WO2009039195A1 - Resveratrol-containing compositions for modulating gene product concentration or activity - Google Patents

Resveratrol-containing compositions for modulating gene product concentration or activity Download PDF

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WO2009039195A1
WO2009039195A1 PCT/US2008/076707 US2008076707W WO2009039195A1 WO 2009039195 A1 WO2009039195 A1 WO 2009039195A1 US 2008076707 W US2008076707 W US 2008076707W WO 2009039195 A1 WO2009039195 A1 WO 2009039195A1
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lgx
res
resveratrol
containing composition
gene
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PCT/US2008/076707
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French (fr)
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William F. Sardi
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Resveratrol Partners, Llc
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Priority to CA2699908A priority Critical patent/CA2699908C/en
Priority to EP08831905A priority patent/EP2197434A4/en
Priority to JP2010525922A priority patent/JP2010540444A/en
Publication of WO2009039195A1 publication Critical patent/WO2009039195A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a resveratrol-containing composition capable of modulating gene expression to an extent greater than that observed with resveratrol alone or with calorie restriction.
  • the invention particularly pertains to such resveratrol-containing compositions that comprise resveratrol, a chelator, hyaluronic acid, and/or vitamin D and which, upon administration to a recipient, 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 cellular damage.
  • the invention particularly relates to the use of such compositions in the treatment or prevention of cancer, cardiovascular disease, diseases associated with aging, and other conditions and illnesses.
  • Resveratrol (3,4',5-trihydroxy-?r ⁇ «,y-stilbene) is a naturally occurring phenolic compound found, for example in grape skins, that has been demonstrated to have beneficial properties relating to health of humans (Das, S. et al. (2007) “Resveratrol: A Therapeutic Promise For Cardiovascular Diseases " Recent Patents Cardiovasc. Drug Discov. 2(2): 133- 138; Mancuso, C. et al. (2007) "Natural antioxidants in Alzheimer's disease " Expert Opin. Investig. Drugs. 16(12): 1921-1931 ; Baumann L. (2007) “Botanical Ingredients In Cosmeceuticals " J. Drugs Dermatol.
  • resveratrol is believed to be beneficial to the functioning of the heart and in extending the life of human cells.
  • Resveratrol when used in dietary supplements, is generally produced as an alcohol extract from plant sources.
  • Calorie restricted diets have been shown to enhance survival and longevity by up- regulating survival/longevity genes or down-regulating genes whose expression enhances cellular damage (Edwards, M.G. et al. (2007) "Gene Expression Profiling Of Aging Reveals Activation Of A P53-Mediated Transcriptional Program," BMC Genomics 8:80; Anderson, R.M. et al. (2006) “Calorie Restriction: Progress During Mid-2005-Mid-2006 " Exp. Gerontol. 41(12): 1247-1249; Weindruch, R. et al. (2001) "Microarray Profiling of Gene Expression in Aging and Its Alteration by Caloric Restriction in Mice," J.
  • mice have been used extensively as a model for genetic expression comparisons with humans. Without limitation, the validity of murine models to human gene expression reflects the fact that 98% of human and murine gene are homologous, and that mice and humans have about the same number of genes (e.g., approximately 30,000).
  • the present invention relates to a resveratrol-containing composition capable of modulating gene expression to an extent greater than that observed with resveratrol alone or with calorie restriction.
  • the invention particularly pertains to such resveratrol-containing compositions that comprise resveratrol, a chelator, hyaluronic acid, and/or vitamin D and which, upon administration to a recipient, 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 cellular damage.
  • the resveratrol-stabilizing composition will comprise the chelator phytic acid (inositol hexaphosphate; IP6), hyaluronic acid, and vitamin D.
  • IP6 chelator phytic acid
  • the invention further pertains to the use of such compositions to up- regulate a survival/longevity gene or down-regulate a gene whose expression enhances cellular damage upon administration to a recipient.
  • the invention particularly relates to the use of such compositions in the treatment or prevention of cancer, cardiovascular disease, diseases associated with aging, and other conditions and illnesses.
  • the invention provides a resveratrol-containing composition that, upon administration to a recipient, modulates the concentration or activity, relative to resveratrol alone or calorie restriction, of the product of a survival/longevity gene or the product of a gene whose expression enhances cellular damage.
  • Administration is preferably by oral ingestion.
  • the invention further provides the embodiments of such compositions wherein the modulation alters:
  • the invention further provides the embodiments of such compositions wherein the gene product is a survival/longevity gene product, and especially wherein the gene product is sSirtuin 1, or the forkhead Foxol transcription factor.
  • the invention further provides the embodiments of such compositions wherein the gene product is a gene product that enhances cellular damage, and especially wherein the gene product is encoded by the uncoupling protein 3, Pgc-1, or pyruvate dehydrogenase kinase 4 genes.
  • composition comprises:
  • the invention further provides a method of ameliorating a symptom associated with an existing disease of an individual or for preventing the onset of the symptom in an individual prior to the occurrence of the disease in the individual, which comprises administering to the individual, a resveratrol-containing composition that modulates the concentration or activity, relative to resveratrol alone or calorie restriction, of the product of a survival/longevity gene or the product of a gene whose expression enhances cellular damage, wherein the resveratrol is provided in an amount effective to cause a modulation of the concentration or activity of the gene that ameliorates the symptom of the disease, and wherein the disease is selected from the group consisting of: cardiovascular disease, cancer, macular degeneration, a disease associated with aging, and inflammation.
  • the invention further provides the embodiments of such method wherein the modulation alters:
  • the invention further provides the embodiments of such method wherein the survival/longevity gene product is Sirtuin 1 or the forkhead Foxol transcription factor.
  • the invention further provides the embodiments of such method wherein the gene whose expression enhances cellular damage encodes uncoupling protein 3 or pyruvate dehydrogenase kinase 4.
  • composition comprises:
  • the invention further provides the embodiments of such method wherein the disease is cancer, or a disease associated with aging (especially a neurodegenerative disease).
  • the invention further provides the embodiments of such method wherein the composition additionally comprises quercetin, hyaluronic acid and/or vitamin D. [0019] The invention further provides the embodiments of such method wherein the modulation is relative to resveratrol alone or wherein the modulation is relative to calorie restriction.
  • the invention further provides the embodiments of such methods wherein the gene product is a survival/longevity gene product, and especially wherein the gene product is Sirtuin 1 , or the forkhead Foxo 1 transcription factor.
  • the invention further provides the embodiments of such methods wherein the gene product is a gene product that enhances cellular damage, and especially wherein the gene product is encoded by the uncoupling protein 3, Pgc-1, or pyruvate dehydrogenase kinase 4 gene.
  • Figure 1 shows the change in body weight of mice administered resveratrol or a composition of the present invention (Longevinex®) relative to control animals and animals maintained on a calorie restricted diet.
  • Figure 2 shows the serum insulin level of mice administered resveratrol or a composition of the present invention (Longevinex®) relative to control animals and animals maintained on a calorie restricted diet.
  • Figure 4 shows a schematic of a mechanism of action that is consistent with the observed biological activities of the compositions of the present invention.
  • the present invention relates to a resveratrol-containing composition (and especially a resveratrol-containing dietary composition (i.e., a composition amenable for oral ingestion by a recipient)) capable of modulating gene expression to an extent greater than that observed with resveratrol alone or with calorie restriction.
  • the invention particularly pertains to such resveratrol-containing compositions that comprise resveratrol, a chelator, hyaluronic acid, and/or vitamin D and which up-regulate a survival/longevity gene or down-regulate a gene whose expression enhances cellular damage upon administration to a recipient.
  • the resveratrol-stabilizing composition will comprise the chelator phytic acid (inositol hexaphosphate; IP6), hyaluronic acid, and vitamin D.
  • IP6 chelator phytic acid
  • the invention further pertains to the use of such compositions to up-regulate a survival/longevity gene or down-regulate a gene whose expression enhances cellular damage upon administration to a recipient.
  • the mineral chelators of the present invention provide anti-aging effects, as evidence in differentiation of the genome.
  • resveratrol refers to the phytoalexin: 3,4',5-trihydroxy- trans-stilbcnc having the structure:
  • Resveratrol has been ascribed multiple beneficial biological effects (see, United States Patent No. 7,345,178, which listing of disclosed effects is herein incorporated by reference), including preventing or treating cardiovascular disease (see, e.g., Das, S. et al. (2007) “Resveratrol: A Therapeutic Promise For Cardiovascular Diseases " Recent Patents Cardiovasc. Drug Discov. 2(2): 133- 138), Opie, L.H. et al. (Epub 2007 Jun 7) "The Red Wine Hypothesis: From Concepts To Protective Signaling Molecules " Eur. Heart J. 28(14): 1683- 1693; Bertelli, A. A.
  • Reseveratrol may be synthesized chemically (Farina, A. et al. (2006) “An Improved Synthesis Of Resveratrol,” Nat. Prod. Res. 20(3):247-252), or, more preferably, may be extracted from plant sources. Resveratrol is found in at least 72 species of plants distributed among 31 genera and 12 families (see, Counet, C. et al. (2006) “Chocolate And Cocoa: New Sources Of Trans-Resveratrol And Trans-Piceid,” Food Chem. 98:649-657; Jang, M. et al.
  • the invention pertains to compositions that, upon administration to a recipient, increase the concentration or activity of a survival/longevity gene product and/or decrease the concentration or activity of a gene product that induces or causes cellular damage.
  • increase (or decrease) in concentration or activity may be accomplished by any mechanism.
  • increase (or decrease) may reflect a modulation of gene expression resulting in either increased (or decreased) expression of the gene encoding the survival/longevity gene product, or a gene that regulates (e.g., induces or represses) or whose product regulates such expression or activity.
  • such increase (or decrease) in concentration or activity may reflect a modulation of the recipient's ability to degrade or stabilize any such gene products.
  • such increase (or decrease) in concentration or activity may reflect a modulation of the recipient's ability to enhance, accelerate, repress or decelerate the activity of any such gene products.
  • the modulation of concentration or activity discussed above may be a modulation of intracellular, intercellular and/or tissue concentration or activity of such survival/longevity gene products or such gene products that induce or cause cellular damage.
  • Such modulation may be identified by assays of DNA expression, assays of gene product activity, assays of the level of gene product, assays of the rate of gene product turnover, etc. conducted in one or more types of cells, tissues, etc.
  • An increase in the concentration of a survival/longevity gene product may result from, for example, increased transcription of the gene that encodes the survival/longevity gene product, increased transcription of a gene that induces the expression of the gene that encodes the survival/longevity gene product, decreased transcription of a gene that represses the expression of the gene that encodes the survival/longevity gene product, decreased degradation or enhanced stabilization of expressed molecules of the survival/longevity gene product (leading to the enhanced accumulation of the survival/longevity gene product).
  • a decrease in the concentration of a survival/longevity gene product may result from, for example, decreased transcription of the gene that encodes the survival/longevity gene product, decreased transcription of a gene that induces the expression of the gene that encodes the survival/longevity gene product, increased transcription of a gene that represses the expression of the gene that encodes the survival/longevity gene product, increased degradation or decreased stabilization of expressed molecules of the survival/longevity gene product (leading to the enhanced dissipation of the survival/longevity gene product).
  • One aspect of the present invention thus relates to the use of resveratrol and resveratrol-containing compositions to modulate gene expression, and in particular, to modulate the expression of "survival/longevity" genes and/or "damage inducing" genes.
  • a compound is said to "modulate” gene expression if its administration results in a change in expression (relative to a control) of such genes of at least 10%. Modulation may involve an increase in expression ("up-regulation") or it may involve a decrease in expression (“down- regulation").
  • up-regulate thus denotes an increase of 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 (relative to a control).
  • down-regulate conversely denotes a decrease of 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 (relative to a control).
  • a second aspect of the present invention thus relates to the use of resveratrol and resveratrol-containing compositions to modulate the concentration or activity of expressed products of "survival/longevity" genes and/or "damage inducing" genes.
  • a compound is said to "modulate” the concentration or activity of such expressed products if its administration results in a change in an intracellular, intercellular or tissue concentration or activity (relative to a control) of such gene products of at least 10%.
  • Modulation may, for example, involve an "enhanced accumulation” or an “enhanced activity” or, for example, it may involve a “diminished accumulation” or a “diminished activity.”
  • the term “enhanced accumulation” (or “enhanced activity”) denotes an increase in concentration (or activity) 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 (relative to a control).
  • diminished accumulation or “diminished activity.” conversely denotes a decrease in concentration (or activity) 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 (relative to a control).
  • a "survival/longevity" gene is a gene whose expression contributes to an increase in the survival or longevity of a subject (e.g., a mammal, and particularly a human) expressing such gene.
  • a “damage inducing” gene is a gene whose expression contributes to DNA, cellular, or tissue damage in such subject.
  • Such genes are responders to biological stressors, they initiate action in response to stressors such as radiation (e.g., sunlight, gamma rays, UV light, etc.), radiomimetic agents (e.g., vitamin D), heat, near starvation (calorie restriction, or its mimetic, resveratrol) by modulating their expression.
  • Examples of survival/longevity genes are provided in Table 1. Examples of genes whose expression enhances cellular damage are provided in Table 2. These Tables provide the gene's NCBI "ENTREZGENE” accession number. Most preferably, such genes are human genes.
  • the Sirtuin 1 gene is known to control the rate of aging of living organisms by virtue of its ability to produce DNA repair enzymes and mimics the beneficial effects of calorie restriction.
  • the trans form of resveratrol (but not czs-resveratrol) activates the Sirtuin 1 gene (Alcendor, R.R.
  • the invention particularly pertains to compositions that increase the concentration of the Sirtuin 1 survival/longevity gene product.
  • the invention further particularly pertains to compositions that increase the concentration of the forkhead Foxol (daf-16, dFoxO) transcription factor survival/longevity gene product.
  • the invention particularly pertains to resveratrol-containing compositions in which the specific activity of the resveratrol has been stabilized or enhanced.
  • specific activity refers to the ratio of the extent of gene modulation (relative to control) per amount (mass) of administered resveratrol.
  • compositions will comprise a chelator, hyaluronic acid, and/or vitamin D.
  • the invention particularly pertains to such compositions that comprise resveratrol (preferably, the compositions of the present invention will provide a composition dosage of from about 10 mg to about 2 g, more preferably from about 100 mg to about 500 mg), and at least one compound selected from the group consisting of an antioxidant (chelator), hyaluronic acid, and vitamin D.
  • the compositions of the present invention will contain resveratrol, an antioxidant, hyaluronic acid, and vitamin D.
  • chelator refers to an organic compound that bonds with and removes free metal ions from solution.
  • suitable chelators include ethylenediaminetetraacetic acid (EDTA), histidine, antibiotic drugs of the tetracycline family, pyridoxal 2-chlorobenzoyl hydrazone, desferrioxamine, dexrazoxane, deferasirox, pyoverdine, pseudan, citrate, NDGA (nordihydroguaiaretic acid: l,4-bis[3,4-dihydroxyphenyl]2,3- dimethylbutane), ferulic acid and phytic acid.
  • the compositions of the present invention will provide a composition dosage of chelator 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 chelator for the purposes of the present invention.
  • the term “phytic acid” refers to inositol hexaphosphate ((2,3,4,5,6- pentaphosphonooxycyclohexyl) dihydrogen phosphate; also known as "IP6";) (see, Thome 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 Inositol,” Nutr. Cancer. 55(2): 109-125; Lopez, M.A. et al.
  • IP6 is an Anti-Neoplastic And Lipid-Lowering Agent
  • IP6 is an Anti-Neoplastic And Lipid-Lowering Agent
  • IP6 is an Anti-Neoplastic And Lipid-Lowering Agent
  • Phytic acid is found in substantial amounts in whole grains, cereals, legumes, nuts, and seeds, and is the primary energy source for the germinating plant (Graf, E. (1983) " Applications of Phytic Acid,” J. Am. Oil. Chem. Soc 60: 1861-1867). Phytic acid and its lower phosphorylated forms (such as IP3) are also found in most mammalian cells, where they assist in regulating a variety of important cellular functions (Szwergold, B. S. 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) "Ferulic Acid' Therapeutic Potential Through Its Antioxidant Property " J. Clin. Biochem. Nutr. (2007) 40(2):92-100; Kim, MJ. et al. (2007) "Ferulic Acid Supplementation Prevents Tnmethyltin-Induced Cognitive Deficits in Mice," Biosci. Biotechnol. Biochem. (2007) 71(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 responsible for cell injury and carcinogenesis (Harland, B.F. et al. (1987) "Phytate In Foods " World Rev. Nutr. Diet 52:235-259).
  • the preferred composition dosage of phytic acid (for example, as rice bran) is in the range of 2000-12,000 mg.
  • hyaluronic acid also known as hyaluronan refers to linear polymer composed of repeating disaccharides of D-glucuronic acid and D-N- acetylglucosamine, linked together via alternating ⁇ -1,4 and ⁇ - 1,3 glycosidic bonds ([- ⁇ (1,4)- GlcUA- ⁇ (l,3)-GlcNAc-] n ).
  • Hyaluronic acid can be 25,000 disaccharide repeats (n) in length:
  • Hyaluronic acid is a water-retaining molecule that is generated naturally in the human body but in decreasing amounts 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 3 different but related enzymes (hyaluronan synthases: HASl, HAS2 and HAS3 (Weigel, P.H. et al. (1997) "Hyaluronan Synthases,” J. Biol. Chem. 272:13997-14000; Tammi, M.I. et al. (2002) “Hyaluronan And Homeostasis' A Balancing Act,” J. Biol. Chem.
  • Hyaluronic acid has been traditionally extracted from rooster combs, from bovine or fish vitreous humor, from microbial production or from other sources (Rangaswamy, V. et al. (Epub 2007 Oct 24) "An Efficient Process For Production And Purification Of Hyaluronic Acid From Streptococcus Equi Subsp. Zooepidemicus," Biotechnol. Lett. 30(3):493-496; Gao, F. et al. (2006) "Preparation And Characterization Of Hyaluronan Oligosaccharides For Angiogenesis Study " J. Biomed. Mater. Res. B Appl. Biomater. 78(2):385-392; Blank, L.M.
  • the hyaluronic acid of the present invention is obtained from rooster combs.
  • Hyaluronic acid is widely available commercially, and such preparations are suitable for the purposes of the present invention.
  • the compositions of the present invention will provide a composition dosage of hyaluronic acid of from about 1 mg to about 400 mg, more preferably from about 50 mg to about 200 mg.
  • Vitamin D refers to a fat-soluble prohormone.
  • vitamin D 2 ergocalciferol
  • vitamin D 3 cholecalciferol
  • Vitamin D exhibits many biological actions. While vitamin D is widely known for its ability to stave off bone disease (rickets in growing children, osteoporosis in senior adults), it is becoming a central player in the battle against cancer. Regarding the role of vitamin D in immunity and cancer, vitamin D improves the chemotactic (affinity for) neutrophils to mobilize and migrate. Patients with rickets due to vitamin D deficiency are observed to have sluggish neutrophils that cannot migrate properly. Vitamin D stimulates the maturation of monocytes to macrophages. This results in an enlarged army of immune fighting cells to mount against tumors. Vitamin D is widely available commercially, and such preparations are suitable for the purposes of the present invention.
  • Vitamin D is essential for optimal muscle, bone, brain, immune and cardiovascular health and is undergoing re-discovery by aging researchers worldwide. Vitamin D supplementation up to 2000 IU has been shown to significantly reduce mortality rates, thus adding vitamin D to the lineup of molecules now considered to be true longevity factors (Autier, P. et al. (2007) "Vitamin D Supplementation And Total Mortality: A Meta-Analysis Of Randomized Controlled Trials," Arch Intern Med. 167(16):1730-1737). Its anti-calcifying properties (Zittermann, A. et al. (2007) “Vitamin D And Vascular Calcification,” Curr. Opin.
  • Lipidology 18(l):41-46) qualify vitamin D as another powerful agent that inhibits progressive overmineralization in the human body with advancing age and parallels the action of other mineral chelators in the compositions of the present invention. While the 1200 IU dose is three times more than the Recommended Daily Allowance, it is well within the Safe Upper Limit established by the National Academy of Sciences (2000 IU) and corresponds with a supplemental dosage recently found to be beneficial in a human clinical trial (Lappe, J.M. et al. (2007) "Vitamin D and calcium supplementation reduces cancer risk: results of a randomized trial," Amer. J. Clin. Nutr. 85(6):1586-1591).
  • compositions of the present invention will provide a composition dosage of vitamin D of from about 100 IU to about 100,000 IU, more preferably from about 1,000 IU to about 50,000 IU.
  • compositions of the present invention may contain additional components, including additional active components that act to enhance resveratrol biological activity and inactive compounds (e.g., flavorants, sweeteners, dyes, vitamins, amino acids (e.g., lysine, proline, etc.), minerals, nutrients, etc.).
  • additional active components e.g., flavorants, sweeteners, dyes, vitamins, amino acids (e.g., lysine, proline, etc.), minerals, nutrients, etc.).
  • quercetin (3,3',4',5,7-pentahydroxy-2-phenylchromen-4-one)
  • Resveratrol is glucuronated in the human liver, which may reduce its bioavailability.
  • Flavonoids such as quercetin, inhibit resveratrol glucuronidation and thus may act to improve resveratrol bioavailability (see, de Santi, C. et al. (2000) "Glucuronidation Of Resveratrol, A Natural Product Present In Grape And Wine, In The Human Liver," Xenobiotica 30(11):1047- 1054); De Santi, C. et al. (2000) “Sulphation Of Resveratrol, A Natural Compound Present In Wine, And Its Inhibition By Natural Flavonoids," Xenobiotica 30(9):857-866; De Santi, C.
  • Quercetin may also act synergistically with resveratrol or independently of resveratrol to provide beneficial function (Kampk ⁇ tter, A. et al. (Epub 2007 Oct 16) "Increase Of Stress Resistance And Lifespan Of Caenorhabditis Elegans By Quercetin,” Comp. Biochem. Physiol. B Biochem. MoI. Biol. 149(2):314-323). (See also, Kaindl, U. et al.
  • Emulsifiers, fillers, binding agents, and the like may also be included in the compositions of the present invention.
  • compositions of the present invention comprise a novel combination of: small molecules such as quercetin and resveratrol with widespread preventive and therapeutic health properties; and/or collagen-building nutrients (such as vitamin C- ascorbate, lysine, proline, etc.); and a glycosaminoglycan such as a shortened (low molecular weight) chain of hyaluronic acid (HA) or its singular components (glucosamine, glucuronate) or chondroitin sulfate, which are linear disaccharides (sugar-like molecules) that serve as structural components of cartilage, but in this combination serve as synergistic co-healing agents in non-cellular (connective) tissue that surrounds living cells.
  • small molecules such as quercetin and resveratrol with widespread preventive and therapeutic health properties
  • collagen-building nutrients such as vitamin C- ascorbate, lysine, proline, etc.
  • a glycosaminoglycan such as a shortened (
  • glycosaminoglycans hyaluronan, glucuronate or chondroitin
  • ascorbate lysine or proline
  • the combination of the present invention is intended for human or animal oral intake as a dietary supplement.
  • such compositions may comprise a combination of resveratrol and hyaluronan in a dietary supplement that serves to heal a variety of illnesses including some cancers.
  • Resveratrol is known to be an anti-cancer molecule and to have other healing and longevity enhancing properties.
  • Hyaluronan (hyaluronic acid, HA) is taken as an oral supplement or can be given intravenously to target cancer cells. When combined with or attached to other molecules, hyaluronan will deliver other anti-cancer and healing agents such as resveratrol to tumor sites.
  • the combination may or may not include a chelating agent, an antioxidant and/or an emulsifier as described in my above-referenced co- pending patent application.
  • resveratrol and HA When encapsulated or otherwise applied together, with or without those additives, resveratrol and HA have powerful healing properties for animals and humans.
  • compositions of the present invention stabilize resveratrol specific activity such that the resveratrol of the compositions has a specific activity that is greater than that of resveratrol maintained in the presence of oxygen gas, or maintained in the absence of a chelator, hyaluronic acid, or vitamin D.
  • the amounts of the non- resveratrol constituents of the compositions will stabilize the composition's resveratrol so that it exhibits at least 10% more activity, at least 20% more activity, at least 50% more activity, at least 2-times the activity, at least 5-times the activity, or at least 10-times the activity of resveratrol maintained in the presence of oxygen gas, or maintained in the absence of a chelator, hyaluronic acid, or vitamin D and so that it remains capable of exhibiting such specific activity over extended periods (for example, 1, 2, 4, 6, 10, 12, 18, 24, or 36 months or longer) at ambient conditions of temperature and humidity (i.e., without need for special precautions as to temperature or humidity).
  • extended periods for example, 1, 2, 4, 6, 10, 12, 18, 24, or 36 months or longer
  • Resveratrol is typically unstable to light and oxidation (Shaanxi University of Science & Technology, Xianyang China (2007) "Study On The Stability Of Resveratrol In 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. It is preferred to prepare, package and/or store resveratrol in low light (or in the dark) and/or in low oxygen, so as to minimize light-induced degradation (e.g., photo- isomerization) or oxygen-induced degradation.
  • compositions of the present invention are formulated as dietary supplements for oral ingestion in the form of a pill, lozenge, capsule, elixir, syrup, etc.
  • Other modalities of administration may alternatively be employed (e.g., intranasal, parenteral, intravenous, intraarterial, topical, etc.).
  • the compositions of the present invention are formulated as air-tight capsules in which encapsulation is conducted so as to prevent or minimize exposure to oxygen.
  • such encapsulation is conducted in an oxygen-free environment.
  • the components of the compositions of the present invention may be inserted into a capsule in an inert gas (e.g., nitrogen, argon, etc.) environment.
  • an inert gas e.g., nitrogen, argon, etc.
  • a nitrogen bubble e.g., 5-20% of the capsule volume
  • That international application has a corresponding U. S. patent application.
  • Suitable capsules useful in the encapsulation of resveratrol and other oxidation prone ingredients of dietary supplements include Licaps® (Capsugel), an air-tight gelatin capsule.
  • Licaps® Capsugel
  • phytic acid which has the ability to protect the components from metal-induced oxidation, augments such anti- oxidation precautions.
  • a particularly preferred example of such a resveratrol-containing composition is Longevinex® (Resveratrol Partners, LLC, San Dimas, CA), which comprises resveratrol and phytic acid.
  • Longevinex® contains as active ingredients (per capsule): 5 mg Vitamin E (as mixed tocopherols), 215 mg total resveratrol (obtained from French red wine and giant knotwood (Polygonum cuspidatum), and providing 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.
  • an oxygen absorbing packette is preferably employed to reduce the presence of free oxygen. Vacuum or nitrogen-flushed packaging (bottles, pill cases, etc.) in air-tight materials is desirable.
