whose expression increases cytotoxicity encodes uncoupling protein 3 or pyruvate dehydrogenase kinase 4. The composition is
Comprising (a) trans-resveratrol, and (b) a metal chelator, wherein said trans-resveratrol is encapsulated, whereby the product of the survival / longevity gene or expression thereof is cytotoxic. 16. The ability of the composition to modulate the product concentration or activity of the gene to be increased substantially protects the trans-resveratrol from loss due to light or oxygen exposure. Use of a composition comprising resveratrol according to claim 1.   Use of a composition comprising resveratrol according to any of claims 12 to 16, wherein the disease is cancer.   Use of a composition comprising resveratrol according to any of claims 12 to 16, wherein the disease is a disease associated with aging.   Use of a composition comprising resveratrol according to claim 18, wherein the disease associated with aging is a neurodegenerative disease.   20. Use of a composition comprising resveratrol according to any of claims 12 to 19, wherein the composition further comprises quercetin, hyaluronic acid and / or vitamin D.

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