  • the components and compositions of the present invention may be prepared as a microencapsulated process (see, generally, Rubiana, M. et al. (2004) "Drug Delivery Systems: Past, Present, and Future," Current Drug Targets, 5(5):449- 455).
  • Micro-encapsulation is a process by which tiny particles or droplets (ranging in size from a few nanometers to one micron) are coated with a protective layer to create small capsules with controlled properties.
  • Suitable micron-sized, encapsulated, preparations can be obtained using the microencapsulation processes of Maxx Performance Inc. (Chester, NY), Blue California (Rancho Santa Margarita, CA), Southwest Research Institute (San Antonio, TX), Coating Place, Inc.
  • the present invention further comprises a practical method of stabilizing quercetin and other easily oxidized dietary supplement ingredients which may come in contact with oxidizing metals.
  • compositions of the present invention enhance resveratrol's specific activity.
  • the compositions of the present invention therefore find utility in the treatment of diseases (or in the amelioration of the symptoms of diseases) such as cardiovascular disease, cancer, macular degeneration, aging, neurodegenerative diseases (e.g., Alzheimer's Disease, Parkinson's Disease, etc.) and inflammation in which the modulation of expression of "survival/longevity" genes and/or “damage inducing" genes is desired.
  • diseases or in the amelioration of the symptoms of diseases
  • diseases such as cardiovascular disease, cancer, macular degeneration, aging, neurodegenerative diseases (e.g., Alzheimer's Disease, Parkinson's Disease, etc.) and inflammation in which the modulation of expression of "survival/longevity" genes and/or “damage inducing” genes is desired.
  • diseases or in the amelioration of the symptoms of diseases
  • diseases such as cardiovascular disease, cancer, macular degeneration, aging, neurodegenerative diseases (e.g., Alzheimer's Disease
  • Macular degeneration is a progressive, age-related disease that can be broken down into four stages:
  • RPE retinal pigment epithelia
  • Bruch's membrane an underlying cellophane -thin retinal layer called Bruch's membrane, which resides between the RPE and the blood supply layer (choroid). While drusen that forms within the retina is partially composed of cholesterol, this lipid does not originate from the blood circulation or the liver where most cholesterol is produced. Calcifications within Bruch's membrane further impairs the exit of lipids (fats), protein, and cellular debris, from the photoreceptor layer, which results in the formation of yellow spots called drusen on the retina. Drusen can be observed during an eye examination using an ophthalmoscope. There is currently no method of removing drusen.
  • the death of the RPE cells is the third stage of this progressive disease. This is sometimes called RPE dropout. As the RPE cells are either impaired or have died, and Bruch's membrane is clogged with calcium, the photoreceptors then cannot be nourished and also begin to die off. There is currently no treatment for stages 1 -3 of macular degeneration. Stage 1-3 is called the "dry" form of macular degeneration because it has not resulted in hemorrhage or edema or new blood vessel formation. About 85% of macular degeneration patients have the "dry" form of this disease. 4.
  • the cell cleansing process facilitated by the lysosomes cannot keep up with the accumulation of metabolic waste over a lifetime.
  • the parafoveal ring where rod cell density is highest, and therefore more discs of used-up vitamin A are shed, is where macular degeneration begins, and where the highest concentration of lipofuscin is observed in the retina.
  • the RPE cells die off with advancing age, which increases the burden on the remaining RPE cells to maintain a healthy retina.
  • lipofuscin has been considered a harmless wear-and-tear byproduct of cellular metabolism.
  • One aspect of the present invention relates to the recognition that lipofuscin, which forms from iron and copper-induced oxidation, and hardens within lysosomal bodies within retinal pigment epithelial cells, sensitizes the retina to damage by mild amounts of radiation and oxidation.
  • the retina becomes increasingly sensitive to blue-light damage with advancing age.
  • Drusen formation within the retina is associated with RPE cell inability to produce superoxide dismutase, an endogenous antioxidant enzyme. Mice deficient in superoxide dismutase develop features that are typical of age-related macular degeneration in humans. Superoxide dismutase protects retinal cells against unbound (free) iron. High iron diets and cellular environments have been shown to reduce superoxide dismutase activity.
  • Retinal photoreceptors and retinal pigment epithelial cells are believed to be especially vulnerable to damage by low-molecular weight complexes of iron. Since antioxidants in the blood circulation may not always be able to cross the blood-retinal barrier, the retina produces its own protective antioxidants that bind iron. Iron chelators inhibit the adverse effects of unbound (free) iron (not bound to proteins). Heme oxygenase also serves in a similar manner to iron chelators to prevent retinal damage induced by loose iron.
  • Sorbinil has been shown to partially reduce lipofuscin deposits in the retinal pigment epithelium cells of rodents.
  • Hydergine is a drug used to treat senile dementia. In a rodent study, hydergine was reported to have reduced brain lipofuscin levels, but also led to the early demise of the animals.
  • the East Indian spice turmeric contains an antioxidant molecule called curcumin. Curcumin has been used in an experimental mouse study to reduce lipofuscin in the brain.
  • Purslane is a flowering plant rich in magnesium, beta carotene and omega-3 oil. The provision of purslane to mice has been shown to reduce lipofuscin deposition in the brain of mice.
  • lipoic acid a natural metabolic antioxidant
  • Lipoic acid a natural antioxidant produced within living tissues, and also available as a dietary supplement, has been shown to protect RPE cells from oxidative damage in lab dish studies.
  • Lipofuscin formation dramatically increases in brain tissues following alcohol consumption. Supplementation with high-dose grape seed flavonols prevents increase lipofuscin formation. Lipofuscin is an end-product of lipid peroxidation which dramatically increases following ethanol consumption.
  • Epigallocatechin-3-gallate (EGCG) the major constituent of green tea, upregulates the activity of heme oxygenase in lab dish studies.
  • Heme oxygenase is a protective enzyme against iron-induced oxidation, which occurs in the retina.
  • U. S Patent No. 5,747,536 describes the combined therapeutic use of L-carnitine, lower alkanoyl L-carnitines or the pharmacologically acceptable salts thereof, with resveratrol, resveratrol derivatives or resveratrol-containing natural products, for producing a medicament for the prophylaxis and treatment of cardiovascular disorders, peripheral vascular diseases and peripheral diabetic neuropathy.
  • Melanin is an iron-binding antioxidant in the retina. As melanin levels decline in the retina with advancing age, there is a greater accumulation of lipofuscin.
  • the present invention relates to a composition comprising a combination of:
  • a chelator such as inositol hexaphosphate (IP6), trans resveratrol, quercetin, or any polyphenol or bioflavonoid for metal(s) such as iron, copper, heavy metals;
  • a calcium chelator such as inositol hexaphosphate (IP6);
  • a heme oxygenase activator such as trans resveratrol, piceatannol, or any of resveratrol's natural analogs, or similar small molecules such as fisetin, myricetin, quercetin or other bioflavonoids;
  • IP6 inositol hexaphosphate
  • antioxidants such as vitamin E, lutein/zeaxanthin, alpha lipoic acid.
  • a major challenge in cancer therapy is to selectively target cytotoxic agents to tumor cells (Luo, Y. et al. (2000) "A Hyaluronic Acid-Taxol Antitumor Bioconjugate Targeted To Cancer Cells " Biomacromolecules l(2):208-218).
  • cytotoxic agents to tumor cells
  • a major challenge in cancer therapy is to selectively target cytotoxic agents to tumor cells (Luo, Y. et al. (2000) "A Hyaluronic Acid-Taxol Antitumor Bioconjugate Targeted To Cancer Cells " Biomacromolecules l(2):208-218).
  • cytotoxic agents to tumor cells
  • a macromolecular carrier and in particular with hyaluronic acid
  • the present invention relates to a resveratrol- and hyaluronic acid-containing composition for the treatment of cancer comprising: resveratrol, hyaluronan, and optionally vitamin D and/or IP6. It is believed that these components act synergistically with one another to mediate an effect in curing and/or in preventing cancer in humans and/or in improving immunity (e.g., immune system response) in patients threatened by tumors.
  • This aspect of the present invention is based in part upon the recognition that natural molecules can boost cancer immunity, possibly in a manner similar to that observed in cancer-proof mice.
  • the sentinels of the innate immune system, dendritic cells can be alerted and neutrophils, macrophages and natural killer cell activity can be significantly enhanced.
  • the enhancement of vitamin D receptors via resveratrol is yet another major advantage of a combination approach to treat or prevent cancer. This approach appears to be more appropriate for senior adults, the highest risk group for cancer, who are often immune-compromised due to poor nutrition or lack of nutrient absorption. The fact that this therapy can now be immediately measured for effectiveness by non-invasive cancer cell counting technology means that expensive and equivocal tests on animals may not be required to prove efficacy.
  • Vitamin D exhibits many biological actions. While vitamin D is widely known for its ability to stave off bone disease (rickets in growing children, osteoporosis in senior adults), it is becoming a central player in the battle against cancer. Only recently is it also gaining attention as an antibiotic. Vitamin D-deficient mice exhibit a defective response from phagocyte cells in the face of infection or inflammation. Vitamin D deficiency is frequently associated with recurrent infections. Only about half of the macrophage cells accumulate at the site of inflammation in vitamin D-deficient animals compared to animals whose vitamin D levels are adequate.
  • vitamin D improves the chemotactic (affinity for) neutrophils to mobilize and migrate. Patients with rickets due to vitamin D deficiency are observed to have sluggish neutrophils that cannot migrate properly. Vitamin D stimulates the maturation of monocytes to macrophages. This results in an enlarged army of immune fighting cells to mount against tumors. Greater attention is now being given to vitamin D as an anti-cancer weapon because of studies which show supplemental vitamin D drastically reduces the risk for all types of cancer. A study that employed 1100 IU of vitamin D3 produced a 60-77% reduction in cancer risk among women in California in just a 4-year period.
  • Vitamin D's ability to inhibit cancer may be heightened when it is aided by weak estrogen-like molecules in the diet.
  • Resveratrol an estrogen-like molecule commonly found in red wine, upregulates the vitamin D receptor in breast cancer cells without increasing cancer growth.
  • Resveratrol in effect, can sensitize breast cancer cells to the anti-cancer properties of vitamin D.
  • resveratrol by itself has been shown to calm the response of phagocytes to foreign invaders like germs and tumor cells. Resveratrol dampens production of reactive oxygen species (free radicals) and normalizes particle ingestion in macrophage cells. Therefore, resveratrol prevents the over-response of immune cells that can produce autoimmunity.
  • Resveratrol blocks cancer in so many ways that it is difficult to find a pathway for cancer that is not obstructed by resveratrol.
  • Resveratrol induces the cell energy compartments in tumor cells, called mitochondria, to release an enzyme called cytochrome C oxidase that usually leads to a cascade of other enzymes that induce programmed cell death, called apoptosis.
  • cytochrome C oxidase an enzyme that usually leads to a cascade of other enzymes that induce programmed cell death, called apoptosis.
  • autophagy a process where enzymes produced inside the tumor cell actually digest its innards (kind of a form of intracellular cannibalism). This is a form of cell suicide that resveratrol activates in tumor cells, but not healthy cells.
  • IP6 inositol hexaphosphate
  • IP6 In examining the immune enhancing properties of IP6 it has been shown that it boosts production of free radicals (superoxide) and the cell digesting action of neutrophils in the presence of bacteria. IP6 increases the release of interleukin-8.
  • the action of natural killer cells, which are involved in tumor cell destruction, is enhanced by IP6.
  • the hyaluronic acid of such composition is conjugated to a chemotherapeutic agent.
  • the invention particularly pertains to such compositions in which the chemotherapeutic agent is taxol.
  • the invention particularly pertains to such compositions that additionally and preferably comprise a chelator, 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.
  • a preferred method of conjugation entails forming an NHS (N-hydroxy- succimimide derivative of the chemotherapeutic agent.
  • NHS N-hydroxy- succimimide derivative of the chemotherapeutic agent.
  • Such a derivative can be made by adding a molar excess of dry pyridine to a stirred solution of Taxol and succinic anhydride in CH 2 CI 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 produced Taxol-2'-hemisuccinate can be purified on silica gel (washed with hexane; eluted with ethyl acetate) to give the desired product (Luo, Y. et al. (1999) "Synthesis And Selective Cytotoxicity Of A Hyaluronic Acid-Antitumor Bioconjugate," Bioconjug. Chem. 10(5):755- 763).
  • N-hydroxy-succimimide derivative of the chemotherapeutic agent is then conjugated to adipic dihydrazido-functionalized hyaluronic acid.
  • Adipic dihydrazido- functionalized hyaluronic acid is preferably prepared as described by Pouyani, T. et al. (1994) ("Functionalized Derivatives Of Hyaluronic Acid Oligosaccharides - Drug Carriers And Novel Biomaterials " Bioconjugate Chem. 5:339-347); Pouyani, T. et al. (1994) ("Novel Hydrogels Of Hyaluronic Acid: Synthesis, Surface Morphology, And Solid-State NMR," J. Am. Chem. Soc.
  • hyaluronic acid is preferably dissolved in water and an excess of adipic dihydrazide (ADH).
  • ADH adipic dihydrazide
  • the pH of the reaction mixture is adjusted to 4.75 by addition acid.
  • 1 equivalent of l-Ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDCI) is added in solid form. The pH of the reaction mixture is maintained at 4.75 by addition of acid.
  • reaction is quenched by addition of 0.1 N NaOH to adjust the pH of reaction mixture to 7.0.
  • the reaction mixture is then transferred to pretreated dialysis tubing (Mw cutoff 3,500) and dialyzed exhaustively against 100 mM NaCl, then 25% EtOH/H2O and finally water.
  • the solution is then filtered through 0.2 m cellulose acetate membrane, flash frozen, and lyophilized (Luo, Y. et al. (1999) "Synthesis And Selective Cytotoxicity Of A Hyaluronic Acid- Antitumor Bioconjugate " Bioconjug. Chem. 10(5):755-763).
  • Calcification and rusting are major accelerators of aging.
  • the human body is composed of cells that must continually be replaced or renewed from within, and a gooey substance that fills space between cells called collagen or connective tissue which also must be continually regenerated.
  • As the human body ages at the cellular level there is a slow accumulation of cellular debris called lipofuscin.
  • the formation of lipofuscin is facilitated by the progressive accumulation of iron and calcium within cell bodies called lysosomes and mitochondria.
  • a cell cleansing and renewal process called autophagy prevents the accumulation of lipofuscin.
  • Progressive inability to remove cellular debris results in declining cell function and then premature death of the cell.
  • a young cell efficiently removes debris from within. An old cell cannot efficiently remove debris and accumulates lipofuscin.
  • compositions of the present invention inhibit and/or reverse cellular aging and/or connective tissue aging, and in particular, inhibit and/or reverse cellular aging and/or connective tissue aging caused by an accumulation of major minerals (e.g., iron, calcium, etc.). As a consequence, recipients of the compositions of the present invention exhibit enhanced longevity and enhanced cellular and connective tissue health and structure.
  • major minerals e.g., iron, calcium, etc.
  • fibroblasts The human body ages within connective tissue by failure of cells called fibroblasts to regenerate collagen and hyaluronic acid, the latter being a space-filling, water-holding molecule. Collagen formation is facilitated by vitamins and amino acids in the diet (vitamin C, lysine, proline). Fibroblasts can be stimulated to produce hyaluronic acid by estrogen, made naturally in the body, and by estrogen-like molecules found in plants, called phytoestrogens, provided in the diet of by hyaluronic acid itself. Young females, by virtue of the ability to produce estrogen, exhibit thicker hair, smoother skin and more flexible joints, due to the abundance of hyaluronic acid. All of these being attributes of youthfulness.
  • the present invention invention addresses both cellular and extracellular (connective tissue) aging, thus (a) preserving youthful function of living cells by removal of excess minerals, largely calcium and iron, from cells, this facilitating autophagy (cleanup of cellular debris, such as lipofuscin, via lysosomal enzymes) and (b) invigorating and preserving production of hyaluronan by stimulation of fibroblasts by HA, phytoestrogens (resveratrol, quercetin, genistein, are a few), to inhibition of degradation of HA by provision of metal chelators, such as phytic acid, ferulate, quercetin, resveratrol, etc.
  • metal chelators such as phytic acid, ferulate, quercetin, resveratrol, etc.
  • the present invention is a dietary supplement that addresses both cellular and non-cellular aging by its ability to:
  • metal chelating molecules that help maintain youthful lysosomal function are identified as antioxidants, like vitamin E or vitamin C, lipoic acid, metal chelators like IP6 phytate, quercetin, bioflavonoids or polyphenols, resveratrol.
  • Resveratrol works by its ability to stimulate production of heme oxygenase, an enzyme that helps to control iron.
  • hyaluronic acid hyaluronic acid, glucosamine, chondroitin, or estrogen-like molecules such as genistein, lignans, hydroxytyrosol, or other molecules configured like estrogen.
  • Orally consumed HA stimulates greater HA and chondroitin synthesis.
  • glucosamine stimulate fibroblasts to produce HA.
  • glucosamine stimulates synovial production of hyaluronic acid, which is primarily responsible for the lubricating and shock- absorbing properties of synovial fluid" (McCarty, M.F. (1998) "Enhanced Synovial Production Of Hyaluronic Acid May Explain Rapid Clinical Response To High-Dose Glucosamine In Osteoarthritis " Medical Hypotheses 50:507-510, 1998).
  • (f) orally consumed molecules that stimulate production of collagen are vitamin C, proline and lysine.
  • the present invention relates to a resveratrol and hyaluronic acid-containing dietary supplement that restores youthful function and appearance to human cells and tissue.
  • the invention particularly pertains to such compositions that additionally comprise a chelator, and/or vitamin D.
  • the composition will comprise the chelator phytic acid (inositol hexaphosphate; IP6).
  • IP6 chelator phytic acid
  • the compositions of the present invention synergistically enhance the specific activity of the resveratrol and/or hyaluronic acid, and thus the compositions of the present invention provide an enhancement of activity above and beyond that obtained with the components administered individually.
  • the invention relates to a method for restoring youthful function and appearance to human cells and tissues comprising the following steps: (a) stimulating renewal of living cells from within via enzymatic degradation of cellular debris by intracellular lysosomal bodies (preferably by providing a metal chelating molecule that helps maintain youthful lysosomal function, such molecules comprising antioxidants, such as vitamin E or vitamin C, lipoic acid, metal chelators like IP6 phytate, quercetin, bioflavonoids or polyphenols, and/or resveratrol); and
  • stimulating fibroblasts to produce hyaluronic acid comprises providing orally consumed molecules that stimulate fibroblasts to produce hyaluronic acid, such orally consumed molecules comprising, for example, hyaluronic acid, glucosamine, chondroitin, and/or estrogen-like molecules such as genistein, lignans, hydroxytyrosol, or other molecules configured like estrogen).
  • such stimulation is achieved by the dietary administration of a composition comprising the stated compounds, more preferably in combination with an orally consumable molecule that stimulates production of collagen, such molecules comprising, for example, vitamin C, proline and/or lysine.
  • a composition comprising the stated compounds, more preferably in combination with an orally consumable molecule that stimulates production of collagen, such molecules comprising, for example, vitamin C, proline and/or lysine.
  • Vitamin D3 works as an agent that mimics the response to a biological stressor, solar radiation.
  • vitamin D3 upregulates protective genes involved in activation of the immune system, particularly neutrophil count and motility, and aids in overcoming the decline in endogenous vitamin D3 production with advancing age due to thickening of the skin, which reduces sun/skin production of vitamin D.
  • vitamin D3 works synergistically to breakdown IP6 to IP3, thought to be a major active molecule.
  • Resveratrol also works synergistically to sensitize cells to vitamin D3 (sensitizes the vitamin D receptor on the cell surface).
  • Vitamin D serves to break down IP6 to IP3, which is its primary active form.
  • Vitamin D is also believed to act as an immune system enhancing agent, boosting innate immunity in humans. In this capacity, vitamin D has been shown experimentally to have important cancer-preventive and cancer-curing properties.
  • Resveratrol increases the sensitivity of the vitamin D receptor on the surface of cells, and thus is believed to act as an enhancing agent for vitamin D and as an anti-cancer agent. Resveratrol up-regulates the vitamin D receptor on the surface of cancer cells, and sensitizes cancer cells to vitamin D (Wietzke, J.A. 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):149-157; Wietzke, J.A. et al.
  • Resveratrol is also believed to be a monoamine oxidase inhibitor (MAO Inhibitor).
  • Hyaluronic acid is the water gelling molecule of the human body which serves as its scaffolding and hydrating agent. As aging progresses, less Hyaluronic acid is produced, resulting in wrinkled skin, thinning hair, unlubricated joints.
  • the chelators of the present composition also help to preserve hyaluronic acid in the body.
  • the hyaluronic acid component and the mineral chelating components e.g., resveratrol, quercetin, phytic acid IP6, ferulate
  • Hyaluronic acid is believed to have an affinity to cancer cells.
  • hyaluronic acid to the compositions of the present invention is believed to activate fibroblast cells in the human body to produce additional hyaluronic acid, thus serving to preserve connective tissue (collagen) in a youthful state (Yadav, A.K. et al. (2008) "An Insight On Hyaluronic Acid In Drug Targeting And Drug Delivery;' J. Drug Target.
  • Phytic Acid preferably in the form of rice bran, is believed to act as an iron and copper chelator and as an inhibitor of calcium cystallization. Phytic Acid also is believed to reduce the availability of metallic minerals that serve as growth factors in tumor cells. It is also believed to serve as a neutrophil priming and motility agent. Additionally, phytic acid has been found to be neuroprotective, and thus to attenuate the severity of conditions associated with neurodegenerative diseases (especially Parkinson's Disease, camptocormia, and Alzheimer's Disease) (Xu, Q. et al.
  • compositions of the present invention are believed to enhance such neuroprotection.
  • the iron chelator, quercetin, if present, is believed to serve to increase immediate bioavailability of resveratrol by permitting more passes through the liver before it is metabolized.
  • the individual components of the composition are believed to act synergistically to enhance the effect of, for example, resveratrol. Without intending to be limited thereby, it is proposed that the body's control or chelation of iron and calcium regulates the rate of aging after full growth has been achieved.
  • all the iron and calcium are directed towards production of new bone and new red blood cells (hemoglobin).
  • the cessation of childhood growth results in excess iron, copper and calcium, which then progressively (a) calcifies and (b) rusts tissues.
  • the lysosomes begin to accumulate iron and calcium, which results in their dysfunction.
  • the mitochondria begin to malfunction as they also progressively rust and calcify.
  • compositions of the present invention are believed to be capable of limiting or slowing the progressive rusting and calcification of cells and cellular organelles to thereby facilitate a slowing or reversal of the aging process.
  • the chelation is what controls the genes. Genes are then favorably upregulated or downregulated. Resveratrol and a copper chelator are believed to act: (1) as controllers of calcium concentration via upregulation of osteocalcin, the hormone that helps retain calcium in bones and (2) as controllers of iron concentration via heme oxygenase, an antioxidant enzyme.
  • MAO inhibitors and iron chelators have been proposed as treatments for Parkinson' s disease (Youdim, M.B. et al. (2004) "Novel Bifunctional Drugs Targeting Monoamine Oxidase Inhibition And Iron Chelation As An Approach To Neuroprotection In Parkinson 's Disease And Other Neurodegenerative Diseases," J. Neural. Transm. 111(10-11): 1455-1471 ; Yanez, M. et al. (2006) "(-)-Trans-Epsilon-Viniferin, A Polyphenol Present In Wines, Is An Inhibitor Of Noradrenaline And 5-Hydroxytryptamine Uptake And Of Monoamine Oxidase Activity " Eur. J. Pharmacol.
  • compositions of the present invention which contain the MAO inhibitor and copper chelator, resveratrol, the iron chelator and MAO inhibitor, quercetin, and the broad metal chelator, phytic acid are particularly preferred for the treatment of neurodegenerative diseases (especially Parkinson's Disease, camptocormia, and Alzheimer's Disease) or in the amelioration of the symptoms of such diseases.
  • compositions of the present invention were more effective than resveratrol alone in mediating a resveratrol biological activity, an analysis of gene expression was conducted, comparing the modulation of gene expression achieved by calorie restriction to the modulation of gene expression achieved by the compositions of the present invention.
  • mice Male B6CHF1 mice (2 months of age) were thus either placed on a 40% calorie restricted diet, provided commercially obtained trans-resveratrol (Sigma Chemical; 1.25 mg/kg per day), provided a resveratrol-containing composition of the present invention (Longevinex®; Resveratrol Associates, LLC; 100 mg ?ra «,y-resveratrol containing capsule per 80 kg human per day (i.e., 2.5 mg/kg per day of resveratrol (1.25 mg/kg per day ?r ⁇ «,y-resveratrol) 0.31 mg/kg per day quercetin dihydrate, 0.94 mg/kg per day rice bran extract, 4.75 mg/kg per day rice bran oil and 0.70 mg/kg per day sunflower lecithin)). The mice were monitored until they had reached five months of age.
  • trans-resveratrol Sigma Chemical
  • a resveratrol-containing composition of the present invention Longevinex®; Resveratrol Associates, LLC;
  • FC is calculated as the mean of the treated group divided by the mean of the control group, and this value is then log-transformed (base 2) for statistical purposes.
  • base 2 a gene that is expressed at 100 in the control and 200 in a treated group would be have an Fc of 2 (i.e., a twofold increase in expression); a gene that is expressed at 100 in the control and 50 in the treated group, would have an Fc of -2 (i.e., a twofold decrease in expression).
  • Probe Set ID Treatment mean mean mean mean mean mean CR Res 4 X Entrez Info
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Abstract

The present invention relates to a resveratrol-containing composition capable of modulating gene expression to an extent greater than that observed with resveratrol alone or with calorie restriction. The invention particularly pertains to such resveratrol-containing compositions that comprise resveratrol, a chelator, hyaluronic acid, and/or vitamin D and which upon administration to a recipient, 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 cellular damage. Most preferably, the resveratrol-stabilizing composition will comprise the chelator phytic acid (inositol hexaphosphate; IP6), hyaluronic acid, and vitamin D. The invention further pertains to the use of such compositions in the treatment or prevention of cancer, cardiovascular disease, diseases associated with aging, and other conditions and illnesses.

Description

RESVERATROL-CONTAINING COMPOSITIONS FOR MODULATING GENE PRODUCT CONCENTRATION OR ACTIVITY
Cross-Reference To Related Applications:
[0001] This application claims priority from United States Patent Application Serial No. 10/971,017 (filed October 25, 2004; pending), which application claims priority to 60/513,225 (filed on October 23, 2003; lapsed), and to United States Patent Applications Serial Nos. 60/973,817 (filed on September 20, 2007; pending), 61/023,227 (filed on January 24, 2008; pending), 61/023,230 (filed on January 24, 2008; pending), 61/023,234 (filed on January 24, 2008; pending), 61/048,756 (filed on April 29, 2008; pending) and ), 61/048,769 (filed on April 29, 2008; pending) all of which applications are herein incorporated by reference in their entirety.
Field of the Invention:
[0002] The present invention relates to a resveratrol-containing composition capable of modulating gene expression to an extent greater than that observed with resveratrol alone or with calorie restriction. The invention particularly pertains to such resveratrol-containing compositions that comprise resveratrol, a chelator, hyaluronic acid, and/or vitamin D and which, upon administration to a recipient, 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 cellular damage. The invention particularly relates to the use of such compositions in the treatment or prevention of cancer, cardiovascular disease, diseases associated with aging, and other conditions and illnesses.
Background of the Invention:
[0003] Despite a high level of risk factors such as cholesterol, diabetes, hypertension and a high intake of saturated fat, French males display the lowest mortality rate from ischaemic heart disease and cardiovascular diseases in Western industrialized nations (36% lower than the USA and 39% lower than the UK). The so-called 'French Paradox' (a low mortality rate specifically from cardiovascular diseases) may be due mainly to the regular consumption of wine (Renaud, S. et al. (1998) "The French Paradox And Wine Drinking " Novartis Found. Symp. 216:208- 222, 152-158). [0004] Resveratrol (3,4',5-trihydroxy-?rα«,y-stilbene) is a naturally occurring phenolic compound found, for example in grape skins, that has been demonstrated to have beneficial properties relating to health of humans (Das, S. et al. (2007) "Resveratrol: A Therapeutic Promise For Cardiovascular Diseases " Recent Patents Cardiovasc. Drug Discov. 2(2): 133- 138; Mancuso, C. et al. (2007) "Natural antioxidants in Alzheimer's disease " Expert Opin. Investig. Drugs. 16(12): 1921-1931 ; Baumann L. (2007) "Botanical Ingredients In Cosmeceuticals " J. Drugs Dermatol. 6(11): 1084-1088; Meeran, S.M. et al. (2008) "Cell Cycle Control As A Basis For Cancer Chemoprevention Through Dietary Agents " Front. Biosci. 13:2191-2202; de la Lastra, CA. et al. (2007) "Resveratrol As An Antioxidant And Pr o-Oxidant Agent: Mechanisms And Clinical Implications," Biochem. Soc. Trans. 35(Pt 5): 1156- 1160; Das, S. et al. (2007) "Anti-Inflammatory Responses Of Resveratrol " Inflamm. Allergy Drug Targets 6(3): 168-173; Cucciolla, V. et al. (Epub 2007 JuI 31) "Resveratrol: From Basic Science To The Clinic," Cell Cycle 6(20):2495-2510; Opie, L.H. et al. (Epub 2007 Jun 7) "The Red Wine Hypothesis : From Concepts To Protective Signaling Molecules," Eur. Heart J. 28(14): 1683-1693; Chen, Y. et al. (2007) "Review. Pro- And Anti-Angiogenesis Effects Of Resveratrol," In Vivo 21(2):365-370 [Erratum in: In Vivo 21(6): 1172 and 21(5):955]; Holme, A.L. et al. (2007) "Resveratrol In Cell Fate Decisions," J. Bioenerg. Biomembr. 39(l):59-63; Athar, M. et al. (Epub 2007 Jan 3) "Resveratrol: A Review Of Preclinical Studies For Human Cancer Prevention," Toxicol. Appl. Pharmacol. 224(3) :274-283). In particular, resveratrol is believed to be beneficial to the functioning of the heart and in extending the life of human cells. Resveratrol, when used in dietary supplements, is generally produced as an alcohol extract from plant sources.
[0005] Calorie restricted diets have been shown to enhance survival and longevity by up- regulating survival/longevity genes or down-regulating genes whose expression enhances cellular damage (Edwards, M.G. et al. (2007) "Gene Expression Profiling Of Aging Reveals Activation Of A P53-Mediated Transcriptional Program," BMC Genomics 8:80; Anderson, R.M. et al. (2006) "Calorie Restriction: Progress During Mid-2005-Mid-2006 " Exp. Gerontol. 41(12): 1247-1249; Weindruch, R. et al. (2001) "Microarray Profiling of Gene Expression in Aging and Its Alteration by Caloric Restriction in Mice," J. Nutrition 131 :918S— 923S; Lee, K.- C. et al. (2003) "Transcriptional Profiles Associated With Aging And Middle Age-Onset Caloric Restriction In Mouse Hearts," Proc. Natl. Acad. Sci. (U.S.A.) 99(23): 14988-14993; Weindruch, R. et al. (2002) "Effects Of Caloric Restriction On Gene Expression," Nestle Nutr. Workshop Ser. Clin. Perform. Programme 6: 17-28; 28-32; Mulligan, J.D. et al. (Epub 2007 Nov 1) "Downregulation Of Plasma Insulin Levels And Hepatic Ppar gamma Expression During The First Week Of Caloric Restriction In Mice," Exp. Gerontol. 43(3): 146-153; Rodgers, J.T. et al. (Epub 2007 Nov 26) "Metabolic Adaptations Through The PGC-I Alpha And SIRTl Pathways," FEBS Lett. 582(l):46-53; Swindell, W.R. (2007) "Gene Expression Profiling Of Long-Lived Dwarf Mice: Longevity-Associated Genes And Relationships With Diet, Gender And Aging," BMC Genomics. 8:353; Zhu, M. et al. (Epub 2007 Jun 6) "Adipogenic Signaling In Rat White Adipose Tissue: Modulation By Aging And Calorie Restriction," Exp. Gerontol. 42(8):733-744; Chiarpotto, E. et al. (2006) "Molecular Mechanisms Of Calorie Restriction 's Protection Against Age-Related Sclerosis," IUBMB Life. 58(12):695-702; Lu, J. et al. (2007) "Different Gene Expression Of Skin Tissues Between Mice With Weight Controlled By Either Calorie Restriction Or Physical Exercise," Exp. Biol. Med. (Maywood). 232(4):473-480; Mastemak, M.M. et al. (2007) "pPARS In Calorie Restricted And Genetically Long-Lived Mice," pPAR Res. 2007:28436; Fu, C. et al. (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 indicated by the above-cited references, mice have been used extensively as a model for genetic expression comparisons with humans. Without limitation, the validity of murine models to human gene expression reflects the fact that 98% of human and murine gene are homologous, and that mice and humans have about the same number of genes (e.g., approximately 30,000).
[0006] Despite the established benefits of a calorie restricted diet, the severity of the required dietary regime has limited adoption of this approach to increasing longevity. It would therefore be desirable to provide an alternative route to obtaining the benefits of calorie restriction that would avoid the need for dietary regulation and that would be amenable to widespread adoption. The present invention is directed to this and other needs.
Summary of the Invention:
[0007] The present invention relates to a resveratrol-containing composition capable of modulating gene expression to an extent greater than that observed with resveratrol alone or with calorie restriction. The invention particularly pertains to such resveratrol-containing compositions that comprise resveratrol, a chelator, hyaluronic acid, and/or vitamin D and which, upon administration to a recipient, 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 cellular damage. Most preferably, the resveratrol-stabilizing composition will comprise the chelator phytic acid (inositol hexaphosphate; IP6), hyaluronic acid, and vitamin D. The invention further pertains to the use of such compositions to up- regulate a survival/longevity gene or down-regulate a gene whose expression enhances cellular damage upon administration to a recipient. The invention particularly relates to the use of such compositions in the treatment or prevention of cancer, cardiovascular disease, diseases associated with aging, and other conditions and illnesses. - A -
[0008] In detail, the invention provides a resveratrol-containing composition that, upon administration to a recipient, modulates the concentration or activity, relative to resveratrol alone or calorie restriction, of the product of a survival/longevity gene or the product of a gene whose expression enhances cellular damage. Administration is preferably by oral ingestion.
[0009] The invention further provides the embodiments of such compositions wherein the modulation alters:
(A) oxidative phosphorylation;
(B) actin filament length or polymerization;
(C) intracellular transport;
(D) organelle biogenesis;
(E) insulin signaling;
(F) glycolysis;
(G) gluconeogenesis; or (H) fatty acid metabolism.
[0010] The invention further provides the embodiments of such compositions wherein the gene product is a survival/longevity gene product, and especially wherein the gene product is sSirtuin 1, or the forkhead Foxol transcription factor.
[0011] The invention further provides the embodiments of such compositions wherein the gene product is a gene product that enhances cellular damage, and especially wherein the gene product is encoded by the uncoupling protein 3, Pgc-1, or pyruvate dehydrogenase kinase 4 genes.
[0012] The invention further provides the embodiments of such compositions wherein the composition comprises:
(a) trans-rcsYcratrol; and
(b) a metal chelating agent; wherein the ?rα«,y-resveratrol is encapsulated to thereby substantially preserve the ability of the composition to modulate the concentration or activity of the product of the survival/longevity gene or the product of the gene whose expression enhances cellular damage, from loss due to exposure of the trαns-resveratrol to light or oxygen.
[0013] The invention further provides a method of ameliorating a symptom associated with an existing disease of an individual or for preventing the onset of the symptom in an individual prior to the occurrence of the disease in the individual, which comprises administering to the individual, a resveratrol-containing composition that modulates the concentration or activity, relative to resveratrol alone or calorie restriction, of the product of a survival/longevity gene or the product of a gene whose expression enhances cellular damage, wherein the resveratrol is provided in an amount effective to cause a modulation of the concentration or activity of the gene that ameliorates the symptom of the disease, and wherein the disease is selected from the group consisting of: cardiovascular disease, cancer, macular degeneration, a disease associated with aging, and inflammation.
[0014] The invention further provides the embodiments of such method wherein the modulation alters:
(A) oxidative phosphorylation;
(B) actin filament length or polymerization;
(C) intracellular transport;
(D) organelle biogenesis;
(E) insulin signaling;
(F) glycolysis;
(G) gluconeogenesis; or (H) fatty acid metabolism.
[0015] The invention further provides the embodiments of such method wherein the survival/longevity gene product is Sirtuin 1 or the forkhead Foxol transcription factor. The invention further provides the embodiments of such method wherein the gene whose expression enhances cellular damage encodes uncoupling protein 3 or pyruvate dehydrogenase kinase 4.
[0016] The invention further provides the embodiments of such method wherein said composition comprises:
(a) trans-resveratrol; and
(b) a metal chelating agent; wherein the ?rα«,y-resveratrol is encapsulated to thereby substantially preserve the ability of the composition to modulate the concentration or activity of the product of the survival/longevity gene or the product of the gene whose expression enhances cellular damage, from loss due to exposure of the ?rα«,y-resveratrol to light or oxygen.
[0017] The invention further provides the embodiments of such method wherein the disease is cancer, or a disease associated with aging (especially a neurodegenerative disease).
[0018] The invention further provides the embodiments of such method wherein the composition additionally comprises quercetin, hyaluronic acid and/or vitamin D. [0019] The invention further provides the embodiments of such method wherein the modulation is relative to resveratrol alone or wherein the modulation is relative to calorie restriction.
[0020] The invention further provides the embodiments of such methods wherein the gene product is a survival/longevity gene product, and especially wherein the gene product is Sirtuin 1 , or the forkhead Foxo 1 transcription factor.
[0021] The invention further provides the embodiments of such methods wherein the gene product is a gene product that enhances cellular damage, and especially wherein the gene product is encoded by the uncoupling protein 3, Pgc-1, or pyruvate dehydrogenase kinase 4 gene.
Brief Description of the Figures:
[0022] Figure 1 shows the change in body weight of mice administered resveratrol or a composition of the present invention (Longevinex®) relative to control animals and animals maintained on a calorie restricted diet.
[0023] Figure 2 shows the serum insulin level of mice administered resveratrol or a composition of the present invention (Longevinex®) relative to control animals and animals maintained on a calorie restricted diet.
[0024] Figure 3 shows the serum glucose level of mice administered resveratrol (P = 0.97) or a composition of the present invention (Longevinex®) (P = 0.07) relative to control animals and animals maintained on a calorie restricted diet (P = 0.10).
[0025] Figure 4 shows a schematic of a mechanism of action that is consistent with the observed biological activities of the compositions of the present invention.
Description of the Preferred Embodiments:
[0026] The present invention relates to a resveratrol-containing composition (and especially a resveratrol-containing dietary composition (i.e., a composition amenable for oral ingestion by a recipient)) capable of modulating gene expression to an extent greater than that observed with resveratrol alone or with calorie restriction. The invention particularly pertains to such resveratrol-containing compositions that comprise resveratrol, a chelator, hyaluronic acid, and/or vitamin D and which up-regulate a survival/longevity gene or down-regulate a gene whose expression enhances cellular damage upon administration to a recipient. Most preferably, the resveratrol-stabilizing composition will comprise the chelator phytic acid (inositol hexaphosphate; IP6), hyaluronic acid, and vitamin D. The invention further pertains to the use of such compositions to up-regulate a survival/longevity gene or down-regulate a gene whose expression enhances cellular damage upon administration to a recipient. The mineral chelators of the present invention provide anti-aging effects, as evidence in differentiation of the genome.
A. Resveratrol
[0027] As used herein, the term "resveratrol" refers to the phytoalexin: 3,4',5-trihydroxy- trans-stilbcnc having the structure:
Figure imgf000008_0001
[0028] Resveratrol has been ascribed multiple beneficial biological effects (see, United States Patent No. 7,345,178, which listing of disclosed effects is herein incorporated by reference), including preventing or treating cardiovascular disease (see, e.g., Das, S. et al. (2007) "Resveratrol: A Therapeutic Promise For Cardiovascular Diseases " Recent Patents Cardiovasc. Drug Discov. 2(2): 133- 138), Opie, L.H. et al. (Epub 2007 Jun 7) "The Red Wine Hypothesis: From Concepts To Protective Signaling Molecules " Eur. Heart J. 28(14): 1683- 1693; Bertelli, A. A. (Epub 2007 May 24) "Wine, Research And Cardiovascular Disease: Instructions For Use " Atherosclerosis 195(2):242-247; Providencia, R. (2006) "Cardiovascular Protection From Alcoholic Drinks: Scientific Basis Of The French Paradox " Rev. Port. Cardiol. 25(11):1043-1058; Maulik, N. (2006) "Reactive Oxygen Species Drives Myocardial AngiogenesisT Antioxid. Redox Signal. 8(11-12):2161-2168; Olas, B. et al. (2005) "Resveratrol, A Phenolic Antioxidant With Effects On Blood Platelet Functions " Platelets 16(5) :251-260; Bradamante, S. et al. (2004) "Cardiovascular Protective Effects Of Resveratrol," Cardiovasc Drug Rev. 22(3):169-188; Hao, H.D. et al. (2004) "Mechanisms Of Cardiovascular Protection By Resveratrol," J. Med. Food 7(3):290-298), preventing or treating cancer (Jang, M. et al. (1997) "Cancer Chemopreventive Activity Of Resveratrol, A Natural Product Derived From Grapes," Science 275:218-220; Das, S. et al. (2007) "Anti- Inflammatory Responses Of Resveratrol," Inflamm. Allergy Drug Targets 6(3): 168-173; de Ia Lastra, CA. et al. (2007) "Resveratrol As An Antioxidant And Pro-Oxidant Agent: Mechanisms And Clinical Implications," Biochem. Soc. Trans. 35(Pt 5): 1156-1160; Athar, M. et al. (Epub 2007 Jan 3) "Resveratrol: A Review Of Preclinical Studies For Human Cancer Prevention," Toxicol. Appl. Pharmacol. 224(3):274-283), Meeran, S.M. et al. (2008) "Ce// Cycle Control As A Basis For Cancer Chemoprevention Through Dietary Agents " Front. Biosci. 13:2191-2202; Shankar, S. et al. (2007) "Chemoprevention By Resveratrol: Molecular Mechanisms And Therapeutic Potential," Front. Biosci. 12:4839-4854; Delmas, D. et al. (2006) "Resveratrol As A Chemopreventive Agent: A Promising Molecule For Fighting Cancer " Curr. Drug Targets 7(4):423-442; Signorelli, P. et al. (2005) "Resveratrol As An Anticancer Nutrient: Molecular Basis, Open Questions And Promises," J. Nutr. Biochem. 16(8):449-466; preventing or treating macular degeneration (United States Patent Application Serial No. 61/023,234; King, R.E. et al. (2005) "Resveratrol reduces oxidation and proliferation of human retinal pigment epithelial cells via extracellular signal-regulated kinase inhibition " Chem. Biol. Interact. 151(2): 143-149; Sparrow, J.R. et al. (2003) "A2E-epoxides damage DNA in retinal pigment epithelial cells. Vitamin E and other antioxidants inhibit A2E-epoxide formation " J. Biol. Chem. 278(20): 18207- 18213), attenuating or preventing diseases associated with aging, and other conditions and illnesses, including the incidence or severity of neurodegenerative diseases such as Alzheimer's Disease and Parkinson's Disease (Baxter, R.A. (2008) "Anti-Aging Properties Of Resveratrol: Review And Report Of A Potent New Antioxidant Skin Care Formulation," J. Cosmet. Dermatol. 7(l):2-7; Engel, N. et al. (2008) "Aging And Anti-Aging: Unexpected Side Effects Of Everyday Medication Through Sirtuinl Modulation," Int. J. MoI. Med. 21(2):223-232; Bickenbach, K.A. et al. (Epub 2007 Dec 21) "Resveratrol Is An Effective Inducer Of Carg-Driven Tnf-Alpha Gene Therapy," Cancer Gene Ther. 15(3): 133- 139; Putics, A. et al. (2008) "Resveratrol Induces The Heat-Shock Response And Protects Human Cells From Severe Heat Stress," Antioxid. Redox Signal. 10(l):65-75; Bass, T.M. et al. (Epub 2007 Aug 14) "Effects Of Resveratrol On Lifespan In Drosophila melanogaster And Caenorhabditis elegans," Mech. Ageing Dev. 128(10):546-552; Stefani, M. et al. (Epub 2007 Sep 5) "The Effect Of Resveratrol On A Cell Model Of Human Aging," Ann. N.Y. Acad. Sci. 1114:407-418; Heiss, E.H. et al. (Epub 2007 JuI 11) "Chronic Treatment With Resveratrol Induces Redox Stress- And Ataxia Telangiectasia-Mutated (Atm)-Dependent Senescence In P53-Positive Cancer Cells " J. 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. et al. (Epub 2007 Jun 21) "Sirtl Deacetylase Protects Against Neurodegeneration In Models For Alzheimer's Disease And Amyotrophic Lateral Sclerosis ," EMBO J. 26(13):3169-3179), and anti-inflammatory activity (Das, S. et al. (2007) "Anti- Inflammatory Responses Of Resveratrol," Inflamm. Allergy Drug Targets 6(3): 168-173).
[0029] Reseveratrol may be synthesized chemically (Farina, A. et al. (2006) "An Improved Synthesis Of Resveratrol," Nat. Prod. Res. 20(3):247-252), or, more preferably, may be extracted from plant sources. Resveratrol is found in at least 72 species of plants distributed among 31 genera and 12 families (see, Counet, C. et al. (2006) "Chocolate And Cocoa: New Sources Of Trans-Resveratrol And Trans-Piceid," Food Chem. 98:649-657; Jang, M. et al. (1997) "Cancer Chemopreventive Activity Of Resveratrol, A Natural Product Derived From Grapes," Science 275:218-220; Wang, Y. et al. (2002) "An LC-MS Method For Analyzing Total Resveratrol In Grape Juice, Cranberry Juice, And In Wine " J. Agricult. Food Chem. 50(3):431-435). All of the families found to contain resveratrol belong to the spermatophytes division: Vitaceae, Myrtaceae, Dipterocarpaceae, Cyperaceae, Gnetaceae, Leguminosae, Pinaceae, Moraceae, Fagaceae, Liliaceae (Langcake, P. et al. (1976) "The Production Of Resveratrol By Vitis Vinifera And Other Members Of The Vitaceae As A Response To Infection Or Injury " Physiol. Plant Pathol. 9:77-86; Yoshiaki, T. et al. (2002) "Biogenic Reactions On Stilbenetetramers From Vitaceaeous Plants " Tetrahedron 58:9265-9271). Resveratrol has most often been reported in nonedible plants: vine, eucalyptus, spruce, and the tropical deciduous tree Bauhinia racemosa, Pterolobium Hexapetallum (Cassady, A. et al. (2000) "Isoflavones, Lignans, And Stilbenes -Origins, Metabolism And Potential Importance To Human Health " J. Science Food Agric. 80:1044-1062; Soleas, G. J. et al. (1997) "Resveratrol: A Molecule Whose Time Has Come? And Gone? " Clin. Biochem. 30:91-113). Reseveratrol is particularly found in grape skins and Giant Knotweed (see, Burns, J. et al. (2002) "Plant Foods and Herbal Sources of Resveratrol " J. Agric. Food Chem. 50(11):3337 -3340), cocoa and chocolate (Counet, C. et al. (2006) "Chocolate And Cocoa: New Sources Of Trans-Resveratrol And Trans-Piceid," Food Chem. 98:649-657). Peanut sprouts are also a rich source of resveratrol.
B. Modulation of Gene Product Concentration or Activity
[0030] The invention pertains to compositions that, upon administration to a recipient, increase the concentration or activity of a survival/longevity gene product and/or decrease the concentration or activity of a gene product that induces or causes cellular damage. As used herein, such increase (or decrease) in concentration or activity may be accomplished by any mechanism. For example, such increase (or decrease) may reflect a modulation of gene expression resulting in either increased (or decreased) expression of the gene encoding the survival/longevity gene product, or a gene that regulates (e.g., induces or represses) or whose product regulates such expression or activity. Alternatively, or conjunctively, such increase (or decrease) in concentration or activity may reflect a modulation of the recipient's ability to degrade or stabilize any such gene products. Alternatively, or conjunctively, such increase (or decrease) in concentration or activity may reflect a modulation of the recipient's ability to enhance, accelerate, repress or decelerate the activity of any such gene products. [0031] The modulation of concentration or activity discussed above may be a modulation of intracellular, intercellular and/or tissue concentration or activity of such survival/longevity gene products or such gene products that induce or cause cellular damage. Such modulation may be identified by assays of DNA expression, assays of gene product activity, assays of the level of gene product, assays of the rate of gene product turnover, etc. conducted in one or more types of cells, tissues, etc.
[0032] An increase in the concentration of a survival/longevity gene product may result from, for example, increased transcription of the gene that encodes the survival/longevity gene product, increased transcription of a gene that induces the expression of the gene that encodes the survival/longevity gene product, decreased transcription of a gene that represses the expression of the gene that encodes the survival/longevity gene product, decreased degradation or enhanced stabilization of expressed molecules of the survival/longevity gene product (leading to the enhanced accumulation of the survival/longevity gene product). Similarly, a decrease in the concentration of a survival/longevity gene product may result from, for example, decreased transcription of the gene that encodes the survival/longevity gene product, decreased transcription of a gene that induces the expression of the gene that encodes the survival/longevity gene product, increased transcription of a gene that represses the expression of the gene that encodes the survival/longevity gene product, increased degradation or decreased stabilization of expressed molecules of the survival/longevity gene product (leading to the enhanced dissipation of the survival/longevity gene product).
[0033] One aspect of the present invention thus relates to the use of resveratrol and resveratrol-containing compositions to modulate gene expression, and in particular, to modulate the expression of "survival/longevity" genes and/or "damage inducing" genes. As used herein, a compound is said to "modulate" gene expression if its administration results in a change in expression (relative to a control) of such genes of at least 10%. Modulation may involve an increase in expression ("up-regulation") or it may involve a decrease in expression ("down- regulation"). The term up-regulate thus denotes an increase of 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 (relative to a control). The term down-regulate conversely denotes a decrease of 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 (relative to a control).
[0034] A second aspect of the present invention thus relates to the use of resveratrol and resveratrol-containing compositions to modulate the concentration or activity of expressed products of "survival/longevity" genes and/or "damage inducing" genes. As used herein, a compound is said to "modulate" the concentration or activity of such expressed products if its administration results in a change in an intracellular, intercellular or tissue concentration or activity (relative to a control) of such gene products of at least 10%. Modulation may, for example, involve an "enhanced accumulation" or an "enhanced activity" or, for example, it may involve a "diminished accumulation" or a "diminished activity." The term "enhanced accumulation" (or "enhanced activity") denotes an increase in concentration (or activity) 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 (relative to a control). The term "diminished accumulation" or "diminished activity." conversely denotes a decrease in concentration (or activity) 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 (relative to a control).
[0035] As used herein, a "survival/longevity" gene is a gene whose expression contributes to an increase in the survival or longevity of a subject (e.g., a mammal, and particularly a human) expressing such gene. Conversely, a "damage inducing" gene is a gene whose expression contributes to DNA, cellular, or tissue damage in such subject. Such genes are responders to biological stressors, they initiate action in response to stressors such as radiation (e.g., sunlight, gamma rays, UV light, etc.), radiomimetic agents (e.g., vitamin D), heat, near starvation (calorie restriction, or its mimetic, resveratrol) by modulating their expression.
[0036] Examples of survival/longevity genes are provided in Table 1. Examples of genes whose expression enhances cellular damage are provided in Table 2. These Tables provide the gene's NCBI "ENTREZGENE" accession number. Most preferably, such genes are human genes. The Sirtuin 1 gene is known to control the rate of aging of living organisms by virtue of its ability to produce DNA repair enzymes and mimics the beneficial effects of calorie restriction. The trans form of resveratrol (but not czs-resveratrol) activates the Sirtuin 1 gene (Alcendor, R.R. (2007) "Sirtl Regulates Aging And Resistance To Oxidative Stress In The Heart " Circulation Research 100(10):1512-1521; Howitz, K.T. et al. (2003) "Small Molecule Activators OfSirtuins Extend Saccharomyces Cerevisiae Lifespan," Nature 425 : 191 - 196). The invention particularly pertains to compositions that increase the concentration of the Sirtuin 1 survival/longevity gene product. The invention further particularly pertains to compositions that increase the concentration of the forkhead Foxol (daf-16, dFoxO) transcription factor survival/longevity gene product.
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000018_0002
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
C. Preferred Compositions of the Present Invention
[0037] The invention particularly pertains to resveratrol-containing compositions in which the specific activity of the resveratrol has been stabilized or enhanced. As used herein, the term "specific activity" refers to the ratio of the extent of gene modulation (relative to control) per amount (mass) of administered resveratrol.
[0038] Preferably, such compositions will comprise a chelator, hyaluronic acid, and/or vitamin D. The invention particularly pertains to such compositions that comprise resveratrol (preferably, the compositions of the present invention will provide a composition dosage of from about 10 mg to about 2 g, more preferably from about 100 mg to about 500 mg), and at least one compound selected from the group consisting of an antioxidant (chelator), hyaluronic acid, and vitamin D. Preferably, the compositions of the present invention will contain resveratrol, an antioxidant, hyaluronic acid, and vitamin D.
[0039] As used herein the term "chelator" refers to an organic compound that bonds with and removes free metal ions from solution. Examples of suitable chelators include ethylenediaminetetraacetic acid (EDTA), histidine, antibiotic drugs of the tetracycline family, pyridoxal 2-chlorobenzoyl hydrazone, desferrioxamine, dexrazoxane, deferasirox, pyoverdine, pseudan, citrate, NDGA (nordihydroguaiaretic acid: l,4-bis[3,4-dihydroxyphenyl]2,3- dimethylbutane), ferulic acid and phytic acid. Preferably, the compositions of the present invention will provide a composition dosage of chelator of from about 1 g to about 15 g, more preferably from about 2 g to about 12 g.
[0040] Phytic acid is a particularly preferred chelator 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, Thome 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 Inositol," Nutr. Cancer. 55(2): 109-125; Lopez, M.A. et al. (2004) "Iron Availability: An Updated Review," Int. J. Food Sci. Nutr. 55(8):597-606; Singh, R.P. et al. (2005) "Prostate Cancer And Inositol Hexaphosphate: Efficacy And Mechanisms," Anticancer Res. 25(4):2891-2903; Vucenik, I. et al. (2006) "Cancer Inhibition By Inositol Hexaphosphate (IP6) And Inositol: From Laboratory To Clinic," J. Nutr. 133(11 Suppl 1):3778S-3784S; Raboy, V. (2003) "Myo-Inositol- 1,2,3,4,5,6-Hexakisphosphate," Phytochemistry. 64(6): 1033-19043; Vohra, A. et al. (2003) "Phytases: Microbial Sources, Production, Purification, And Potential Biotechnological Applications," Crit. Rev. Biotechnol. 23(l):29-60; Fox, CH. et al. (2002) "Phytic Acid (IP6), Novel Broad Spectrum Anti-Neoplastic Agent: A Systematic Review," Complement Ther. Med. 10(4):229-234; Grases, F. et al. (1999) "Phytate (IP6) Is A Powerful Agent For Preventing Calcifications In Biological Fluids: Usefulness In Renal Lithiasis Treatment," Anticancer Res. 19(5a):3717-3722; Jariwalla, RJ. (1999) "Inositol Hexaphosphate (IP6) As An Anti-Neoplastic And Lipid-Lowering Agent," Anticancer Res. 19(5a):3699-3702; Katayama T. (1999) "Hypolipidemic Action Of Phytic Acid (IP6): Prevention Of Fatty Liver," Anticancer Res. 19(5a):3695-3698). The structure of phytic acid is provided below:
Figure imgf000025_0001
[0041] Phytic acid is found in substantial amounts in whole grains, cereals, legumes, nuts, and seeds, and is the primary energy source for the germinating plant (Graf, E. (1983) " Applications of Phytic Acid," J. Am. Oil. Chem. Soc 60: 1861-1867). Phytic acid and its lower phosphorylated forms (such as IP3) are also found in most mammalian cells, where they assist in regulating a variety of important cellular functions (Szwergold, B. S. et al. (1987) "Observation Of Inositol Pentakis- And Hexakisphosphates In Mammalian Tissues By 31P NMR," Biochem. Biophys. Res. Commun. 264:874-881). Phytic Acid is preferably provided in the form of rice bran (Srinivasan, M. (2007) "Ferulic Acid' Therapeutic Potential Through Its Antioxidant Property " J. Clin. Biochem. Nutr. (2007) 40(2):92-100; Kim, MJ. et al. (2007) "Ferulic Acid Supplementation Prevents Tnmethyltin-Induced Cognitive Deficits in Mice," Biosci. Biotechnol. Biochem. (2007) 71(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 responsible for cell injury and carcinogenesis (Harland, B.F. et al. (1987) "Phytate In Foods " World Rev. Nutr. Diet 52:235-259). The preferred composition dosage of phytic acid (for example, as rice bran) is in the range of 2000-12,000 mg.
[0042] As used herein, the term "hyaluronic acid" (also known as hyaluronan) refers to linear polymer composed of repeating disaccharides of D-glucuronic acid and D-N- acetylglucosamine, linked together via alternating β-1,4 and β- 1,3 glycosidic bonds ([-β (1,4)- GlcUA-β (l,3)-GlcNAc-]n). Hyaluronic acid can be 25,000 disaccharide repeats (n) in length:
Figure imgf000026_0001
[0043] Hyaluronic acid is a water-retaining molecule that is generated naturally in the human body but in decreasing amounts 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 3 different but related enzymes (hyaluronan synthases: HASl, HAS2 and HAS3 (Weigel, P.H. et al. (1997) "Hyaluronan Synthases," J. Biol. Chem. 272:13997-14000; Tammi, M.I. et al. (2002) "Hyaluronan And Homeostasis' A Balancing Act," J. Biol. Chem. 277:4581- 4584; Kakehi, K. et al. (2003) "Hyaluronic Acid' Separation And Biological Implications," J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 797(l-2):347-355; Radaeva, LF. et al. (1997) "Hyaluronic Acid' Biological Role, Structure, Synthesis, Isolation, Purification, And Application (Review)" Prikl. Biokhim. Mikrobiol. 33(2): 133-137; Stoolmiller, A.C. et al. (1970) "The Biosynthesis Of Hyaluronic Acid By Group A Streptococcus," Expos. Annu. Biochim. Med. 30:65-78). U.S. Patent Application Publication 2004/0234497 discloses the use of hyaluronic acid for cancer drug delivery. The entire disclosure of that publication is incorporated herein by reference.
[0044] Hyaluronic acid has been traditionally extracted from rooster combs, from bovine or fish vitreous humor, from microbial production or from other sources (Rangaswamy, V. et al. (Epub 2007 Oct 24) "An Efficient Process For Production And Purification Of Hyaluronic Acid From Streptococcus Equi Subsp. Zooepidemicus," Biotechnol. Lett. 30(3):493-496; Gao, F. et al. (2006) "Preparation And Characterization Of Hyaluronan Oligosaccharides For Angiogenesis Study " J. Biomed. Mater. Res. B Appl. Biomater. 78(2):385-392; Blank, L.M. et al. (2005) "Stable Production Of Hyaluronic Acid In Streptococcus Zooepidemicus Chemostats Operated At High Dilution Rate " Biotechnol. Bioeng. 90(6):685-693; Kakehi, K. et al. (2003) "Hyaluronic Acid: Separation And Biological Implications " J. Chromatogr. B Analyt. Technol. Biomed Life Sci. 797(1 -2):347-355; Volpi, N. et al. (2003) "Purification And Characterization Of Hyaluronic Acid From The Mollusc Bivalve Mytilus Galloprovincialis " Biochimie 85(6):619-625; Tawada, A. et al. (2002) "Large-Scale Preparation, Purification, And Characterization Of Hyaluronan Oligosaccharides From 4-Mers To 52-Mers " Glycobiology. 2002 Jul;12(7):421-426; Mahoney, DJ. et al. (2001) "Novel Methods For The Preparation And Characterization Of Hyaluronan Oligosaccharides Of Defined Length " Glycobiology. 11(12): 1025-1033; Mcdonald, J. et al. (2002) "Hyaluronan Minireview Series " J. Biol. Chem. 277(7):4575-4579; Radaeva, LF. et al. (1997) "Hyaluronic Acid: Biological Role, Structure, Synthesis, Isolation, Purification, And Application (Review)" Prikl. Biokhim. Mikrobiol. 33(2): 133-137). Most preferably, the hyaluronic acid of the present invention is obtained from rooster combs. Hyaluronic acid is widely available commercially, and such preparations are suitable for the purposes of the present invention. Preferably, the compositions of the present invention will provide a composition dosage of hyaluronic acid of from about 1 mg to about 400 mg, more preferably from about 50 mg to about 200 mg.
[0045] As used herein, the term "Vitamin D" refers to a fat-soluble prohormone. Two major forms of vitamin D are vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) (DeLuca, H.F. et al. (1998) "Mechanisms And Functions Of Vitamin D " Nutr. Rev. 56:S4- SlO):
Figure imgf000028_0001
Ergocalcif erol (Vitamin D2) Cholecalciferol (Vitamin D3)
[0046] Vitamin D exhibits many biological actions. While vitamin D is widely known for its ability to stave off bone disease (rickets in growing children, osteoporosis in senior adults), it is becoming a central player in the battle against cancer. Regarding the role of vitamin D in immunity and cancer, vitamin D improves the chemotactic (affinity for) neutrophils to mobilize and migrate. Patients with rickets due to vitamin D deficiency are observed to have sluggish neutrophils that cannot migrate properly. Vitamin D stimulates the maturation of monocytes to macrophages. This results in an enlarged army of immune fighting cells to mount against tumors. Vitamin D is widely available commercially, and such preparations are suitable for the purposes of the present invention.
[0047] Vitamin D is essential for optimal muscle, bone, brain, immune and cardiovascular health and is undergoing re-discovery by aging researchers worldwide. Vitamin D supplementation up to 2000 IU has been shown to significantly reduce mortality rates, thus adding vitamin D to the lineup of molecules now considered to be true longevity factors (Autier, P. et al. (2007) "Vitamin D Supplementation And Total Mortality: A Meta-Analysis Of Randomized Controlled Trials," Arch Intern Med. 167(16):1730-1737). Its anti-calcifying properties (Zittermann, A. et al. (2007) "Vitamin D And Vascular Calcification," Curr. Opin. Lipidology 18(l):41-46) qualify vitamin D as another powerful agent that inhibits progressive overmineralization in the human body with advancing age and parallels the action of other mineral chelators in the compositions of the present invention. While the 1200 IU dose is three times more than the Recommended Daily Allowance, it is well within the Safe Upper Limit established by the National Academy of Sciences (2000 IU) and corresponds with a supplemental dosage recently found to be beneficial in a human clinical trial (Lappe, J.M. et al. (2007) "Vitamin D and calcium supplementation reduces cancer risk: results of a randomized trial," Amer. J. Clin. Nutr. 85(6):1586-1591). A 2,000 IU dosage is roughly equivalent the natural vitamin D3 produced by 15-30 minutes of total-body summer sun exposure at noontime at a southern latitude, for which no side effects have been reported. Preferably, the compositions of the present invention will provide a composition dosage of vitamin D of from about 100 IU to about 100,000 IU, more preferably from about 1,000 IU to about 50,000 IU.
[0048] The compositions of the present invention may contain additional components, including additional active components that act to enhance resveratrol biological activity and inactive compounds (e.g., flavorants, sweeteners, dyes, vitamins, amino acids (e.g., lysine, proline, etc.), minerals, nutrients, etc.).
[0049] In particular, quercetin (3,3',4',5,7-pentahydroxy-2-phenylchromen-4-one)
Figure imgf000029_0001
Quercetin
[0050] butein, fisetin, myricetin, kaempferol, cis-resveratrol or piceatannol may be added to the compositions.
Figure imgf000029_0002
Butein [3 ,4,2 ' ,4 ' -tetrahydroxychalcone] Fisetin [3,3 '4',7-Tetrahydroxyflavone]
Figure imgf000029_0003
Myricetin [3,5, 7-Trihydroxy-2-(3,4,5- Kaempferol [3,5,7-trihydroxy-2- trihy droxypheny 1) -4-chromenone] (4-hydroxyphenyl)-4H- 1 - benzopyran-4-one]
Figure imgf000030_0001
Piceatannol [5-[(E)-2-(3,4-dihydroxyphenyl)vinyl]benzene-l,3-diol] [also known as 3,4,3',5'-tetrahydroxy-trans-stilbene]
[0051] Such compounds are described, for example by Stecher, G. et al. (2001) ("Determination Of Flavonoids And Stilbenes In Red Wine And Related Biological Products By HPLC And HPLC-ESI-MS-MS," Fresenius J. Anal Chem. 2001 Sep;371(l):73-80).
[0052] Resveratrol is glucuronated in the human liver, which may reduce its bioavailability. Flavonoids, such as quercetin, inhibit resveratrol glucuronidation and thus may act to improve resveratrol bioavailability (see, de Santi, C. et al. (2000) "Glucuronidation Of Resveratrol, A Natural Product Present In Grape And Wine, In The Human Liver," Xenobiotica 30(11):1047- 1054); De Santi, C. et al. (2000) "Sulphation Of Resveratrol, A Natural Compound Present In Wine, And Its Inhibition By Natural Flavonoids," Xenobiotica 30(9):857-866; De Santi, C. et al. (2000) "Sulphation Of Resveratrol, A Natural Product Present In Grapes And Wine, In The Human Liver And Duodenum," Xenobiotica 30(6):609-617). Quercetin may also act synergistically with resveratrol or independently of resveratrol to provide beneficial function (Kampkδtter, A. et al. (Epub 2007 Oct 16) "Increase Of Stress Resistance And Lifespan Of Caenorhabditis Elegans By Quercetin," Comp. Biochem. Physiol. B Biochem. MoI. Biol. 149(2):314-323). (See also, Kaindl, U. et al. (2008) "The Dietary Antioxidants Resveratrol And Quercetin Protect Cells From Exogenous Pro-Oxidative Damage, " Food Chem. Toxicol. 46(4): 1320-1326; Melzig, M.F et al. (2002) "Induction Of Neutral Endopeptidase And Angiotensin-Converting Enzyme Activity Of Sk-N-Sh Cells In Vitro By Quercetin And Resveratrol," Pharmazie. 57(8):556-558; Hsu, CL. et al. (2006) "Induction Of Cell Apoptosis In 3T3-L1 Pre-Adipocytes By Flavonoids Is Associated With Their Antioxidant Activity," Molec. Nutr. Food Res. 50(11): 1072-1079; Chan, M.M. et al. (2000) "Synergy Between Ethanol And Grape Polyphenols, Quercetin, And Resveratrol, In The Inhibition Of The Inducible Nitric Oxide Synthase Pathway," Biochem. Pharmacol. 60(10): 1539-1548; Nicholson S. K. et al. (2008) "Effects Of Dietary Polyphenols On Gene Expression In Human Vascular Endothelial Cells " Proc. Nutr. Soc. 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. Nutr 46(6):329-336). [0053] Emulsifiers, fillers, binding agents, and the like may also be included in the compositions of the present invention.
[0054] In one embodiment, the compositions of the present invention comprise a novel combination of: small molecules such as quercetin and resveratrol with widespread preventive and therapeutic health properties; and/or collagen-building nutrients (such as vitamin C- ascorbate, lysine, proline, etc.); and a glycosaminoglycan such as a shortened (low molecular weight) chain of hyaluronic acid (HA) or its singular components (glucosamine, glucuronate) or chondroitin sulfate, which are linear disaccharides (sugar-like molecules) that serve as structural components of cartilage, but in this combination serve as synergistic co-healing agents in non-cellular (connective) tissue that surrounds living cells. The combination of glycosaminoglycans (hyaluronan, glucuronate or chondroitin) and/or ascorbate, lysine or proline, to encourage the generation of collagen, and small molecules, that operate on intracellular basis, comprise therapeutic or preventive action that encompasses the total tissue matrix. The combination of the present invention is intended for human or animal oral intake as a dietary supplement. In a sub-embodiment thereof, such compositions may comprise a combination of resveratrol and hyaluronan in a dietary supplement that serves to heal a variety of illnesses including some cancers. Resveratrol is known to be an anti-cancer molecule and to have other healing and longevity enhancing properties. Hyaluronan (hyaluronic acid, HA) is taken as an oral supplement or can be given intravenously to target cancer cells. When combined with or attached to other molecules, hyaluronan will deliver other anti-cancer and healing agents such as resveratrol to tumor sites. The combination may or may not include a chelating agent, an antioxidant and/or an emulsifier as described in my above-referenced co- pending patent application. When encapsulated or otherwise applied together, with or without those additives, resveratrol and HA have powerful healing properties for animals and humans.
[0055] Most preferably, the compositions of the present invention stabilize resveratrol specific activity such that the resveratrol of the compositions has a specific activity that is greater than that of resveratrol maintained in the presence of oxygen gas, or maintained in the absence of a chelator, hyaluronic acid, or vitamin D. Preferably, the amounts of the non- resveratrol constituents of the compositions will stabilize the composition's resveratrol so that it exhibits at least 10% more activity, at least 20% more activity, at least 50% more activity, at least 2-times the activity, at least 5-times the activity, or at least 10-times the activity of resveratrol maintained in the presence of oxygen gas, or maintained in the absence of a chelator, hyaluronic acid, or vitamin D and so that it remains capable of exhibiting such specific activity over extended periods (for example, 1, 2, 4, 6, 10, 12, 18, 24, or 36 months or longer) at ambient conditions of temperature and humidity (i.e., without need for special precautions as to temperature or humidity).
D. Packaging of the Preferred Compositions of the Present Invention
[0056] Resveratrol is typically unstable to light and oxidation (Shaanxi University of Science & Technology, Xianyang China (2007) "Study On The Stability Of Resveratrol In 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. It is preferred to prepare, package and/or store resveratrol in low light (or in the dark) and/or in low oxygen, so as to minimize light-induced degradation (e.g., photo- isomerization) or oxygen-induced degradation. The preferred compositions of the present invention are formulated as dietary supplements for oral ingestion in the form of a pill, lozenge, capsule, elixir, syrup, etc. Other modalities of administration may alternatively be employed (e.g., intranasal, parenteral, intravenous, intraarterial, topical, etc.).
[0057] In a first example of such preferred packaging, the compositions of the present invention are formulated as air-tight capsules in which encapsulation is conducted so as to prevent or minimize exposure to oxygen. In one embodiment, such encapsulation is conducted in an oxygen-free environment. For example, the components of the compositions of the present invention may be inserted into a capsule in an inert gas (e.g., nitrogen, argon, etc.) environment. Preferably, a nitrogen bubble (e.g., 5-20% of the capsule volume) may be introduced into the capsule to further stabilize and protect the components against oxidation (see, PCT Publication No. WO 01/08631, herein incorporated by reference). That international application has a corresponding U. S. patent application. Suitable capsules useful in the encapsulation of resveratrol and other oxidation prone ingredients of dietary supplements include Licaps® (Capsugel), an air-tight gelatin capsule. The presence of phytic acid, which has the ability to protect the components from metal-induced oxidation, augments such anti- oxidation precautions. A particularly preferred example of such a resveratrol-containing composition is Longevinex® (Resveratrol Partners, LLC, San Dimas, CA), which comprises resveratrol and phytic acid. Longevinex® contains as active ingredients (per capsule): 5 mg Vitamin E (as mixed tocopherols), 215 mg total resveratrol (obtained from French red wine and giant knotwood (Polygonum cuspidatum), and providing 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.
[0058] Once a composition has been sealed into an air-tight capsule, it is important to maintain a low or no-oxygen environment in the packaging surrounding the capsules in order to protect the composition from oxidation should a break or leak occur in the sealed capsule. Therefore, an oxygen absorbing packette is preferably employed to reduce the presence of free oxygen. Vacuum or nitrogen-flushed packaging (bottles, pill cases, etc.) in air-tight materials is desirable.
[0059] In an alternative embodiment, the components and compositions of the present invention may be prepared as a microencapsulated process (see, generally, Rubiana, M. et al. (2004) "Drug Delivery Systems: Past, Present, and Future," Current Drug Targets, 5(5):449- 455). Micro-encapsulation is a process by which tiny particles or droplets (ranging in size from a few nanometers to one micron) are coated with a protective layer to create small capsules with controlled properties. Suitable micron-sized, encapsulated, preparations can be obtained using the microencapsulation processes of 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. (Bethlehem, PA), etc. 3rd-generation Longevinex® ("Longevinex-3®") (Resveratrol Partners, LLC), which contains Vitamin D3, Vitamin E, Resveratrol, Quercetin, and Phytic Acid is a particularly preferred microencapsulated form of the compositions of the present invention.
[0060] The present invention further comprises a practical method of stabilizing quercetin and other easily oxidized dietary supplement ingredients which may come in contact with oxidizing metals.
E. Utility of the Compositions of the Present Invention
[0061] The compositions of the present invention enhance resveratrol's specific activity. The compositions of the present invention therefore find utility in the treatment of diseases (or in the amelioration of the symptoms of diseases) such as cardiovascular disease, cancer, macular degeneration, aging, neurodegenerative diseases (e.g., Alzheimer's Disease, Parkinson's Disease, etc.) and inflammation in which the modulation of expression of "survival/longevity" genes and/or "damage inducing" genes is desired. Over time, as minerals such as calcium and iron accumulate in the human body, genes respond in deleterious ways. Liu, Y. et al. (2005) "Global Genomic Approaches To The Iron-Regulated Proteome," Ann. Clin. Lab. Sci. 35(3):230-239; Templeton, D.M. et al. (2003) "Genetic Regulation Of Cell Function In Response To Iron Overload Or Chelation," Biochim. Biophys. Acta. 1619(2): 113- 124; Ikeda, H. et al. (1992) "Evidence That An Iron Chelator Regulates Collagen Synthesis By Decreasing The Stability Of Procollagen mRNA " Hepatology 15(2):282-287. The present invention has particular utility in the treatment of macular degeneration, cancer and the conditions of aging. 1. Macular Degeneration
[0062] The prolongation of the human lifespan over the past few decades in the US has spawned the proliferation of macular degeneration, an age-related eye disease. While not resulting in total vision loss, the disease robs older adults 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 macula is part of the retina where color- vision cells (cones) are located.
[0063] Macular degeneration is a progressive, age-related disease that can be broken down into four stages:
1. Beginning in about the third decade of life, the inability of the "garbage cleaning" cells, called the retinal pigment epithelia (RPE), to engulf and remove cellular debris from the back of the eyes, results in the formation of small microscopic deposits called lipofuscin (ly-poh-fus-kin). Lipofuscin is from by iron and copper-induced oxidation of cellular debris and its accumulation correlates with premature aging and shortened lifespan of organisms. The prevalence of macular degeneration is greater in Caucasians than persons with darkly-pigmented skin and Caucasians have more lipofuscin deposits in their retinas. Some of this cellular debris in the retina is comprised of used-up vitamin A that is shed from night- vision (rod) cells each morning in the human eye. The failure of the RPE cells to function results from accumulation of iron and calcium within the RPE.
2. Later, in about the fifth decade of life, there is progressive calcification of an underlying cellophane -thin retinal layer called Bruch's membrane, which resides between the RPE and the blood supply layer (choroid). While drusen that forms within the retina is partially composed of cholesterol, this lipid does not originate from the blood circulation or the liver where most cholesterol is produced. Calcifications within Bruch's membrane further impairs the exit of lipids (fats), protein, and cellular debris, from the photoreceptor layer, which results in the formation of yellow spots called drusen on the retina. Drusen can be observed during an eye examination using an ophthalmoscope. There is currently no method of removing drusen.
3. The death of the RPE cells is the third stage of this progressive disease. This is sometimes called RPE dropout. As the RPE cells are either impaired or have died, and Bruch's membrane is clogged with calcium, the photoreceptors then cannot be nourished and also begin to die off. There is currently no treatment for stages 1 -3 of macular degeneration. Stage 1-3 is called the "dry" form of macular degeneration because it has not resulted in hemorrhage or edema or new blood vessel formation. About 85% of macular degeneration patients have the "dry" form of this disease. 4. As breaks in Bruch's membrane occur, or Bruch's membrane becomes totally calcified, the photoreceptor layer is deprived of oxygen and new blood vessels form (called neovascularization) which can invade the photoreceptor layer in the macula and impair vision; or there may be leakage of blood serum or frank release of red blood cells, which results in edema or hemorrhage. This is the more advanced and sight- threatening form of macular degeneration, often called "wet" macular degeneration because of the presence of the leakage of blood serum or red blood cells into the photoreceptor layer. This stage of the disease, if caught early, can be treated with laser beams, which can seal up leaky blood vessels. However, this treatment is only effective in delaying the progression of the disease, not curing it.
[0064] The cell cleansing process facilitated by the lysosomes cannot keep up with the accumulation of metabolic waste over a lifetime. The parafoveal ring, where rod cell density is highest, and therefore more discs of used-up vitamin A are shed, is where macular degeneration begins, and where the highest concentration of lipofuscin is observed in the retina. Eventually, the RPE cells die off with advancing age, which increases the burden on the remaining RPE cells to maintain a healthy retina.
[0065] In the past, lipofuscin has been considered a harmless wear-and-tear byproduct of cellular metabolism. One aspect of the present invention relates to the recognition that lipofuscin, which forms from iron and copper-induced oxidation, and hardens within lysosomal bodies within retinal pigment epithelial cells, sensitizes the retina to damage by mild amounts of radiation and oxidation. The retina becomes increasingly sensitive to blue-light damage with advancing age. Drusen formation within the retina is associated with RPE cell inability to produce superoxide dismutase, an endogenous antioxidant enzyme. Mice deficient in superoxide dismutase develop features that are typical of age-related macular degeneration in humans. Superoxide dismutase protects retinal cells against unbound (free) iron. High iron diets and cellular environments have been shown to reduce superoxide dismutase activity.
[0066] Retinal photoreceptors and retinal pigment epithelial cells are believed to be especially vulnerable to damage by low-molecular weight complexes of iron. Since antioxidants in the blood circulation may not always be able to cross the blood-retinal barrier, the retina produces its own protective antioxidants that bind iron. Iron chelators inhibit the adverse effects of unbound (free) iron (not bound to proteins). Heme oxygenase also serves in a similar manner to iron chelators to prevent retinal damage induced by loose iron.
[0067] Numerous agents have been used experimentally to clear up lipofuscin and drusen. Statin drugs, commonly used to reduce blood serum levels of cholesterol, have also been tested to prevent lipofuscin deposits in animals. Statin drugs reduced lipofuscin formation but were toxic to the liver and brought about the early death of these animals. Piracetam, a derivative of the neurotransmitter GABA, now available as a dietary supplement, has been used successfully to reduce lipofuscin formation in brain tissues. Sorbinil is an enzyme inhibiting drug (aklose reductase inhibitor) that underwent unsuccessful human trials in the 1990s to prevent retinal problems associated with diabetes. Sorbinil has been shown to partially reduce lipofuscin deposits in the retinal pigment epithelium cells of rodents. Hydergine is a drug used to treat senile dementia. In a rodent study, hydergine was reported to have reduced brain lipofuscin levels, but also led to the early demise of the animals. The East Indian spice turmeric contains an antioxidant molecule called curcumin. Curcumin has been used in an experimental mouse study to reduce lipofuscin in the brain. Purslane is a flowering plant rich in magnesium, beta carotene and omega-3 oil. The provision of purslane to mice has been shown to reduce lipofuscin deposition in the brain of mice.
[0068] In a lab dish study, sulforaphane, an antioxidant molecule found in Brussels sprouts and broccoli in 1992, has been used successfully to reduce lipofuscin deposits in RPE cells exposed to blue light.
[0069] Intraperitoneal administration of lipoic acid to aged rats leads to a reduction and elevation in lipofuscin and enzyme activity, respectively, in the cortex, cerebellum, striatum, hippocampus, and hypothalamus of the brain. These results suggest that lipoic acid, a natural metabolic antioxidant, should be useful as a therapeutic tool in preventing neuronal dysfunction in aged individuals. Lipoic acid, a natural antioxidant produced within living tissues, and also available as a dietary supplement, has been shown to protect RPE cells from oxidative damage in lab dish studies.
[0070] Lipofuscin formation dramatically increases in brain tissues following alcohol consumption. Supplementation with high-dose grape seed flavonols prevents increase lipofuscin formation. Lipofuscin is an end-product of lipid peroxidation which dramatically increases following ethanol consumption.
[0071] Oolong and green tea drinks reverse the cognitive impairment and lipofuscin formation in mice. Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, upregulates the activity of heme oxygenase in lab dish studies. Heme oxygenase is a protective enzyme against iron-induced oxidation, which occurs in the retina.
[0072] It has been shown that the provision of supplemental estrogen decreases lipofuscin deposition in brain tissues. In a lab dish study, the provision of lutein and zeaxanthin to RPE cells reduced lipofuscin formation. In rodents given supplemental acetyl-L-carnitine, a decline in lipofuscin deposits has been measured in brain cells.
[0073] U. S Patent No. 5,747,536 describes the combined therapeutic use of L-carnitine, lower alkanoyl L-carnitines or the pharmacologically acceptable salts thereof, with resveratrol, resveratrol derivatives or resveratrol-containing natural products, for producing a medicament for the prophylaxis and treatment of cardiovascular disorders, peripheral vascular diseases and peripheral diabetic neuropathy.
[0074] Melanin is an iron-binding antioxidant in the retina. As melanin levels decline in the retina with advancing age, there is a greater accumulation of lipofuscin.
[0075] In one embodiment, the present invention relates to a composition comprising a combination of:
(a) A chelator such as inositol hexaphosphate (IP6), trans resveratrol, quercetin, or any polyphenol or bioflavonoid for metal(s) such as iron, copper, heavy metals;
(b) A calcium chelator, such as inositol hexaphosphate (IP6);
(c) A heme oxygenase activator, such as trans resveratrol, piceatannol, or any of resveratrol's natural analogs, or similar small molecules such as fisetin, myricetin, quercetin or other bioflavonoids;
(d) An agent that lowers 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.
[0076] The formulation functions to:
(1) Limit oxidation in retinal tissues (photoreceptors, retinal pigment epithelial cells (RPE), choroid, specifically mitochondria and lysosomes in RPE cells);
(2) Inhibit accumulation of lipofuscin deposits;
(3) Inhibit formation of drusen; and
(4) Limit calcifications to retinal tissues, especially Bruch's membrane.
2. Cancer
[0077] A major challenge in cancer therapy is to selectively target cytotoxic agents to tumor cells (Luo, Y. et al. (2000) "A Hyaluronic Acid-Taxol Antitumor Bioconjugate Targeted To Cancer Cells " Biomacromolecules l(2):208-218). To decrease undesirable side effects of small molecule anticancer agents, many targeting approaches have been examined. One of the most promising methods involves the combination or covalent attachment of the cytotoxin with a macromolecular carrier, and in particular with 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 Antitumor Bioconjugate Targeted To Cancer Cells " Biomacromolecules l(2):208-218; Luo, Y. et al. (1999) "Hyaluronic Acid-N-Hydroxysuccinimide: A Useful Intermediate For Bioconjugation " Bioconjug. Chem. 12(6): 1085-1088; Luo, Y. et al. (2002) "Targeted Delivery Of Doxorubicin By HPMA Copolymer-Hyaluronan Bioconjugates ," Pharm. Res. 19(4):396-402).
[0078] In one embodiment, the present invention relates to a resveratrol- and hyaluronic acid-containing composition for the treatment of cancer comprising: resveratrol, hyaluronan, and optionally vitamin D and/or IP6. It is believed that these components act synergistically with one another to mediate an effect in curing and/or in preventing cancer in humans and/or in improving immunity (e.g., immune system response) in patients threatened by tumors. This aspect of the present invention is based in part upon the recognition that natural molecules can boost cancer immunity, possibly in a manner similar to that observed in cancer-proof mice.
[0079] Upon provision with such composition, the sentinels of the innate immune system, dendritic cells, can be alerted and neutrophils, macrophages and natural killer cell activity can be significantly enhanced. The enhancement of vitamin D receptors via resveratrol is yet another major advantage of a combination approach to treat or prevent cancer. This approach appears to be more appropriate for senior adults, the highest risk group for cancer, who are often immune-compromised due to poor nutrition or lack of nutrient absorption. The fact that this therapy can now be immediately measured for effectiveness by non-invasive cancer cell counting technology means that expensive and equivocal tests on animals may not be required to prove efficacy.
[0080] Vitamin D exhibits many biological actions. While vitamin D is widely known for its ability to stave off bone disease (rickets in growing children, osteoporosis in senior adults), it is becoming a central player in the battle against cancer. Only recently is it also gaining attention as an antibiotic. Vitamin D-deficient mice exhibit a defective response from phagocyte cells in the face of infection or inflammation. Vitamin D deficiency is frequently associated with recurrent infections. Only about half of the macrophage cells accumulate at the site of inflammation in vitamin D-deficient animals compared to animals whose vitamin D levels are adequate.
[0081] To delve deeper into the role of vitamin D in immunity and cancer, vitamin D improves the chemotactic (affinity for) neutrophils to mobilize and migrate. Patients with rickets due to vitamin D deficiency are observed to have sluggish neutrophils that cannot migrate properly. Vitamin D stimulates the maturation of monocytes to macrophages. This results in an enlarged army of immune fighting cells to mount against tumors. Greater attention is now being given to vitamin D as an anti-cancer weapon because of studies which show supplemental vitamin D drastically reduces the risk for all types of cancer. A study that employed 1100 IU of vitamin D3 produced a 60-77% reduction in cancer risk among women in Nebraska in just a 4-year period.
[0082] Even though cancer risk is lowest in sunnier and Equatorial areas geographically, where vitamin D levels are higher in sun-exposed populations, the protective effect of vitamin D against cancer has been repeatedly dismissed or discounted. The consumption of vitamin D orally eliminates the concern of skin cancer emanating from overexposure to unfiltered sun rays. One of the latest analyses shows that the risk of colon cancer can be halved by taking 2000 IU of vitamin D per day and that the risk for breast cancer can be halved by taking 3500 IU of vitamin D per day. The median dietary intake of vitamin D is only about 230 IU per day, so the prospect of food fortification or supplementation to prevent or treat cancer now becomes real.
[0083] In order for tissues to utilize and benefit from vitamin D they must have proteins in their outer coat (cell membrane) that are designed to receive and bind to vitamin D. For example, about 80% of human breast tumors produce vitamin D cell receptors, though gene expression (production) of vitamin D receptor is at low levels.
[0084] Vitamin D's ability to inhibit cancer may be heightened when it is aided by weak estrogen-like molecules in the diet. Resveratrol, an estrogen-like molecule commonly found in red wine, upregulates the vitamin D receptor in breast cancer cells without increasing cancer growth. Resveratrol, in effect, can sensitize breast cancer cells to the anti-cancer properties of vitamin D.
[0085] Laboratory experiments show that low-dose vitamin D3 does not reduce breast tumor cell growth but when combined with resveratrol, tumor cell numbers declines by 40%. At higher concentrations vitamin D3 reduces the number of breast cancer cells in a lab dish by about 25%, and this decline improves to 50% when combined with resveratrol. Whereas estrogen increases vitamin D receptor gene expression, it also stimulates breast tumor growth. Resveratrol does not have this drawback. Resveratrol potentiates or "weaponizes" the cancer- inhibiting effect of vitamin D.
[0086] Furthermore, resveratrol by itself has been shown to calm the response of phagocytes to foreign invaders like germs and tumor cells. Resveratrol dampens production of reactive oxygen species (free radicals) and normalizes particle ingestion in macrophage cells. Therefore, resveratrol prevents the over-response of immune cells that can produce autoimmunity.
[0087] Resveratrol blocks cancer in so many ways that it is difficult to find a pathway for cancer that is not obstructed by resveratrol. Resveratrol induces the cell energy compartments in tumor cells, called mitochondria, to release an enzyme called cytochrome C oxidase that usually leads to a cascade of other enzymes that induce programmed cell death, called apoptosis. But a recent experiment also shows that resveratrol releases cytochrome C from ovarian tumor cells that leads to rapid cell death via a process called autophagy, a process where enzymes produced inside the tumor cell actually digest its innards (kind of a form of intracellular cannibalism). This is a form of cell suicide that resveratrol activates in tumor cells, but not healthy cells.
[0088] The contribution of innate immunity in surveillance of tumors is comparatively neglected in cancer biology. Phagocytosis, or "cell eating" is the cornerstone of the innate immune response. Focus has been directed to dendritic cells which are believed to be sentinels of the innate immune response. A limited number of immune -boosting agents have been investigated.
[0089] Skepticism surrounds interest in innate immune approaches to cancer treatment. For example, patients taking immune-suppressing don't necessarily develop cancer with more frequency. However, this may be misunderstood. An over-responsive immune system may lead to more tissue and organ damage that can be mortal to cancer patients. Most of the drugs used for breast cancer therapy induce immune suppression.
[0090] Nature's most potent iron chelator is inositol hexaphosphate (IP6), which is found in seeds and the bran fraction of whole grains. A low dosage of IP6 has been found to suppress the growth of rhabdomyosarcoma cells by 50%. Removal of IP6 allows these tumor cells to recover and grow once again. IP6-treated mice with injected tumors exhibit tumors that are 50 times smaller than non-treated mice. IP6 has also been shown to reduce the growth of injected fibrosarcoma cells in mice and prolong their survival.
[0091] In examining the immune enhancing properties of IP6 it has been shown that it boosts production of free radicals (superoxide) and the cell digesting action of neutrophils in the presence of bacteria. IP6 increases the release of interleukin-8.
[0092] The action of natural killer cells, which are involved in tumor cell destruction, is enhanced by IP6. [0093] In one embodiment, the hyaluronic acid of such composition is conjugated to a chemotherapeutic agent. The invention particularly pertains to such compositions in which the chemotherapeutic agent is taxol. The invention particularly pertains to such compositions that additionally and preferably comprise a chelator, 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 facilitates invasion (Hua, Q. et al. (1993) "Internalization Of Hyaluronan By Chondrocytes Occurs Via Receptor-Mediated Endocytosis," J. Cell. Sci. 106(Pt l):365-375; Luo, Y. et al. (2000) "A Hyaluronic Acid-Taxol Antitumor Bioconjugate Targeted To Cancer Cells," Biomacromolecules l(2):208-218). Thus chemotherapeutic agents that are conjugated to Hyaluronic Acid target tumor cells, and can provide an effective anti-tumor dosage at lower overall concentration.
[0094] In brief, a preferred method of conjugation entails forming an NHS (N-hydroxy- succimimide derivative of the chemotherapeutic agent. Such a derivative can be made by adding a molar excess of dry pyridine to a stirred solution of Taxol and succinic anhydride in CH2CI2 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 CH2Cl2 and the produced Taxol-2'-hemisuccinate can be purified on silica gel (washed with hexane; eluted with ethyl acetate) to give the desired product (Luo, Y. et al. (1999) "Synthesis And Selective Cytotoxicity Of A Hyaluronic Acid-Antitumor Bioconjugate," Bioconjug. Chem. 10(5):755- 763).
[0095] The N-hydroxy-succimimide derivative of the chemotherapeutic agent is then conjugated to adipic dihydrazido-functionalized hyaluronic acid. Adipic dihydrazido- functionalized hyaluronic acid is preferably prepared as described by Pouyani, T. et al. (1994) ("Functionalized Derivatives Of Hyaluronic Acid Oligosaccharides - Drug Carriers And Novel Biomaterials " Bioconjugate Chem. 5:339-347); Pouyani, T. et al. (1994) ("Novel Hydrogels Of Hyaluronic Acid: Synthesis, Surface Morphology, And Solid-State NMR," J. Am. Chem. Soc. 116:7515-7522); Vercruysse, K.P. et al. (1997) ("Synthesis And In Vitro Degradation Of New Polyvalent Hydrazide Cross-Linked Hydrogels Of Hyaluronic Acid," Bioconjugate Chem. 8:686-694). Thus, hyaluronic acid is preferably dissolved in water and an excess of adipic dihydrazide (ADH). The pH of the reaction mixture is adjusted to 4.75 by addition acid. Next, 1 equivalent of l-Ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDCI) is added in solid form. The pH of the reaction mixture is maintained at 4.75 by addition of acid. The reaction is quenched by addition of 0.1 N NaOH to adjust the pH of reaction mixture to 7.0. The reaction mixture is then transferred to pretreated dialysis tubing (Mw cutoff 3,500) and dialyzed exhaustively against 100 mM NaCl, then 25% EtOH/H2O and finally water. The solution is then filtered through 0.2 m cellulose acetate membrane, flash frozen, and lyophilized (Luo, Y. et al. (1999) "Synthesis And Selective Cytotoxicity Of A Hyaluronic Acid- Antitumor Bioconjugate " Bioconjug. Chem. 10(5):755-763).
3. Aging
[0096] Calcification and rusting are major accelerators of aging. The human body is composed of cells that must continually be replaced or renewed from within, and a gooey substance that fills space between cells called collagen or connective tissue which also must be continually regenerated. As the human body ages at the cellular level, there is a slow accumulation of cellular debris called lipofuscin. The formation of lipofuscin is facilitated by the progressive accumulation of iron and calcium within cell bodies called lysosomes and mitochondria. A cell cleansing and renewal process called autophagy prevents the accumulation of lipofuscin. Progressive inability to remove cellular debris results in declining cell function and then premature death of the cell. A young cell efficiently removes debris from within. An old cell cannot efficiently remove debris and accumulates lipofuscin.
[0097] Calcification and rusting of cells impairs the cleansing of cellular debris (lipofuscin) from cells by enzymes produced by lysosomes, and results in impairment of cellular energy (ATP) produced by the mitochondria within cells. The compositions of the present invention inhibit and/or reverse cellular aging and/or connective tissue aging, and in particular, inhibit and/or reverse cellular aging and/or connective tissue aging caused by an accumulation of major minerals (e.g., iron, calcium, etc.). As a consequence, recipients of the compositions of the present invention exhibit enhanced longevity and enhanced cellular and connective tissue health and structure.
[0098] The human body ages at the cellular level by the slow accumulation of cellular debris called lipofuscin, which is facilitated by the progressive accumulation of iron and calcium within cell bodies called lysosomes and mitochondria. A cell cleansing and renewal process called autophagy prevents the accumulation of lipofuscin during the years of youthful growth, but this lysosomal mechanism declines once full growth is achieved due to accumulation of intracellular iron and calcium. Progressive inability to remove cellular debris results declining cell function and then premature death of the cell. A young cell efficiently removes debris from within. An old cell cannot efficiently remove debris and accumulates lipofuscin. The mitochondria, which provides cellular energy for lysosomal bodies to perform their cell cleansing activity, also becomes progressively calcified and ironized once childhood growth ceases. Only about 5% of mitochondria are functioning by age 80. Iron and calcium chelators are proposed to remedy mitochondrial aging which impacts cellular functions such as lysosomal enzymatic activity
[0099] The human body ages within connective tissue by failure of cells called fibroblasts to regenerate collagen and hyaluronic acid, the latter being a space-filling, water-holding molecule. Collagen formation is facilitated by vitamins and amino acids in the diet (vitamin C, lysine, proline). Fibroblasts can be stimulated to produce hyaluronic acid by estrogen, made naturally in the body, and by estrogen-like molecules found in plants, called phytoestrogens, provided in the diet of by hyaluronic acid itself. Young females, by virtue of the ability to produce estrogen, exhibit thicker hair, smoother skin and more flexible joints, due to the abundance of hyaluronic acid. All of these being attributes of youthfulness.
[00100] The inability to regenerate hyaluronic acid results in tissues losing their physical integrity by virtue of loss of the space-filling properties of hyaluronic acid. Without adequate hyaluronic acid, a dehydrated state results and tissues shrink and shrivel up. For example, skin that is lacking hyaluronic acid will appear wrinkled and dry. Joint spaces will lack the cushioning and space-filling needed to prevent bone from rubbing on bone. The eyes will begin to shrink in size. Hair will thin due to the lack of hydration. These are the most prominent visible or cosmetic signs of aging.
[00101] In one embodiment, the present invention invention addresses both cellular and extracellular (connective tissue) aging, thus (a) preserving youthful function of living cells by removal of excess minerals, largely calcium and iron, from cells, this facilitating autophagy (cleanup of cellular debris, such as lipofuscin, via lysosomal enzymes) and (b) invigorating and preserving production of hyaluronan by stimulation of fibroblasts by HA, phytoestrogens (resveratrol, quercetin, genistein, are a few), to inhibition of degradation of HA by provision of metal chelators, such as phytic acid, ferulate, quercetin, resveratrol, etc.
[00102] In one embodiment, the present invention is a dietary supplement that addresses both cellular and non-cellular aging by its ability to:
(a) stimulate renewal of living cells from within via enzymatic degradation of cellular debris by intracellular lysosomal bodies. This is facilitated by the inclusion of metal (iron, copper, heavy metal) and calcium chelating molecules within the formula. Lysosomes lose their ability to enzymatically digest cellular debris with the progressive accumulation of iron, copper and other metals, and the crystallization of calcium. (b) stimulate fibroblasts to produce hyaluronic acid at youthful levels again. This is accomplished by provision of orally-consumed molecules that stimulate fibroblasts to produce hyaluronic acid.
(c) metal chelating molecules that help maintain youthful lysosomal function are identified as antioxidants, like vitamin E or vitamin C, lipoic acid, metal chelators like IP6 phytate, quercetin, bioflavonoids or polyphenols, resveratrol. Resveratrol works by its ability to stimulate production of heme oxygenase, an enzyme that helps to control iron.
(d) molecules that inhibit crystallization of calcium 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, lignans, hydroxytyrosol, or other molecules configured like estrogen. Orally consumed HA stimulates greater HA and chondroitin synthesis. Similarly, glucosamine stimulate fibroblasts to produce HA. Alternatively, or additionally, glucosamine stimulates synovial production of hyaluronic acid, which is primarily responsible for the lubricating and shock- absorbing properties of synovial fluid" (McCarty, M.F. (1998) "Enhanced Synovial Production Of Hyaluronic Acid May Explain Rapid Clinical Response To High-Dose Glucosamine In Osteoarthritis " Medical Hypotheses 50:507-510, 1998).
(f) orally consumed molecules that stimulate production of collagen are vitamin C, proline and lysine.
[00103] In such embodiment, the present invention relates to a resveratrol and hyaluronic acid-containing dietary supplement that restores youthful function and appearance to human cells and tissue. The invention particularly pertains to such compositions that additionally comprise a chelator, and/or vitamin D. Most preferably, the composition will comprise the chelator phytic acid (inositol hexaphosphate; IP6). The compositions of the present invention synergistically enhance the specific activity of the resveratrol and/or hyaluronic acid, and thus the compositions of the present invention provide an enhancement of activity above and beyond that obtained with the components administered individually. In such embodiment, the invention relates to a method for restoring youthful function and appearance to human cells and tissues comprising the following steps: (a) stimulating renewal of living cells from within via enzymatic degradation of cellular debris by intracellular lysosomal bodies (preferably by providing a metal chelating molecule that helps maintain youthful lysosomal function, such molecules comprising antioxidants, such as vitamin E or vitamin C, lipoic acid, metal chelators like IP6 phytate, quercetin, bioflavonoids or polyphenols, and/or resveratrol); and
(b) stimulating fibroblasts to produce hyaluronic acid (comprises providing orally consumed molecules that stimulate fibroblasts to produce hyaluronic acid, such orally consumed molecules comprising, for example, hyaluronic acid, glucosamine, chondroitin, and/or estrogen-like molecules such as genistein, lignans, hydroxytyrosol, or other molecules configured like estrogen).
Preferably, such stimulation is achieved by the dietary administration of a composition comprising the stated compounds, more preferably in combination with an orally consumable molecule that stimulates production of collagen, such molecules comprising, for example, vitamin C, proline and/or lysine.
[00104] Without intending to be bound by any mechanism, when administered together, the four preferred components are believed to act in the following manner:
(a) Vitamin D: Vitamin D3 works as an agent that mimics the response to a biological stressor, solar radiation. In particular, vitamin D3 upregulates protective genes involved in activation of the immune system, particularly neutrophil count and motility, and aids in overcoming the decline in endogenous vitamin D3 production with advancing age due to thickening of the skin, which reduces sun/skin production of vitamin D. Furthermore, vitamin D3 works synergistically to breakdown IP6 to IP3, thought to be a major active molecule. Resveratrol also works synergistically to sensitize cells to vitamin D3 (sensitizes the vitamin D receptor on the cell surface). Vitamin D serves to break down IP6 to IP3, which is its primary active form. Vitamin D is also believed to act as an immune system enhancing agent, boosting innate immunity in humans. In this capacity, vitamin D has been shown experimentally to have important cancer-preventive and cancer-curing properties.
(b) Resveratrol: Resveratrol increases the sensitivity of the vitamin D receptor on the surface of cells, and thus is believed to act as an enhancing agent for vitamin D and as an anti-cancer agent. Resveratrol up-regulates the vitamin D receptor on the surface of cancer cells, and sensitizes cancer cells to vitamin D (Wietzke, J.A. 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):149-157; Wietzke, J.A. et al. (2005) "Regulation Of The Human Vitamin D3 Receptor Promoter In Breast Cancer Cells Is Mediated Through SpI Sites " Molec. Cell. Endocrinol. 230(1-2): 59-68). Resveratrol is also believed to be a monoamine oxidase inhibitor (MAO Inhibitor).
(c) Hyaluronic acid: Hyaluronic acid is the water gelling molecule of the human body which serves as its scaffolding and hydrating agent. As aging progresses, less Hyaluronic acid is produced, resulting in wrinkled skin, thinning hair, unlubricated joints. The chelators of the present composition also help to preserve hyaluronic acid in the body. The hyaluronic acid component and the mineral chelating components (e.g., resveratrol, quercetin, phytic acid IP6, ferulate) work as a total anti-aging strategy to maintain youthful function within cells and connective tissues. Hyaluronic acid is believed to have an affinity to cancer cells. It is believed to serve as a delivery and targeting (drug delivery agent) molecule in blood circulation and to address aging of the connective tissue. The collapse and loss of integrity of connective tissue between cells provides the signs of aging (e.g., skin wrinkling, hair thinning, joint stiffness, loss of stature, etc.). The addition of hyaluronic acid to the compositions of the present invention is believed to activate fibroblast cells in the human body to produce additional hyaluronic acid, thus serving to preserve connective tissue (collagen) in a youthful state (Yadav, A.K. et al. (2008) "An Insight On Hyaluronic Acid In Drug Targeting And Drug Delivery;' J. Drug Target. 16(2):91-107; Liao, Y.H. et al. (2005) "Hyaluronan: Pharmaceutical Characterization And Drug Delivery " Drug Deliv. 12(6):327-342; Joddar, B. et al. (2006) "Elastogenic Effects Of Exogenous Hyaluronan Oligosaccharides On Vascular Smooth Muscle Cells " Biomaterials 27(33):5698-5707; Girish, K.S. et al. (2007) "The Magic Glue Hyaluronan And Its Eraser Hyaluronidase: A 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 an iron and copper chelator and as an inhibitor of calcium cystallization. Phytic Acid also is believed to reduce the availability of metallic minerals that serve as growth factors in tumor cells. It is also believed to serve as a neutrophil priming and motility agent. Additionally, phytic acid has been found to be neuroprotective, and thus to attenuate the severity of conditions associated with neurodegenerative diseases (especially Parkinson's Disease, camptocormia, and Alzheimer's Disease) (Xu, Q. et al. (Epub 2007 Dec 27) "Neuroprotective Effect Of The Natural Iron Chelator, Phytic Acid In A Cell Culture Model Of Parkinson's Disease," Toxicology 245(1-2): 101-108). The components of the compositions of the present invention are believed to enhance such neuroprotection. [00105] The iron chelator, quercetin, if present, is believed to serve to increase immediate bioavailability of resveratrol by permitting more passes through the liver before it is metabolized.
[00106] The individual components of the composition are believed to act synergistically to enhance the effect of, for example, resveratrol. Without intending to be limited thereby, it is proposed that the body's control or chelation of iron and calcium regulates the rate of aging after full growth has been achieved. During childhood growth all the iron and calcium are directed towards production of new bone and new red blood cells (hemoglobin). The cessation of childhood growth results in excess iron, copper and calcium, which then progressively (a) calcifies and (b) rusts tissues. The lysosomes begin to accumulate iron and calcium, which results in their dysfunction. The mitochondria begin to malfunction as they also progressively rust and calcify. The compositions of the present invention are believed to be capable of limiting or slowing the progressive rusting and calcification of cells and cellular organelles to thereby facilitate a slowing or reversal of the aging process. The chelation is what controls the genes. Genes are then favorably upregulated or downregulated. Resveratrol and a copper chelator are believed to act: (1) as controllers of calcium concentration via upregulation of osteocalcin, the hormone that helps retain calcium in bones and (2) as controllers of iron concentration via heme oxygenase, an antioxidant enzyme.
[00107] MAO inhibitors and iron chelators have been proposed as treatments for Parkinson' s disease (Youdim, M.B. et al. (2004) "Novel Bifunctional Drugs Targeting Monoamine Oxidase Inhibition And Iron Chelation As An Approach To Neuroprotection In Parkinson 's Disease And Other Neurodegenerative Diseases," J. Neural. Transm. 111(10-11): 1455-1471 ; Yanez, M. et al. (2006) "(-)-Trans-Epsilon-Viniferin, A Polyphenol Present In Wines, Is An Inhibitor Of Noradrenaline And 5-Hydroxytryptamine Uptake And Of Monoamine Oxidase Activity " Eur. J. Pharmacol. 542(l-3):54-60; Bureau, G. et al. (2008) "Resveratrol And Quercetin, Two Natural Polyphenols, Reduce Apoptotic Neuronal Cell Death Induced By Neuroinflammation " J. Neurosci. Res. 86(2):403-410; Singh, A. et al. (2003) "Quercetin Potentiates L-Dopa Reversal Of Drug-Induced Catalepsy In Rats: Possible COMT/MAO Inhibition;" Pharmacol. 68(2):81- 88; Gao, X. et al. (2007) "Prospective Study Of Dietary Pattern And Risk Of Parkinson Disease " Am. J. 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). The compositions of the present invention which contain the MAO inhibitor and copper chelator, resveratrol, the iron chelator and MAO inhibitor, quercetin, and the broad metal chelator, phytic acid are particularly preferred for the treatment of neurodegenerative diseases (especially Parkinson's Disease, camptocormia, and Alzheimer's Disease) or in the amelioration of the symptoms of such diseases.
[00108] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention unless specified.
Example 1 Comparative Effects of Resveratrol and the Compositions of the Present Invention
[00109] In order to determine if the compositions of the present invention were more effective than resveratrol alone in mediating a resveratrol biological activity, an analysis of gene expression was conducted, comparing the modulation of gene expression achieved by calorie restriction to the modulation of gene expression achieved by the compositions of the present invention.
[00110] Accordingly, the ability of resveratrol alone and the resveratrol-containing compositions of the present invention to up-regulate survival/longevity genes or down-regulate genes whose expression enhances cellular damage was compared using the expression profile of a calorie restricted ("CR") animal as a positive control and the expression profile of a normally fed animal as a negative control. Male B6CHF1 mice (2 months of age) were thus either placed on a 40% calorie restricted diet, provided commercially obtained trans-resveratrol (Sigma Chemical; 1.25 mg/kg per day), provided a resveratrol-containing composition of the present invention (Longevinex®; Resveratrol Associates, LLC; 100 mg ?ra«,y-resveratrol containing capsule per 80 kg human per day (i.e., 2.5 mg/kg per day of resveratrol (1.25 mg/kg per day ?rα«,y-resveratrol) 0.31 mg/kg per day quercetin dihydrate, 0.94 mg/kg per day rice bran extract, 4.75 mg/kg per day rice bran oil and 0.70 mg/kg per day sunflower lecithin)). The mice were monitored until they had reached five months of age.
[00111] Body weight, serum glucose levels, serum insulin levels and lipid peroxidation in brain and muscle tissue were measured. The results showed that Longevinex® did not result in an increase in weight that was distinguishable from control animals (Figure 1). Serum insulin levels were found to be approximately the same as that observed in the calorie restricted animals (Figure 2). Serum glucose levels were found to be lower than that observed in the calorie restricted animals (Figure 3). Example 2
Comparative Effects of Resveratrol and the Compositions of the Present Invention on
Gene Expression in Cardiac Tissue
[00112] The profile of expressed genes in the cardiac tissue of mice receiving resveratrol or a composition of the present invention (Longevinex®) was compared to that of mice placed on a calorie restricted diet and control mice. Gene expression was monitored using an Affymetrix MG430 2.0 Array, containing 45,101 probe sets per array. In cases in which the array represented the same gene with multiple probes, the probe set with the highest signal intensity was employed. Unknown genes (including uncharacterized ESTs and cDNA sequences were not analyzed. Thus, the array provided a means for analyzing 20,341 genes having a single Entrez Gene ID. Analysis was conducted substantially as described by Lee, C-K. et al. (2002) "Transcriptional Profiles Associated With Aging And Middle Age-Onset Caloric Restriction In Mouse Hearts;' Proc. Natl. Acad. Sci. (U.S.A.) 99: 14988-14993, herein incorporated by reference. The mean of all arrays in a group were calculated. The means of treated groups were compared to the mean of the control group, and the statistical significance of any differences were determined using two-tailed ϊ-tests (P < 0.01). The results of the analysis are presented in Table 3 (CO, control; CR, calorie restricted; RES, resveratrol; LGX, Longevinex®; FC, fold change. FC is calculated as the mean of the treated group divided by the mean of the control group, and this value is then log-transformed (base 2) for statistical purposes. As an example, a gene that is expressed at 100 in the control and 200 in a treated group would be have an Fc of 2 (i.e., a twofold increase in expression); a gene that is expressed at 100 in the control and 50 in the treated group, would have an Fc of -2 (i.e., a twofold decrease in expression).
[00113] Treatment of human umbilical vein epithelial cells with ferulic acid, quercetin or resveratrol has been reported to result in changes to gene expression of greater than 2-fold down-regulation of 363 genes, and greater than 2-fold up-regulation of 233 genes of 10,000 genes probed (Nicholson, S. K. et al. (2008) "Effects Of Dietary Polyphenols On Gene Expression In Human Vascular Endothelial Cells " Proc. Nutr. Soc. 67(l):42-47). In contrast, Table 3 shows that 2,829 genes were found to exhibit a statistically significant change in expression in treated vs. control mice. Of these genes, 7% were found to exhibit altered expression in mice that had been subjected to only calorie restriction; 8% were found to exhibit altered expression in mice subjected only to resveratrol. Combining calorie restriction with resveratrol administration failed to alter the expression of any additional genes. In contrast, administration of Longevinex® was found to alter the expression of 61% of the 2,829 genes. Administration of Longevinex® to calorie restricted mice was found to alter the expression of an additional 2% of the genes. Administration of Longevinex® to mice receiving resveratrol was found to alter the expression of an additional 21% of the genes. Thus, Longevinex® alone or in combination with other regimens was found to affect 85% (2,406) of the total genes showing altered expression.
Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1415670 at Lgx only 987 1139 1187 1378 1 15 1 20 1 40 Copg
1415671 at Res & Lgx 2454 2539 2030 2071 1 03 -1 21 -1 19 Atp6v0d1
1415672 at Res & Lgx 3213 2819 2637 2262 -1 14 -1 22 -1 42 Golga7
1415677 at Res & Lgx 706 747 933 934 1 06 1 32 1 32 Dhrsi
1415679 at Lgx only 4825 4129 3776 3154 -1 17 -1 28 -1 53 Psenen
1415684 at Lgx only 638 573 490 381 -1 11 -1 30 -1 67 Atg5
1415696 at Res only 6629 7063 5491 5988 1 07 -1 21 -1 11 SaMa
1415700 a at Res & Lgx 5461 5179 3791 2603 -1 05 -1 44 -2 10 Ssr3
1415704 a at Lgx only 5010 4472 3850 3285 -1 12 -1 30 -1 52 Cdv3
1415707 at Lgx only 1111 1317 1468 1535 1 19 1 32 1 38 Anapc2
1415714 a at Res & Lgx 4941 4632 2720 2379 -1 07 -1 82 -2 08 2610209M04Rιk
1415723 at Res & Lgx 4272 3466 3385 3062 -1 23 -1 26 -1 40 Eιf5
1415733 a at Lgx only 13615 13963 9719 9991 1 03 -1 40 -1 36 1110019J04Rιk
1415735 at Lgx only 2025 2617 3355 3642 1 29 1 66 1 80 Ddb1
1415736 at Res & Lgx 4957 3953 2536 1805 -1 25 -1 95 -2 75 Pfdn5
1415738 at Res & Lgx 1914 1941 1575 1395 1 01 -1 22 -1 37 Txndc12
1415742 at Lgx only 749 837 856 987 1 12 1 14 1 32 Aup1
1415746 at Lgx only 726 949 1075 1501 1 31 1 48 207 Cic
1415749 a at Lgx only 2368 2397 2111 1912 1 01 -1 12 -1 24 Rragc
1415754 at Lgx only 3512 3195 3099 2411 -1 10 -1 13 -1 46 Polr2f
1415755 a at Lgx only 2983 3602 3890 4068 1 21 1 30 1 36 Ube2v1
1415756 a at Res & Lgx 3581 3290 2710 2706 -1 09 -1 32 -1 32 Snapap
1415757 at Lgx only 901 1073 1189 1337 1 19 1 32 1 48 Gbf1
1415764 at Lgx only 3381 3218 2545 2419 -1 05 -1 33 -1 40 Zc3h11a
1415783 at Lgx only 4869 4728 4237 3960 -1 03 -1 15 -1 23 Vps35
1415788 at Lgx only 1490 1572 1169 1046 1 05 -1 27 -1 42 Ublcpi
1415791 at Res & Lgx 2983 2870 2364 1805 -1 04 -1 26 -1 65 Rnf34
1415797 at Lgx only 319 488 522 805 1 53 1 64 2 53 Ddr1
1415802 at Res & Lgx 8566 8379 4763 4548 -1 02 -1 80 -1 88 SId6a1
1415812 at Lgx only 31926 36029 41778 58920 1 13 1 31 1 85 Gsn
1415814 at CR only 1760 2239 1514 1433 1 27 -1 16 -1 23 Atp6v1b2
1415816 at Res only 5617 5279 4428 5553 -1 06 -1 27 -1 01 Cct7
1415818 at Lgx only 2554 2785 2838 3335 1 09 1 11 1 31 Anxa6
1415830 at Lgx only 586 494 424 318 -1 19 -1 38 -1 84 Orc5l
1415834 at Res only 579 703 419 430 1 21 -1 38 -1 35 Dusp6
1415840 at Res & Lgx 1636 1461 974 856 -1 12 -1 68 -1 91 Elovl5
1415850 at Res & Lgx 824 880 1129 1093 1 07 1 37 1 33 Rasa3
1415856 at Lgx only 148 168 172 242 1 14 1 16 1 63 Emb
1415875 at Res only 83 38 34 51 -2 19 -243 -1 63 3010003L21Rιk
1415876 a at Lgx only 13888 14344 16149 18965 1 03 1 16 1 37 Rps26
1415879 a at Lgx only 33385 37008 41034 49898 1 11 1 23 1 49 Rplp2
1415882 at Res only 20588 18774 17207 17741 -1 10 -1 20 -1 16 Ghitm
1415886 at All 637 966 1017 1132 1 51 1 60 1 78 Sh2d3c
1415901 at Lgx only 1435 1699 1682 1903 1 18 1 17 1 33 Plod3
1415907 at Lgx only 1656 1817 2329 2446 1 10 1 41 1 48 Ccnd3
1415909 at Res & Lgx 1788 1949 1414 1451 1 09 -1 26 -1 23 Stip1 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1415915 at Res & Lgx 2843 3059 3559 3668 1 08 1 25 1 29 Ddx1
1415930 a at Lgx only 14546 13685 14019 11677 -1 06 -1 04 -1 25 Map1 lc3b
1415935 at Res & Lgx 1687 1988 201 1 2446 1 18 1 19 1 45 Smoc2
1415947 at CR only 7499 6219 8373 7163 -1 21 1 12 -1 05 Cregi
1415951 at Lgx only 496 588 587 717 1 19 1 18 1 45 FkbpiO
1415961 at Lgx only 2152 2177 2503 3192 1 01 1 16 1 48 Itm2c
1415966 a at Lgx only 29789 33932 35207 35979 1 14 1 18 1 21 Ndufvi
1415971 at CR only 1825 1273 1649 1440 -1 43 -1 11 -1 27 Marcks
1415974 at Lgx only 1694 2075 2211 2392 1 22 1 31 1 41 Map2k2
1415977 at Lgx only 444 603 658 715 1 36 1 48 1 61 Isynal
1415987 at Lgx only 8838 9568 10218 11 109 1 08 1 16 1 26 Hdlbp
1415990 at CR & Lgx 27024 32941 29714 35847 1 22 1 10 1 33 Vdac2
1415991 a at Res & Lgx 3051 3384 4293 4892 1 11 1 41 1 60 Klhdc3
1415996 at All 10650 22628 23188 27989 2 12 2 18 2 63 Txnip
1415998 at Lgx only 33371 40497 35239 46095 1 21 1 06 1 38 Vdad
1416013 at Res & Lgx 926 1 171 1314 1772 1 26 1 42 1 91 Pld3
1416014 at All 1518 981 801 662 -1 55 -1 90 -2 29 Abcei
1416016 at Res & Lgx 232 236 387 358 1 02 1 67 1 55 Tap1
1416019 at Res only 336 291 275 269 -1 15 -1 22 -1 25 Dr1
1416027 at Res & Lgx 2414 2298 1834 1569 -1 05 -1 32 -1 54 Pdcd6
1416032 at Lgx only 1053 1425 1776 1638 1 35 1 69 1 56 Tmem109
1416046 a at Lgx only 4982 5500 3916 3426 1 10 -1 27 -1 45 Fuca2
1416048 at Res & Lgx 1089 1 174 1589 1635 1 08 1 46 1 50 Phc2
1416050 a at Lgx only 903 1023 1209 1283 1 13 1 34 1 42 Scarbi
1416051 at Lgx only 128 165 178 252 1 28 1 39 1 97 C2
1416061 at Lgx only 1318 1255 1052 964 -1 05 -1 25 -1 37 Tbc1 d15
1416064 a at Res only 12341 12004 9273 10433 -1 03 -1 33 -1 18 Hspa5
1416069 at CR only 1055 1396 1197 1313 1 32 1 13 1 24 Pfkp
1416079 a at Lgx only 1323 1494 1456 161 1 1 13 1 10 1 22 Arpda
1416082 at Res & Lgx 13983 13415 1 1321 10270 -1 04 -1 24 -1 36 Rab1
1416091 at Lgx only 1996 2372 2622 3315 1 19 1 31 1 66 Mtap4
1416106 at Lgx only 1108 939 1035 775 -1 18 -1 07 -1 43 Kti 12
14161 11 at CR & Lgx 368 212 288 138 -1 74 -1 28 -2 68 Cd83
14161 12 at Lgx only 7587 9334 8583 12194 1 23 1 13 1 61 Coxδa
14161 13 at Lgx only 1810 1965 2224 2559 1 09 1 23 1 41 Fkbpδ
1416125 at Lgx only 550 764 772 809 1 39 1 40 1 47 Fkbp5
1416129 at Lgx only 1270 1 118 1078 734 -1 14 -1 18 -1 73 Errfil
1416140 a at Lgx only 1672 1904 1977 1952 1 14 1 18 1 17 Dhx30
1416142 at Lgx only 614 690 511 333 1 12 -1 20 -1 85 Rps6
1416155 at Res & Lgx 3723 3890 2824 2551 1 04 -1 32 -1 46 Hmgb3
1416175 a at Res & Lgx 21409 19416 15845 13003 -1 10 -1 35 -1 65 Vdac3
1416176 at Lgx only 2064 1801 1869 1358 -1 15 -1 10 -1 52 Hmgbi
1416177 at Res & Lgx 884 734 669 666 -1 20 -1 32 -1 33 Rbmxrt
1416181 at Res only 1982 1958 1504 1705 -1 01 -1 32 -1 16 Mesdc2
1416183 a at Lgx only 40384 48786 54318 64528 1 21 1 35 1 60 Ldhb
1416185 a at Lgx only 4447 4224 3331 2724 -1 05 -1 33 -1 63 Ad h5
Figure imgf000053_0001
Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1416452 at Lgx only 5918 6059 6368 7090 1 02 1 08 1 20 Oat
1416455 a at Lgx only 49845 53641 55445 64019 1 08 1 1 1 1 28 Cryab
1416457 at Lgx only 1208 1356 1592 1930 1 12 1 32 1 60 Ddah2
1416462 at Res & Lgx 3796 3321 2539 2621 -1 14 -1 49 -1 45 Caprini
1416478 a at Lgx only 49370 54196 49980 56965 1 10 1 01 1 15 Mdh2
1416479 a at Lgx only 3314 4024 3824 5065 1 21 1 15 1 53 Tmem14c
1416494 at Lgx only 24034 24945 28712 32059 1 04 1 19 1 33 Ndufs5
1416496 at CR & Lgx 9192 10747 9867 10827 1 17 1 07 1 18 Mrfapi
1416498 at CR & Lgx 1234 1020 1151 968 -1 21 -1 07 -1 28 Ppic
1416502 a at Res & Lgx 1327 1624 1659 1918 1 22 1 25 1 45 Preb
1416506 at Lgx only 5718 5208 4876 4228 -1 10 -1 17 -1 35 Psma6
1416510 at Lgx only 5555 6339 5912 6679 1 14 1 06 1 20 Mrpl4
1416513 at Lgx only 1582 2144 2762 2762 1 35 1 75 1 75 Lamb2
1416514 a at Lgx only 888 805 648 439 -1 10 -1 37 -2 02 Fscni
1416517 at Lgx only 411 441 486 580 1 07 1 18 1 41 Pnpla6
1416524 at Lgx only 4034 4167 4527 5256 1 03 1 12 1 30 Spop
1416540 at Lgx only 1017 1340 1386 1769 1 32 1 36 1 74 Hgs
1416547 at Lgx only 12925 12431 1 1457 10620 -1 04 -1 13 -1 22 Ndufb3
1416555 at Res & Lgx 2837 2868 2221 2008 1 01 -1 28 -1 41 Eι24
1416563 at Lgx only 1374 1532 1043 788 1 11 -1 32 -1 74 Ctps
1416576 at Lgx only 230 236 432 441 1 02 1 88 1 92 Socs3
1416587 a at Res & Lgx 372 605 623 822 1 63 1 68 2 21 Xrcd
1416595 at Lgx only 1722 1750 1272 1269 1 02 -1 35 -1 36 Mrps22
1416604 at Lgx only 55121 62820 59589 71 198 1 14 1 08 1 29 Cyd
1416612 at Lgx only 424 520 635 826 1 23 1 50 1 95 Cyp1 b1
1416621 at Res & Lgx 869 1007 1063 1287 1 16 1 22 1 48 LIgM
1416629 at Lgx only 412 524 548 620 1 27 1 33 1 50 Slc1a5
1416634 at Res & Lgx 3167 2719 1899 1420 -1 16 -1 67 -2 23 5730536A07Rιk
1416635 at Res & Lgx 4444 3643 3384 2684 -1 22 -1 31 -1 66 Smpdl3a
1416637 at Lgx only 525 603 661 780 1 15 1 26 1 49 Slc4a2
1416647 at Lgx only 5775 6096 7099 7557 1 06 1 23 1 31 Bckdha
1416648 at Lgx only 2599 3174 3890 4330 1 22 1 50 1 67 Dynd hi
1416656 at Lgx only 1412 1659 1596 1864 1 18 1 13 1 32 did
1416668 at Lgx only 10958 10417 8267 7237 -1 05 -1 33 -1 51 Ttc35
1416680 at Res & Lgx 2599 1810 1455 813 -1 44 -1 79 -3 20 Ube3a
1416683 at Lgx only 1687 2209 2493 2585 1 31 1 48 1 53 Plxnb2
1416690 at Res & Lgx 1379 1680 1929 1834 1 22 1 40 1 33 Gtpbp2
1416699 at Res only 6446 6175 4968 5208 -1 04 -1 30 -1 24 11 10008F13Rιk
1416703 at Lgx only 2541 231 1 2251 1680 -1 10 -1 13 -1 51 Mapk14
1416706 at Res only 408 412 258 254 1 01 -1 58 -1 61 Rpe
1416708 a at Lgx only 807 815 975 1044 1 01 1 21 1 29 Gramdia
1416709 a at Res & Lgx 1287 1148 920 920 -1 12 -1 40 -1 40 Ngrn
1416713 at Lgx only 920 1 162 1424 2002 1 26 1 55 2 18 Tppp3
1416730 at Lgx only 436 492 540 650 1 13 1 24 1 49 RcM
1416731 at Lgx only 1678 1349 1389 1 118 -1 24 -1 21 -1 50 Top2b
1416737 at Lgx only 1600 2387 2293 2936 1 49 1 43 1 84 Gys1
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1418048 at Lgx only 2291 1894 1822 1403 -1 21 -1 26 -1 63 11 10059G10Rιk
1418049 at Lgx only 564 707 732 893 1 25 1 30 1 58 Ltbp3
1418058 at Lgx only 1896 1822 1556 1532 -1 04 -1 22 -1 24 EltcM
1418085 at Lgx only 1 19 169 209 189 1 42 1 76 1 59 Prkcz
1418090 at Lgx only 393 473 619 747 1 20 1 58 1 90 Plvap
1418093 a at Lgx only 180 168 242 294 -1 07 1 34 1 63 Egf
1418124 at Res & Lgx 3982 4177 4816 5241 1 05 1 21 1 32 Tmem85
1418128 at Lgx only 2612 3481 3429 3842 1 33 1 31 1 47 Adcy6
1418148 at Res & Lgx 1168 915 1700 1493 -1 28 1 46 1 28 Abhdi
1418181 at Lgx only 6615 7164 10513 10742 1 08 1 59 1 62 Ptp4a3
1418183 a at Lgx only 2041 1857 1603 1381 -1 10 -1 27 -1 48 Pscdi
1418186 at Lgx only 1322 1528 1723 2106 1 16 1 30 1 59 Gstti
1418187 at Lgx only 2289 2761 3034 3198 1 21 1 33 1 40 Ramp2
1418209 a at Res & Lgx 3685 3274 2816 2402 -1 13 -1 31 -1 53 Pfn2
1418223 at Res & Lgx 2693 2594 2189 2015 -1 04 -1 23 -1 34 Sec1 1a
1418228 at Lgx only 3494 3467 2926 2282 -1 01 -1 19 -1 53 Nfu 1
1418244 at Res & Lgx 3970 4253 2852 2563 1 07 -1 39 -1 55 Nat5
1418261 at All 412 712 724 860 1 73 1 76 2 09 Syk
1418275 a at Lgx only 977 937 845 806 -1 04 -1 16 -1 21 Elf2
1418277 at Lgx only 5601 5071 4822 3833 -1 10 -1 16 -1 46 rp9
1418296 at Lgx only 743 895 936 1 162 1 20 1 26 1 56 Fxyd5
1418302 at Res & Lgx 1279 1585 1792 2129 1 24 1 40 1 66 Ppt2
1418306 at CR only 212 99 186 157 -2 15 -1 14 -1 35 Crybbi
1418308 at Lgx only 312 316 235 144 1 02 -1 33 -2 17 Hus1
1418310 a at CR only 27 74 31 71 2 71 1 13 2 59 Rlbpi
1418325 at Res only 530 450 302 356 -1 18 -1 75 -1 49 Sephs2
1418327 at Lgx only 6638 6594 5327 4629 -1 01 -1 25 -1 43 11 10058L19Rιk
1418328 at Lgx only 9663 10359 12241 14054 1 07 1 27 1 45 Cpti b
1418364 a at Res & Lgx 6214 6280 8701 10097 1 01 1 40 1 62 FtM
1418373 at Res & Lgx 20321 25840 28141 36744 1 27 1 38 1 81 Pgam2
1418384 at Lgx only 2697 2580 2446 2128 -1 05 -1 10 -1 27 Apool
1418394 a at All 1313 1651 1851 2002 1 26 1 41 1 52 Cd97
1418395 at Res only 2083 2050 2622 2487 -1 02 1 26 1 19 Slc47a1
1418421 at Lgx only 409 447 356 219 1 09 -1 15 -1 87 Bcl6b
1418427 at Lgx only 4160 3689 3518 2931 -1 13 -1 18 -1 42 Kιf5b
1418433 at Lgx only 2097 2223 2417 2813 1 06 1 15 1 34 Cab39
1418456 a at Lgx only 1322 1 134 961 777 -1 17 -1 37 -1 70 Cxcl14
1418461 at Lgx only 137 207 161 226 1 52 1 18 1 65 Sh3d19
1418462 at Lgx only 938 943 764 665 1 01 -1 23 -1 41 Exosc9
1418464 at Lgx only 54 55 98 186 1 02 1 80 3 42 Matn4
1418467 at CR & Lgx 1961 2626 2477 2773 1 34 1 26 1 41 Smarcd3
1418479 at CR & Lgx 971 752 799 628 -1 29 -1 22 -1 55 Vps54
1418483 a at Lgx only 801 694 755 588 -1 15 -1 06 -1 36 Ggtai
1418495 at Res only 310 349 161 194 1 13 -1 93 -1 60 Zc3h8
1418506 a at Res only 20857 23088 24680 25457 1 1 1 1 18 1 22 Prdx2
1418518 at Lgx only 701 821 854 1036 1 17 1 22 1 48 Fuπn
Figure imgf000060_0001
Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1418926 at Res & Lgx 3012 2659 2326 2255 -1 13 -1 29 -1 34 Zeb1
1418928 a at Lgx only 958 1057 1 130 1663 1 10 1 18 1 74 2310038H17Rιk
1418929 at Lgx only 410 472 537 581 1 15 1 31 1 42 Ift57
1418933 at Res only 8 28 16 13 3 76 2 08 1 68 SId a6
1418947 at CR only 247 343 289 319 1 39 1 17 1 29 Nek3
1418952 at Res & Lgx 9461 9278 12035 12244 -1 02 1 27 1 29 Txlnb
1418967 a at Lgx only 302 261 219 209 -1 16 -1 38 -1 45 St7
1418968 at Lgx only 2166 1884 1630 1473 -1 15 -1 33 -1 47 Rb1 cc1
1418986 a at Lgx only 1097 1006 854 642 -1 09 -1 28 -1 71 Uxt
1418987 at Res only 161 117 260 269 -1 38 1 61 1 67 Pla2g2d
1418988 at Res & Lgx 2768 2378 2028 1916 -1 16 -1 36 -1 44 Pex7
1418996 a at Lgx only 3436 3503 3043 2693 1 02 -1 13 -1 28 Lyrm5
1419013 at Res only 438 501 605 499 1 14 1 38 1 14 GpatcM
1419026 at Lgx only 910 978 1211 1 131 1 08 1 33 1 24 Daxx
1419037 at Lgx only 1248 1228 1010 1005 -1 02 -1 24 -1 24 Csnk2a1
1419062 at Lgx only 646 672 717 873 1 04 1 1 1 1 35 Epb4 113
1419070 at CR only 274 536 312 423 1 96 1 14 1 54 Cys1
1419072 at Lgx only 1253 1214 1154 1035 -1 03 -1 09 -1 21 Gstm7
1419074 at Lgx only 669 635 434 385 -1 05 -1 54 -1 74 Chac2
1419081 at Lgx only 620 617 514 392 -1 00 -1 21 -1 58 Atg10
1419109 at Res & Lgx 19560 22347 25933 31691 1 14 1 33 1 62 H re
1419131 at CR only 10 65 30 43 6 52 2 99 4 25 F13b
1419144 at Lgx only 328 358 484 596 1 09 1 48 1 81 Cd 163
1419158 a at CR only 760 935 726 788 1 23 -1 05 1 04 Hars2
1419164 at Res & Lgx 1598 1612 1126 698 1 01 -1 42 -2 29 Zfp260
1419169 at Lgx only 11 18 1026 941 817 -1 09 -1 19 -1 37 Mapk6
1419170 at Lgx only 2926 2624 2049 1889 -1 12 -1 43 -1 55 Tmem157
1419174 at Lgx only 1433 1234 1044 1065 -1 16 -1 37 -1 34 2410004B18Rιk
1419182 at Lgx only 525 545 632 749 1 04 1 20 1 43 Svepi
1419186 a at Res & Lgx 779 684 511 470 -1 14 -1 52 -1 66 St8sιa4
1419214 at Res only 146 199 310 284 1 36 2 12 1 94 TnfrsfH a
1419238 at Lgx only 154 255 316 301 1 66 2 06 1 96 Abca7
1419258 at Lgx only 1711 1423 1294 1 11 1 -1 20 -1 32 -1 54 Tceai
1419272 at Res only 428 435 585 498 1 02 1 37 1 16 Myd88
1419292 at Lgx only 3158 3740 4270 4656 1 18 1 35 1 47 Htra3
1419295 at Res only 209 21 1 411 276 1 01 1 96 1 32 Creb3l1
1419297 at CR only 149 66 122 147 -2 25 -1 22 -1 01 H2-Oa
1419302 at Res & Lgx 794 919 1108 1247 1 16 1 40 1 57 Heyl
1419333 at Lgx only 392 461 414 572 1 18 1 06 1 46 11 10008J03Rιk
1419352 at Lgx only 1989 1829 1498 1340 -1 09 -1 33 -1 48 l7Rn6
1419354 at Lgx only 1196 1 159 1578 1790 -1 03 1 32 1 50 Klf7
1419358 at Lgx only 207 323 185 348 1 56 -1 11 1 68 Sorcs2
1419366 at Res & Lgx 642 733 782 1009 1 14 1 22 1 57 Zmat5
1419375 at Res & Lgx 1257 121 1 921 872 -1 04 -1 36 -1 44 Wbp4
1419398 a at Lgx only 10797 11078 10041 8759 1 03 -1 08 -1 23 Reep5
1419415 a at CR only 1168 905 1009 964 -1 29 -1 16 -1 21 Rarg Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1419428 a at Lgx only 1374 1692 1993 2038 1 23 1 45 1 48 Gaa
1419429 at Res & Lgx 79 137 161 223 1 74 2 04 2 83 Cntfr
1419452 at Lgx only 1034 914 835 680 -1 13 -1 24 -1 52 Uchl5
1419455 at Lgx only 6361 6701 5697 5310 1 05 -1 12 -1 20 111 Orb
1419470 at Res & Lgx 865 849 644 649 -1 02 -1 34 -1 33 Gnb4
1419477 at Res only 35 98 190 144 2 81 5 44 4 12 Clec2d
1419484 a at Lgx only 20734 22346 1 1461 5733 1 08 -1 81 -3 62 Gbas
1419491 at Res only 13 40 52 67 3 14 4 08 5 30 Defbi
1419495 at Lgx only 2055 1752 1771 1374 -1 17 -1 16 -1 50 Immp2l
1419499 at Lgx only 7940 7261 10653 11012 -1 09 1 34 1 39 Gpam
1419518 at All 7504 12289 3758 4518 1 64 -2 00 -1 66 Tubaδ
1419527 at Lgx only 403 437 548 829 1 08 1 36 2 06 Comp
1419550 a at Lgx only 2491 2352 1855 1537 -1 06 -1 34 -1 62 Stk39
1419569 a at Res only 681 700 1171 1519 1 03 1 72 2 23 lsg20
1419584 at Lgx only 521 505 846 843 -1 03 1 62 1 62 Ttc28
1419609 at CR only 53 133 65 112 2 53 1 24 2 13 Ccr1
1419630 a at CR & Lgx 549 668 630 755 1 22 1 15 1 37 Trim 11
1419631 at Res & Lgx 1 19 146 218 228 1 23 1 84 1 92 Was
1419645 at Lgx only 463 390 335 288 -1 19 -1 38 -1 61 Cstf2
1419657 a at CR & Lgx 1163 874 858 623 -1 33 -1 36 -1 87 Slc25a36
1419660 at Lgx only 4264 3723 4383 3253 -1 15 1 03 -1 31 1600012F09Rιk
1419687 at Lgx only 8909 9784 12046 13075 1 10 1 35 1 47 Macrodi
1419736 a at Res & Lgx 3408 3084 2455 1934 -1 10 -1 39 -1 76 Eif lay
1419762 at Res & Lgx 38 84 155 154 2 18 4 03 4 01 Ubd
1419787 a at Lgx only 312 385 461 581 1 24 1 48 1 86 Zfp628
1419824 a at Res & Lgx 455 428 270 228 -1 06 -1 69 -2 00 A230062G08Rιk
1419952 at Lgx only 27 55 105 168 2 03 3 90 6 26 1700023D09Rιk
1420099 at CR only 203 41 1 349 286 2 02 1 72 1 40 D13Ertd787e
1420123 at Res & Lgx 3511 3401 5048 5686 -1 03 1 44 1 62 Tcta
1420183 at Lgx only 234 252 360 451 1 08 1 54 1 93 Lor
1420325 at Res only 57 130 144 132 2 26 2 52 2 30 Crampi l
1420329 at Lgx only 567 517 392 323 -1 10 -1 45 -1 76 4930455C21 Rιk
1420339 at Res only 309 242 217 225 -1 28 -1 43 -1 37 LOC100047915
1420374 at Res & Lgx 758 989 1238 1240 1 31 1 63 1 64 Foxj2
1420375 at Res & Lgx 401 402 615 593 1 00 1 53 1 48 Kιf3a
1420377 at Lgx only 89 105 134 173 1 18 1 50 1 94 St8sιa2
1420387 at Res & Lgx 2021 2029 1347 1414 1 00 -1 50 -1 43 Mpv17
1420388 at Lgx only 160 221 219 350 1 38 1 37 2 19 Prss12
1420405 at Lgx only 181 177 327 423 -1 02 1 81 2 34 Slco1 a4
1420427 a at CR only 1499 1159 1676 1564 -1 29 1 12 1 04 Dhx32
1420497 a at Res only 446 471 707 61 1 1 06 1 58 1 37 Cebpz
1420502 at Lgx only 1203 971 967 740 -1 24 -1 24 -1 63 Sat1
1420507 a at Res & Lgx 787 742 523 391 -1 06 -1 50 -2 01 31 10031 B13Rιk
1420513 at Res & Lgx 4347 3651 3096 2640 -1 19 -1 40 -1 65 Efcab2
1420580 at Res only 108 171 205 173 1 59 1 90 1 60 4930429B21 Rik
1420617 at CR & Lgx 1660 2030 1377 1316 1 22 -1 21 -1 26 Cpeb4 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1420619 a at Lgx only 9782 11497 12736 14220 1 18 1 30 1 45 Aes
1420654 a at Lgx only 836 1010 530 414 1 21 -1 58 -2 02 Gbe1
1420657 at CR & Lgx 978 1762 1973 2727 1 80 2 02 2 79 Ucp3
1420684 at Lgx only 184 272 311 338 1 48 1 70 1 84 Acox3
1420693 at Lgx only 12144 13502 14705 16745 1 11 1 21 1 38 Myomi
1420703 at Lgx only 230 242 309 412 1 05 1 34 1 79 Csf2ra
1420707 a at Res only 15 45 86 56 303 5 79 3 77 Traip
1420711 a at Res & Lgx 1740 1664 1104 1160 -1 05 -1 58 -1 50 Pxmp3
1420715 a at Res only 264 353 394 398 1 34 1 49 1 51 Pparg
1420727 a at Lgx only 834 685 683 642 -1 22 -1 22 -1 30 Tmlhe
1420770 at Lgx only 119 204 196 229 1 71 1 64 1 92 Klk1b24
1420812 at Res only 636 684 880 887 1 08 1 38 1 39 Hdac7a
1420815 at Res & Lgx 6531 5930 4529 3754 -1 10 -1 44 -1 74 Gdι2
1420829 a at Res & Lgx 1575 1422 1004 915 -1 11 -1 57 -1 72 Ywhaq
1420850 at Lgx only 811 697 641 504 -1 16 -1 27 -1 61 CrnkM
1420851 at Lgx only 742 595 613 560 -1 25 -1 21 -1 32 Pard6g
1420858 at Lgx only 4637 3856 3968 3017 -1 20 -1 17 -1 54 Pkia
1420886 a at Res & Lgx 3739 3833 4963 5400 1 03 1 33 1 44 Xbp1
1420890 at Res & Lgx 5214 4425 2406 1933 -1 18 -2 17 -2 70 Hccs
1420895 at Lgx only 917 770 710 650 -1 19 -1 29 -1 41 TgfbM
1420909 at Lgx only 2678 2641 1885 1364 -1 01 -1 42 -1 96 Vegfa
1420911 a at Lgx only 3466 3820 3678 4193 1 10 1 06 1 21 Mfgeδ
1420925 at Lgx only 72 124 151 175 1 71 2 08 242 Tub
1420960 at Res only 96 110 192 231 1 15 1 99 2 40 Fancg
1420965 a at Lgx only 874 1154 1487 1587 1 32 1 70 1 82 End
1420969 at Lgx only 466 483 535 830 1 04 1 15 1 78 Btbd14b
1420981 a at Lgx only 827 972 964 1187 1 18 1 17 1 44 Lmo4
1420990 at Res only 312 320 517 236 1 02 1 65 -1 32 Chd1
1420991 at Lgx only 53384 52666 61602 72604 -1 01 1 15 1 36 Ankrdi
1421019 at Lgx only 4065 3950 3600 3047 -1 03 -1 13 -1 33 1700021 F05Rιk
1421025 at Lgx only 849 1055 1040 1273 1 24 1 22 1 50 Agpatl
1421027 a at Res & Lgx 1926 1835 1240 1194 -1 05 -1 55 -1 61 Mef2c
1421042 at Res only 445 458 615 583 1 03 1 38 1 31 Arhgef2
1421054 at Lgx only 720 688 568 429 -1 05 -1 27 -1 68 Xpo4
1421087 at CR & Lgx 659 948 669 1049 1 44 1 02 1 59 Per3
1421096 at Res & Lgx 343 231 143 153 -1 48 -2 39 -2 24 Trpd
1421099 at Lgx only 201 206 279 348 1 03 1 39 1 74 Bhlhb3
1421140 a at Lgx only 966 1184 1048 1522 1 23 1 09 1 57 Foxpi
1421164 a at Lgx only 790 1029 1101 1483 1 30 1 39 1 88 Arhgefi
1421174 at CR only 170 74 105 131 -229 -1 62 -1 30 Irf4
1421254 a at Res & Lgx 8190 8322 11932 11052 1 02 1 46 1 35 Sgcg
1421265 a at Lgx only 2787 3702 3156 4067 1 33 1 13 1 46 Rbm38
1421287 a at Res & Lgx 1918 2165 2346 2394 1 13 1 22 1 25 Pecami
1421292 a at Lgx only 1159 1309 1372 1641 1 13 1 18 1 42 A730008L03Rιk
1421301 at Res & Lgx 25 50 65 79 2 01 2 64 320 Zιc2
1421361 at Res only 7 12 14 17 1 71 1 97 2 36 Grk1 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1421373 at Lgx only 150 180 225 291 1 20 1 50 1 94 Cox4ι2
1421374 a at Res & Lgx 22933 26328 30566 3771 1 1 15 1 33 1 64 Fxydi
1421425 a at Lgx only 16449 16822 17946 19197 1 02 1 09 1 17 Rcan2
1421444 at Res only 74 60 18 28 -1 23 -4 13 -2 61 Pg r
1421468 at Lgx only 901 957 947 1325 1 06 1 05 1 47 Kcnj3
1421530 a at Lgx only 27 71 53 97 2 66 1 98 3 62 Grm8
1421534 at Lgx only 811 795 808 606 -1 02 -1 00 -1 34 Dfna5h
1421541 a at Lgx only 21 44 48 100 2 09 2 27 4 69 Mef2b
1421654 a at Lgx only 1679 1993 2038 2187 1 19 1 21 1 30 Lmna
1421657 a at Lgx only 586 697 816 882 1 19 1 39 1 51 Sox 17
1421712 at Res only 44 104 121 85 2 35 2 74 1 93 SeIe
1421729 a at Res & Lgx 681 584 500 370 -1 17 -1 36 -1 84 Fert2
1421733 a at Res & Lgx 1129 1 104 1432 1650 -1 02 1 27 1 46 Tpsti
1421743 a at Res & Lgx 9703 10330 1 1559 11327 1 06 1 19 1 17 Pcbp2
1421750 a at Lgx only 2246 2231 1829 1446 -1 01 -1 23 -1 55 Vbp1
1421797 a at Lgx only 557 651 646 772 1 17 1 16 1 39 Snx12
1421808 at Res only 72 37 25 26 -1 92 -2 88 -2 71 Defb5
1421810 at Lgx only 868 921 932 1 143 1 06 1 07 1 32 Dgcr2
1421813 a at Lgx only 18627 21854 22085 24924 1 17 1 19 1 34 Psap
1421820 a at Lgx only 2152 2587 2644 3110 1 20 1 23 1 45 Nf2
1421826 at Lgx only 702 623 580 445 -1 13 -1 21 -1 58 DII4
1421861 at Lgx only 1285 1602 1652 1882 1 25 1 28 1 46 Clstni
1421871 at Lgx only 1104 837 695 549 -1 32 -1 59 -2 01 Sh3bgrl
1421872 at Lgx only 911 910 719 556 -1 00 -1 27 -1 64 Rab24
1421880 at Res only 970 1061 1162 1 121 1 09 1 20 1 16 Mtmri
1421887 a at Lgx only 10860 11944 14384 16154 1 10 1 32 1 49 ApI p2
1421894 a at Lgx only 1285 1 11 1 1077 876 -1 16 -1 19 -1 47 Tpp2
1421900 at Lgx only 704 851 937 1086 1 21 1 33 1 54 Eιf2ak1
1421910 at CR & Lgx 616 466 496 388 -1 32 -1 24 -1 58 Tcf20
1421929 at Res & Lgx 584 771 1070 101 1 1 32 1 83 1 73 Epha4
1421960 at Lgx only 251 313 373 451 1 25 1 48 1 79 Adcy3
1421985 a at Lgx only 1206 1 190 1259 970 -1 01 1 04 -1 24 Eιf4e2
1422063 a at Res & Lgx 303 432 514 657 1 43 1 70 2 17 Pex5
1422085 at Res only 89 141 178 209 1 58 2 00 2 34 Tbx 19
1422122 at Lgx only 45 52 102 145 1 15 2 26 3 23 Fcer2a
1422157 a at Res & Lgx 1891 1621 1425 1408 -1 17 -1 33 -1 34 Itgb1 bp1
1422160 at Res & Lgx 194 137 98 86 -1 42 -1 99 -2 24 H2-T24
1422183 a at Res & Lgx 456 433 718 774 -1 05 1 57 1 70 Adral b
1422185 a at Lgx only 2280 2364 3023 3264 1 04 1 33 1 43 Cyb5r3
1422202 at All 187 261 266 299 1 39 1 42 1 60 Thrb
1422250 at Res only 74 95 175 109 1 29 2 36 1 47 Map3k2
1422253 at Res only 72 82 22 25 1 14 -3 30 -2 84 CoMOaI
1422273 at CR only 17 64 65 79 3 66 3 75 4 54 Mmpi b
1422303 a at Lgx only 1 15 142 161 192 1 24 1 40 1 67 Tnfrsf18
1422325 at CR only 99 54 92 79 -1 85 -1 08 -1 25 Magea5
1422349 at Res only 108 91 204 164 -1 19 1 88 1 51 Ccr1 l1
Figure imgf000065_0001
Figure imgf000066_0001
Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1423365 at Lgx only 220 341 321 487 1 55 1 46 2 22 Cacnaig
1423368 at Res only 26862 24889 23668 24004 -1 08 -1 13 -1 12 Laptm4a
1423369 at Lgx only 512 450 400 277 -1 14 -1 28 -1 84 Fmr1
1423373 at Lgx only 1662 1543 1231 1041 -1 08 -1 35 -1 60 Rpp30
1423383 a at Lgx only 3010 3451 3134 3991 1 15 1 04 1 33 Osbpl9
1423393 at Res & Lgx 9550 11356 12552 12982 1 19 1 31 1 36 Chc4
1423407 a at Res & Lgx 1285 1527 1976 2493 1 19 1 54 1 94 Fbln2
1423423 at Res & Lgx 3996 3941 3047 3212 -1 01 -1 31 -1 24 Pdιa3
1423425 at Lgx only 639 706 761 947 1 10 1 19 1 48 1300012G16Rιk
1423431 a at Res & Lgx 1593 1952 2320 2336 1 23 1 46 1 47 Mybbpi a
1423440 at Lgx only 631 546 529 467 -1 15 -1 19 -1 35 11 10001A07Rιk
1423441 at Lgx only 4060 3903 3850 3452 -1 04 -1 05 -1 18 Tfb2m
1423448 at Res & Lgx 4469 4669 6840 6850 1 04 1 53 1 53 Rab11 b
1423449 a at Lgx only 1823 2337 2121 2533 1 28 1 16 1 39 Actn4
1423459 at Lgx only 3896 3424 3126 2505 -1 14 -1 25 -1 56 Cops2
1423474 at Res & Lgx 2213 2036 1686 1390 -1 09 -1 31 -1 59 Topi
1423485 at Lgx only 523 580 631 930 1 11 1 21 1 78 Rad54l2
1423486 at Res & Lgx 41 16 3309 2762 2491 -1 24 -1 49 -1 65 Cript
1423490 at Lgx only 3791 3965 3044 2767 1 05 -1 25 -1 37 Fbxo3
1423494 at CR only 38 105 93 45 2 79 2 46 1 20 2310042E22Rιk
1423512 at Lgx only 664 554 571 494 -1 20 -1 16 -1 35 AW209491
1423529 at Lgx only 76 62 55 20 -1 22 -1 38 -3 78 G6pc2
1423535 at Lgx only 4603 3572 3482 2393 -1 29 -1 32 -1 92 LOC100047794
1423557 at Res & Lgx 4123 4659 2949 2995 1 13 -1 40 -1 38 Ifngr2
1423565 at Lgx only 6741 7261 5935 5619 1 08 -1 14 -1 20 Paics
1423577 at CR only 866 691 713 759 -1 25 -1 22 -1 14 Ankrd32
1423588 at Res & Lgx 1204 1 118 984 929 -1 08 -1 22 -1 30 Arpc4
1423599 a at Lgx only 741 579 555 467 -1 28 -1 33 -1 59 Pdcl
1423609 a at Lgx only 2102 2341 2391 3062 1 11 1 14 1 46 Mgati
1423611 at CR only 736 949 973 1010 1 29 1 32 1 37 Akp2
1423620 at Lgx only 428 413 295 207 -1 04 -1 45 -2 07 Cenpq
1423629 at Lgx only 842 1015 1126 1457 1 21 1 34 1 73 Dnm2
1423642 at CR & Res 1 1579 15048 8041 9198 1 30 -1 44 -1 26 Tubb2c
1423643 at Lgx only 1071 1 196 843 821 1 12 -1 27 -1 30 Ddx39
1423647 a at Res only 344 396 605 457 1 15 1 76 1 33 Zdhhc3
1423648 at CR only 1771 2210 1681 1872 1 25 -1 05 1 06 Pdιa6
1423657 at Res & Lgx 1515 1485 1236 1 166 -1 02 -1 23 -1 30 Cdipt
1423662 at Res & Lgx 1443 1423 991 902 -1 01 -1 46 -1 60 Atp6ap2
1423663 at CR only 1057 1384 1080 1 178 1 31 1 02 1 12 Flcn
1423667 at All 8373 71 12 6577 5613 -1 18 -1 27 -1 49 Mat2a
1423669 at Lgx only 1075 1350 1555 2209 1 26 1 45 2 05 Col1a1
1423670 a at CR & Lgx 3169 3700 3314 3615 1 17 1 05 1 14 Srpr
1423676 at Lgx only 58661 62536 61203 80373 1 07 1 04 1 37 Atp5h
1423685 at Lgx only 1108 1452 1375 1574 1 31 1 24 1 42 Aars
1423694 at CR only 1790 2077 2089 1818 1 16 1 17 1 02 Kctdi O
1423697 at Lgx only 6981 6288 6277 5248 -1 11 -1 11 -1 33 Psmd6 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1423710 at Lgx only 9222 11 179 10001 11429 1 21 1 08 1 24 Dlst
1423711 at Res & Lgx 2316 2307 1924 1627 -1 00 -1 20 -1 42 Ndufafi
1423725 at Lgx only 985 705 674 762 -1 40 -1 46 -1 29 Pls3
1423734 at Lgx only 5006 5612 5707 6496 1 12 1 14 1 30 Rac1
1423737 at Lgx only 24235 26450 27218 29730 1 09 1 12 1 23 Ndufs3
1423753 at CR only 11 18 734 898 856 -1 52 -1 24 -1 31 Bambi
1423759 a at Res & Lgx 4031 3513 2876 2736 -1 15 -1 40 -1 47 Tmcoi
1423766 at Res & Lgx 1610 1380 1025 987 -1 17 -1 57 -1 63 Pak1 ιp1
1423767 at Lgx only 2146 2025 1774 1497 -1 06 -1 21 -1 43 2810410M20Rιk
1423771 at Lgx only 815 750 811 1094 -1 09 -1 00 1 34 Prkcdbp
1423773 at Lgx only 4627 3985 3629 2816 -1 16 -1 28 -1 64 Gpbpi
1423780 at Lgx only 10898 11906 12709 12518 1 09 1 17 1 15 Hibadh
1423785 at Lgx only 1 1606 13426 1 1821 15052 1 16 1 02 1 30 EgInI
1423790 at Lgx only 1487 1751 2032 2310 1 18 1 37 1 55 Dap
1423793 at Res & Lgx 936 1073 1319 1580 1 15 1 41 1 69 D2Ertd391 e
1423810 at CR only 1783 2180 1746 1816 1 22 -1 02 1 02 Ppmei
1423822 a at Lgx only 730 653 569 558 -1 12 -1 28 -1 31 Tmem168
1423845 at Lgx only 460 477 805 739 1 04 1 75 1 61 Csdc2
1423847 at Res & Lgx 231 270 401 391 1 17 1 73 1 69 Ncapd2
1423849 a at Lgx only 616 713 916 1030 1 16 1 49 1 67 Clk3
1423852 at Lgx only 50 52 68 154 1 03 1 36 3 07 Tmem46
1423857 at Res & Lgx 13121 11472 8712 6533 -1 14 -1 51 -2 01 Mrpl30
1423881 at Lgx only 1929 2007 2122 2477 1 04 1 10 1 28 Saps3
1423882 at Lgx only 933 840 739 618 -1 11 -1 26 -1 51 Rfwd 3
1423883 at Lgx only 21573 22608 26023 29695 1 05 1 21 1 38 AcsM
1423892 at CR only 3733 2926 4320 4066 -1 28 1 16 1 09 Apbbi
1423896 a at Lgx only 7953 9503 10177 13002 1 19 1 28 1 63 Rnf187
1423907 a at Lgx only 21309 20866 21974 17327 -1 02 1 03 -1 23 Ndufsδ
1423909 at Lgx only 670 657 729 1002 -1 02 1 09 1 50 Tmem176a
1423919 at Lgx only 445 348 353 351 -1 28 -1 26 -1 27 BC023882
1423927 at Res & Lgx 510 626 737 770 1 23 1 45 1 51 Slc35b2
1423939 a at Lgx only 6662 5968 5430 4027 -1 12 -1 23 -1 65 Yιf1 a
1423947 at Res & Lgx 1479 1457 2188 2164 -1 01 1 48 1 46 1 110008P14Rιk
1423951 at Lgx only 936 768 718 654 -1 22 -1 30 -1 43 Tm2d3
1423958 a at Lgx only 1601 1398 1213 998 -1 15 -1 32 -1 60 Ttc33
1423960 at Res & Lgx 1368 1371 1946 2010 1 00 1 42 1 47 Mboat5
1423961 at Res & Lgx 1414 1 142 839 602 -1 24 -1 69 -2 35 LOC100045629
1423967 at Lgx only 903 1044 1073 1359 1 16 1 19 1 51 Palm
1423969 at All 723 538 512 442 -1 34 -1 41 -1 64 Nup37
1423972 at Lgx only 31981 34252 35126 43175 1 07 1 10 1 35 Etfa
1423973 a at Lgx only 644 716 819 1000 1 11 1 27 1 55 Arf3
1423978 at Lgx only 920 1504 1209 1467 1 64 1 31 1 59 SbM
1423991 at Lgx only 392 425 448 564 1 08 1 14 1 44 Nol14
1423993 at Lgx only 4392 3844 3630 3056 -1 14 -1 21 -1 44 Atp6v1f
1424000 a at Lgx only 20066 22495 23422 27778 1 12 1 17 1 38 Rps11
1424002 at Res & Lgx 2495 2175 1827 1785 -1 15 -1 37 -1 40 Pdcl3 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1424005 at Lgx only 3828 3557 3275 3040 -1 08 -1 17 -1 26 B230219D22Rιk
1424010 at Lgx only 168 233 382 732 1 38 2 27 4 36 Mfap4
1424025 at Res & Lgx 5858 5688 4312 4383 -1 03 -1 36 -1 34 BC013529
1424027 at Res only 619 902 924 853 1 46 1 49 1 38 Pxn
1424028 at Lgx only 874 861 614 481 -1 02 -1 42 -1 82 583045701ORik
1424033 at Res & Lgx 935 735 479 332 -1 27 -1 95 -2 82 Sfrs7
1424036 at Lgx only 1422 1354 1874 1752 -1 05 1 32 1 23 Prpf6
1424038 a at Lgx only 2801 3278 3181 3602 1 17 1 14 1 29 2310044H10Rιk
1424040 at Lgx only 5989 7521 7112 8135 1 26 1 19 1 36 Mtap7d1
1424053 a at Res & Lgx 5291 5313 6583 6840 1 00 1 24 1 29 Tcf25
1424054 at Res & Lgx 1063 1467 1588 1882 1 38 1 49 1 77 Btbd2
1424058 at Lgx only 640 771 767 944 1 21 1 20 1 48 Prrd
1424066 at Lgx only 990 1038 1084 1324 1 05 1 09 1 34 Dus3l
1424077 at Res only 654 544 493 479 -1 20 -1 33 -1 37 Gdpdi
1424081 at Res & Lgx 380 271 291 277 -1 40 -1 31 -1 37 Pcgf6
1424099 at Lgx only 2023 1663 1332 1265 -1 22 -1 52 -1 60 2310016C16Rιk
1424101 at Res & Lgx 874 934 533 583 1 07 -1 64 -1 50 Hnrpl
1424105 a at Lgx only 3516 2620 3158 2273 -1 34 -1 11 -1 55 Pttgi
1424109 a at Res & Lgx 6904 7061 6174 6463 1 02 -1 12 -1 07 Glo1
1424115 at CR & Lgx 1970 2313 2291 2496 1 17 1 16 1 27 Ppp5c
1424121 at Lgx only 986 890 677 523 -1 11 -1 46 -1 88 Commdi
1424126 at CR & Res 6092 9782 4437 5967 1 61 -1 37 -1 02 Alasi
1424134 at Lgx only 646 529 499 436 -1 22 -1 29 -1 48 Rspryl
1424138 at Lgx only 1703 1927 1979 2218 1 13 1 16 1 30 Rhbdfi
1424139 at Lgx only 5935 4960 4416 3478 -1 20 -1 34 -1 71 Rap1a
1424140 at Res only 146 192 243 196 1 32 1 67 1 35 Gale
1424141 at Lgx only 2745 3324 3767 4195 1 21 1 37 1 53 Hectdi
1424147 at Res & Lgx 3868 4234 2946 2570 1 09 -1 31 -1 50 Ahsai
1424149 at CR only 435 531 523 573 1 22 1 20 1 32 Nsmce2
1424150 at Lgx only 438 540 561 688 1 23 1 28 1 57 Gdpd5
1424151 at Res & Lgx 2327 2380 1795 1679 1 02 -1 30 -1 39 Jtv1
1424154 a at Lgx only 4155 3735 3761 3368 -1 11 -1 10 -1 23 Isca2
1424159 at Res & Lgx 618 578 459 454 -1 07 -1 34 -1 36 1300010M03Rιk
1424160 at Lgx only 563 486 499 420 -1 16 -1 13 -1 34 Alg5
1424162 at CR only 115 37 144 125 -3 15 1 25 1 08 Trιm29
1424163 at Lgx only 615 642 714 803 1 04 1 16 1 31 Rmnd5b
1424167 a at Lgx only 367 456 568 734 1 24 1 55 2 00 Pmm1
1424175 at CR & Lgx 4138 5751 4597 7356 1 39 1 11 1 78 Tef
1424178 at Lgx only 8368 8313 7872 6995 -1 01 -1 06 -1 20 Tmem38a
1424179 at Lgx only 642 594 541 431 -1 08 -1 19 -1 49 Plekhji
1424184 at Lgx only 33037 35163 37503 45711 1 06 1 14 1 38 Acadvl
1424191 a at Lgx only 613 688 706 837 1 12 1 15 1 36 Tmem41a
1424209 at Lgx only 1349 1298 1166 988 -1 04 -1 16 -1 37 Rars2
1424210 at Res & Lgx 892 764 657 670 -1 17 -1 36 -1 33 Erlini
1424211 at CR & Lgx 1496 2374 1415 1975 1 59 -1 06 1 32 Slc25a33
1424216 a at Lgx only 1633 1442 1293 1181 -1 13 -1 26 -1 38 Papola Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1424223 at Lgx only 7099 8501 6040 6025 1 20 -1 18 -1 18 1700020C11Rιk
1424236 at Lgx only 741 789 838 950 1 06 1 13 1 28 TbddlOb
1424237 at Res only 1047 1010 1262 1184 -1 04 1 21 1 13 Zfp639
1424247 at Lgx only 543 591 600 802 1 09 1 10 1 48 Erc1
1424249 a at All 434 271 226 252 -1 60 -1 92 -1 72 Arhgapθ
1424255 at Res & Lgx 2458 2688 3230 3339 1 09 1 31 1 36 Supt5h
1424258 at Lgx only 955 696 690 604 -1 37 -1 38 -1 58 Polr2d
1424261 at Res & Lgx 1144 871 714 580 -1 31 -1 60 -1 97 Zfp672
1424274 at Res & Lgx 1795 1574 1426 1084 -1 14 -1 26 -1 66 Vdp
1424276 at Lgx only 410 338 308 258 -1 22 -1 33 -1 59 Snx16
1424280 at Res & Lgx 2125 1767 1658 1182 -1 20 -1 28 -1 80 Mospdi
1424303 at Lgx only 90 76 59 26 -1 19 -1 53 -3 47 Depdc7
1424309 a at Res & Lgx 2945 2961 2220 1975 1 01 -1 33 -1 49 Mocs2
1424318 at Lgx only 2148 2040 1851 1802 -1 05 -1 16 -1 19 1110067D22Rιk
1424321 at Lgx only 351 263 228 169 -1 34 -1 54 -207 Rfc4
1424324 at Lgx only 956 712 539 403 -1 34 -1 77 -2 37 Escol
1424346 at Lgx only 2287 1920 1820 1658 -1 19 -1 26 -1 38 Ppp6c
1424349 a at Res & Lgx 2326 1877 1516 1263 -1 24 -1 53 -1 84 Lpgatl
1424356 a at Lgx only 770 697 694 603 -1 10 -1 11 -1 28 Metrnl
1424359 at Lgx only 1036 1229 2242 2519 1 19 2 16 2 43 Oplah
1424361 at Lgx only 294 408 530 653 1 39 1 80 2 22 BC019943
1424372 at Res & Lgx 3282 2988 2215 2091 -1 10 -1 48 -1 57 Mrpl32
1424374 at Lgx only 1936 1845 1535 1363 -1 05 -1 26 -1 42 Gιmap4
1424377 at Lgx only 1007 971 895 801 -1 04 -1 12 -1 26 BC003885
1424380 at Lgx only 434 432 512 559 -1 01 1 18 1 29 Vps37b
1424384 a at Lgx only 726 981 1007 1092 1 35 1 39 1 50 Znrf1
1424390 at Lgx only 597 493 481 472 -1 21 -1 24 -1 26 NupM
1424391 at CR & Lgx 3544 3195 3213 2673 -1 11 -1 10 -1 33 Nrd1
1424403 a at Lgx only 254 221 348 376 -1 15 1 37 1 48 Rufy3
1424406 at Lgx only 1180 1319 1489 1520 1 12 1 26 1 29 BCI2I13
1424408 at Lgx only 3655 3925 4168 4751 1 07 1 14 1 30 Lιms2
1424416 at Lgx only 2199 2535 2429 2712 1 15 1 10 1 23 Nkιras2
1424424 at Lgx only 1819 1642 1309 892 -1 11 -1 39 -2 04 Slc39a1
1424430 at Res only 647 598 475 466 -1 08 -1 36 -1 39 Mterfd2
1424433 at Res & Lgx 3043 3150 2599 2241 1 04 -1 17 -1 36 Msrb2
1424434 at Res & Lgx 2817 2667 2219 2042 -1 06 -1 27 -1 38 BC024814
1424447 at Lgx only 404 418 462 566 1 03 1 14 1 40 1700030K09Rιk
1424461 at Res & Lgx 4865 4886 3994 3716 1 00 -1 22 -1 31 Dctn2
1424463 at Res & Lgx 1299 1043 945 877 -1 24 -1 37 -1 48 2210010L05Rιk
1424465 at Lgx only 957 881 710 583 -1 09 -1 35 -1 64 Ccdc58
1424467 at Lgx only 1725 2308 2228 2625 1 34 1 29 1 52 Phldbi
1424473 at Lgx only 911 823 725 502 -1 11 -1 26 -1 81 Polr2h
1424479 at Lgx only 39 46 64 119 1 18 1 64 3 02 Cst8
1424500 at Lgx only 1020 947 744 637 -1 08 -1 37 -1 60 Utp6
1424505 at Lgx only 1309 1213 1172 895 -1 08 -1 12 -1 46 Rmndi
1424510 at Lgx only 783 733 627 574 -1 07 -1 25 -1 36 Nudt6
Figure imgf000071_0001
Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1424873 at Res & Lgx 906 801 637 641 -1 13 -1 42 -1 41 Rnf2
1424878 at CR & Lgx 255 508 463 632 1 99 1 82 2 48 Lrch4
1424898 at CR only 144 51 82 114 -2 84 -1 76 -1 27 SId OaI
1424912 at Lgx only 417 508 500 569 1 22 1 20 1 36 Slc25a17
1424918 at Res & Lgx 893 857 631 559 -1 04 -1 42 -1 60 Tbc1 d19
1424929 a at Lgx only 239 284 385 449 1 19 1 61 1 88 Trιm26
1424942 a at CR only 55 147 154 55 2 69 2 81 1 00 My c
1424954 a at Lgx only 657 753 904 1030 1 15 1 38 1 57 Pιp5k1c
1424956 at Lgx only 246 31 1 380 513 1 26 1 55 2 09 Ahdd
1424978 at Res only 23 79 87 88 344 3 76 3 82 Odf4
1424988 at Lgx only 1652 1684 1806 2065 1 02 1 09 1 25 Mylip
1424990 at Lgx only 890 960 1037 1205 1 08 1 17 1 35 Tmem142a
1424996 at Lgx only 650 688 746 875 1 06 1 15 1 35 Cflar
1425024 at CR only 104 28 94 50 -3 79 -1 11 -2 09 E430018J23Rιk
1425057 at Res only 88 164 237 176 1 85 2 68 1 98 PbId
1425079 at CR only 40 118 72 60 2 96 1 79 1 51 Tm6sf2
14251 14 at Res & Lgx 795 765 561 544 -1 04 -1 42 -1 46 Rbbp6
1425143 a at Lgx only 25337 27260 28070 34606 1 08 1 1 1 1 37 Ndufsi
1425158 at Res & Lgx 1320 1302 1751 1907 -1 01 1 33 1 44 Tbx20
1425164 a at Lgx only 265 328 383 483 1 24 1 44 1 82 Phkgi
1425189 a at Res & Lgx 3583 3540 2889 2383 -1 01 -1 24 -1 50 Mrpl15
1425214 at Lgx only 324 237 207 133 -1 37 -1 57 -2 44 P2ry6
1425225 at Lgx only 94 142 190 249 1 51 2 02 2 64 Fcgr4
1425228 a at Lgx only 1536 1652 1255 1078 1 08 -1 22 -1 43 Dguok
1425243 at Lgx only 1 15 120 196 325 1 04 1 70 2 82 Cd207
1425257 at Lgx only 7 19 41 16 2 89 6 34 2 39 Acot5
1425270 at Res & Lgx 3017 2790 21 11 1975 -1 08 -1 43 -1 53 Kifl b
1425274 at Res & Lgx 4607 4976 3462 3212 1 08 -1 33 -1 43 Asph
1425314 at Res only 27 57 96 118 2 10 3 57 4 36 Gpr98
1425332 at Lgx only 1 1194 12729 12529 16323 1 14 1 12 1 46 Zfp106
1425333 at Lgx only 410 538 624 856 1 31 1 52 2 09 Rab43
1425340 a at CR & Lgx 1281 1481 1447 1573 1 16 1 13 1 23 Ptpra
1425341 at Lgx only 2691 3341 3786 4027 1 24 1 41 1 50 Kcnk3
1425350 a at Res & Lgx 106 147 186 212 1 38 1 75 2 00 Myef2
1425455 a at Lgx only 4248 4028 3479 3392 -1 05 -1 22 -1 25 Churd
1425480 at Res & Lgx 21 13 2091 1616 1298 -1 01 -1 31 -1 63 Cnot6l
1425492 at Res & Lgx 2530 2180 1751 1792 -1 16 -1 44 -1 41 Bmpri a
1425519 a at Res & Lgx 1769 1610 2963 2821 -1 10 1 67 1 59 Cd74
1425521 at Lgx only 337 279 205 188 -1 21 -1 64 -1 79 Paιp1
1425558 at Res only 136 59 28 101 -2 32 -4 94 -1 34 Klc3
1425589 at Lgx only 148 11 1 123 38 -1 34 -1 21 -3 88 Hsd17b13
1425617 at Lgx only 243 296 355 486 1 22 1 46 1 99 Dhx9
1425639 at Lgx only 209 21 1 342 342 1 01 1 63 1 64 Centa2
1425646 at Lgx only 141 96 96 68 -1 46 -1 47 -2 08 BC016495
1425674 a at Lgx only 2104 1965 1606 1495 -1 07 -1 31 -1 41 Ssu72
1425677 a at Lgx only 1 1130 10594 9470 8378 -1 05 -1 18 -1 33 Ank1 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1425682 a at Lgx only 518 419 373 288 -1 24 -1 39 -1 80 Tprkb
1425702 a at Lgx only 574 571 734 946 -1 01 1 28 1 65 Enpp5
1425706 a at Lgx only 371 337 391 239 -1 10 1 05 -1 55 Ddb2
1425718 a at Lgx only 33649 29835 25700 23101 -1 13 -1 31 -1 46 Ivnsiabp
1425742 a at Lgx only 11487 11684 12973 13668 1 02 1 13 1 19 Tsc22d1
1425753 a at Res only 701 793 434 484 1 13 -1 61 -1 45 Ung
1425760 a at Lgx only 378 450 540 627 1 19 1 43 1 66 Pitpnmi
1425764 a at Lgx only 2958 3344 3747 4049 1 13 1 27 1 37 Bcat2
1425780 a at Lgx only 1362 1035 1202 963 -1 32 -1 13 -1 41 Tmem167
1425792 a at Res & Lgx 599 793 1003 995 1 32 1 67 1 66 Rorc
1425795 a at Res & Lgx 2655 2581 1359 1207 -1 03 -1 95 -2 20 Map3k7
1425826 a at Lgx only 6893 8508 10213 13158 1 23 1 48 1 91 Sorbsi
1425894 at CR only 315 164 315 398 -1 92 1 00 1 26 Mrgprf
1425895 a at Lgx only 2855 3457 3880 4028 1 21 1 36 1 41 Id1
1425904 at Res only 34 52 104 62 1 53 3 06 1 82 Satb2
1425930 a at Lgx only 698 638 680 563 -1 09 -1 03 -1 24 Mix
1425933 a at Res & Lgx 1038 1091 859 854 1 05 -1 21 -1 21 Nt5c2
1425940 a at Lgx only 184 208 306 319 1 13 1 66 1 73 Ssbp3
1425978 at Res & Lgx 1850 1652 1140 1026 -1 12 -1 62 -1 80 Myocd
1425993 a at Res & Lgx 1379 1079 897 848 -1 28 -1 54 -1 63 Hsp110
1426000 at Lgx only 164 196 167 317 1 19 1 02 1 93 Oxtr
1426016 a at Lgx only 55 65 130 164 1 18 2 39 300 Tro
1426068 at Lgx only 935 868 767 692 -1 08 -1 22 -1 35 Slc7a4
1426089 a at Lgx only 1823 1590 1565 1314 -1 15 -1 17 -1 39 BC003331
1426100 a at Lgx only 218 225 310 338 1 03 1 42 1 55 Tk2
1426114 at Res & Lgx 5491 5041 29756 25956 -1 09 542 4 73 Hnrpab
1426118 a at Lgx only 1454 1702 1714 1820 1 17 1 18 1 25 Tomm40
1426179 a at Lgx only 910 1026 1204 1351 1 13 1 32 1 48 Twsgi
1426187 a at Lgx only 1603 1568 1266 1056 -1 02 -1 27 -1 52 Hax1
1426195 a at Lgx only 29737 32186 35405 39796 1 08 1 19 1 34 Cst3
1426235 a at Res & Lgx 2374 2866 5732 6147 1 21 241 2 59 GIuI
1426241 a at Lgx only 771 982 908 1062 1 27 1 18 1 38 Scmhi
1426249 at Res & Lgx 1302 1459 1804 2108 1 12 1 39 1 62 Adrbki
1426254 at Lgx only 1276 1145 917 731 -1 12 -1 39 -1 75 Tm2d1
1426257 a at Res & Lgx 1948 2192 2318 2595 1 13 1 19 1 33 Sars
1426263 at Lgx only 1203 1175 1108 911 -1 02 -1 09 -1 32 Cadm4
1426269 at Res & Lgx 684 560 450 371 -1 22 -1 52 -1 85 Sybil
1426277 at Lgx only 386 336 445 519 -1 15 1 15 1 34 C730025P13Rιk
1426279 at Lgx only 770 726 591 516 -1 06 -1 30 -1 49 5830415L20Rιk
1426285 at Lgx only 2453 2580 2755 3223 1 05 1 12 1 31 Lama2
1426286 at Lgx only 335 275 211 119 -1 22 -1 59 -281 Noc3l
1426297 at Lgx only 466 452 754 680 -1 03 1 62 1 46 Tcfe2a
1426307 at Res & Lgx 1936 1817 1474 1311 -1 07 -1 31 -1 48 Cyb5r4
1426337 a at CR & Lgx 32 84 49 110 264 1 55 3 45 Tead4
1426344 at Lgx only 475 506 511 653 1 07 1 08 1 38 GIeII
1426347 at CR only 783 635 743 686 -1 23 -1 05 -1 14 2010321M09Rιk Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1426353 at Lgx only 1157 1203 1210 1377 1 04 1 05 1 19state
1426380 at Lgx only 4215 4149 4647 5122 -1 02 1 10 1 22 Eιf4b
1426386 at Res & Lgx 1464 1483 1190 993 1 01 -1 23 -1 47 Rpl7l1
1426390 a at Lgx only 22906 23329 19114 18319 1 02 -1 20 -1 25 ArM
1426398 at Lgx only 989 893 750 661 -1 11 -1 32 -1 50 Ube2w
1426400 a at Res & Lgx 9253 10079 10609 11708 1 09 1 15 1 27 Capnsi
1426406 at Res & Lgx 2553 2947 3706 4584 1 15 1 45 1 80 Setdδ
1426416 a at Res & Lgx 1595 1370 1005 953 -1 16 -1 59 -1 67 Yιpf4
1426423 at Res & Lgx 820 1004 1130 1251 1 22 1 38 1 53 Shmt2
1426436 at Lgx only 760 764 675 568 1 00 -1 13 -1 34 Tmem159
1426440 at Lgx only 2254 2820 2177 2947 1 25 -1 04 1 31 Dhrs7
1426444 at Res only 314 254 467 460 -1 24 1 48 1 46 Rhbdd2
1426445 at Lgx only 1978 2219 2502 2536 1 12 1 26 1 28 Ctage5
1426446 at Lgx only 312 312 438 458 1 00 1 40 1 47 6430548M08Rιk
1426452 a at Lgx only 202 256 129 77 1 26 -1 57 -2 64 Rab30
1426455 at Lgx only 402 394 369 274 -1 02 -1 09 -1 47 SdccagiO
1426457 at Lgx only 9329 9212 7693 6876 -1 01 -1 21 -1 36 Slmap
1426468 at Res only 1223 1032 910 976 -1 18 -1 34 -1 25 0610037L13Rιk
1426477 at Lgx only 1012 893 905 676 -1 13 -1 12 -1 50 Rasai
1426480 at Lgx only 2278 2147 2786 3030 -1 06 1 22 1 33 Sbds
1426481 at Res & Lgx 1112 1028 1505 1624 -1 08 1 35 1 46 Klhl22
1426482 at Lgx only 3505 2977 2762 2410 -1 18 -1 27 -1 45 Prkrir
1426495 at Lgx only 348 251 218 208 -1 39 -1 59 -1 68 2410042D21Rιk
1426539 at Lgx only 295 365 390 465 1 24 1 32 1 58 Usp11
1426567 a at Res only 434 445 254 264 1 02 -1 71 -1 64 PqId
1426586 at Lgx only 17315 21329 19691 21472 1 23 1 14 1 24 Slc25a11
1426596 a at All 539 412 417 219 -1 31 -1 29 -2 46 Smn1
1426607 at Lgx only 304 267 198 175 -1 14 -1 54 -1 73 EG633640
1426613 a at Lgx only 1480 1201 1246 838 -1 23 -1 19 -1 77 Snrpb2
1426614 at Lgx only 741 922 870 1015 1 25 1 18 1 37 Prkcbpi
1426624 a at Res & Lgx 2908 3005 4008 4108 1 03 1 38 1 41 Ypel3
1426629 at Lgx only 450 528 534 613 1 17 1 19 1 36 Dhx8
1426643 at Lgx only 959 1059 1176 1265 1 10 1 23 1 32 Elp3
1426646 at Lgx only 3289 3191 2689 2507 -1 03 -1 22 -1 31 9130011J15Rιk
1426648 at Lgx only 3467 4510 4856 5396 1 30 1 40 1 56 Mapkapk2
1426670 at Res & Lgx 701 789 1130 1113 1 13 1 61 1 59 Agrn
1426671 a at Lgx only 1307 1327 1062 766 1 02 -1 23 -1 71 Rbm39
1426675 at Lgx only 1376 1180 884 626 -1 17 -1 56 -2 20 Tomm70a
1426681 at Lgx only 284 309 369 440 1 09 1 30 1 55 Unk
1426682 at Lgx only 1328 1222 1113 852 -1 09 -1 19 -1 56 LOC100046343
1426688 at Lgx only 30012 35064 33818 42859 1 17 1 13 1 43 Sdha
1426690 a at Res & Lgx 1211 1395 1784 2490 1 15 1 47 2 06 Srebfi
1426691 at Lgx only 818 960 944 1189 1 17 1 15 1 45 Tjapi
1426700 a at Lgx only 310 327 417 406 1 06 1 35 1 31 Usp52
1426717 at Res & Lgx 1834 1766 1219 1092 -1 04 -1 50 -1 68 Nιpa2
1426718 at Res & Lgx 972 829 713 726 -1 17 -1 36 -1 34 Skιv2l2 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1426731 at Lgx only 15714 18402 19327 23750 1 17 1 23 1 51 Des
1426741 a at Lgx only 1254 1098 1044 860 -1 14 -1 20 -1 46 Fastkd2
1426743 at Lgx only 3106 3174 2664 2485 1 02 -1 17 -1 25 Appl2
1426752 at Res only 1165 945 826 990 -1 23 -1 41 -1 18 Phf17
1426760 at Res & Lgx 1194 1065 823 781 -1 12 -1 45 -1 53 Ipo8
1426773 at Lgx only 6830 8123 9064 9354 1 19 1 33 1 37 Mf n1
1426774 at Res & Lgx 607 595 485 347 -1 02 -1 25 -1 75 Parp12
1426794 at Lgx only 934 1249 1207 1600 1 34 1 29 1 71 Ptprs
1426799 at Lgx only 826 683 610 489 -1 21 -1 35 -1 69 Rabδb
1426819 at Lgx only 9939 6798 6585 3448 -1 46 -1 51 -2 88 LOC100048439
1426820 at Lgx only 5084 5617 5406 6361 1 10 1 06 1 25 2610507B1 1 Rιk
1426830 a at Lgx only 3633 3395 3097 2585 -1 07 -1 17 -1 41 AhcyM
1426833 at Lgx only 846 870 1103 1089 1 03 1 30 1 29 Eιf4g3
1426854 a at Lgx only 4159 4032 3462 3261 -1 03 -1 20 -1 28 Set
1426857 a at Lgx only 5854 4850 5610 4740 -1 21 -1 04 -1 23 Hsdl2
1426866 at Lgx only 571 766 674 802 1 34 1 18 1 41 Chsti 4
1426886 at Res & Lgx 872 844 723 633 -1 03 -1 21 -1 38 Cln5
1426895 at Res & Lgx 897 707 604 450 -1 27 -1 48 -1 99 Zfp191
1426898 at Res only 391 401 533 528 1 03 1 36 1 35 Map3k7ιp1
1426900 at Lgx only 986 807 644 600 -1 22 -1 53 -1 64 Jmjdi c
1426948 at Res & Lgx 3583 4241 4782 5859 1 18 1 33 1 64 Tpr
1426952 at Lgx only 524 503 478 259 -1 04 -1 10 -2 02 Arhgap18
1426964 at Lgx only 1856 1813 1314 1265 -1 02 -1 41 -1 47 31 10003A17Rιk
1426965 at Lgx only 402 405 499 568 1 01 1 24 1 41 Rap2a
1426969 at Res & Lgx 551 378 376 287 -1 46 -1 46 -1 92 Trιm23
1426976 at Lgx only 3711 3721 4100 4747 1 00 1 10 1 28 Usp47
1426979 at Lgx only 993 1 192 1354 1332 1 20 1 36 1 34 Mlxip
1426982 at Res & Lgx 535 607 880 853 1 13 1 65 1 59 Flywchi
1426984 at Res & Lgx 291 540 554 854 1 86 1 91 2 94 2310067B10Rιk
1426992 at Res & Lgx 1745 1757 1076 1052 1 01 -1 62 -1 66 XpM
1427028 at Lgx only 556 635 615 891 1 14 1 11 1 60 Lg r6
1427039 at Lgx only 1834 2329 2407 2983 1 27 1 31 1 63 Epn1
1427040 at Lgx only 1252 987 952 855 -1 27 -1 31 -1 46 Mdfic
1427045 at Lgx only 778 817 1248 1424 1 05 1 60 1 83 Synpo
1427051 at Res & Lgx 454 548 673 770 1 21 1 48 1 70 Tnks1 bp1
1427058 at Res only 8484 8530 6619 7155 1 01 -1 28 -1 19 Eιf4a1
1427073 at Res only 1956 2206 1536 1648 1 13 -1 27 -1 19 Lacei
1427084 a at Lgx only 686 606 508 463 -1 13 -1 35 -1 48 Map4k5
1427099 at Res only 320 486 646 461 1 52 2 02 1 44 Maz
14271 17 at Lgx only 619 616 790 979 -1 01 1 28 1 58 Mtmr3
1427120 at Lgx only 835 688 573 468 -1 21 -1 46 -1 78 Zfp26
1427129 a at Lgx only 1545 1252 1132 930 -1 23 -1 36 -1 66 Hnrpr
1427132 at Lgx only 348 463 524 564 1 33 1 51 1 62 Sbf2
1427139 at Lgx only 795 940 925 1054 1 18 1 16 1 33 Adamtsi O
1427144 at Res & Lgx 1131 1047 680 637 -1 08 -1 66 -1 78 Hnrpll
1427146 at Res & Lgx 673 839 1204 1613 1 25 1 79 2 40 AI790298 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1427165 at Res & Lgx 1308 1233 814 792 -1 06 -1 61 -1 65 Il13ra1
1427166 a at Lgx only 1401 1747 2035 2399 1 25 1 45 1 71 Spg7
1427170 at All 74 25 24 13 -2 94 -3 10 -5 66 Psmaδ
1427197 at Lgx only 496 530 403 303 1 07 -1 23 -1 64 Atr
1427201 at Lgx only 551 457 565 769 -1 20 1 03 1 40 Mustnl
1427228 at Lgx only 2099 2379 3387 3964 1 13 1 61 1 89 PaIId
1427239 at CR & Lgx 552 808 637 680 1 46 1 15 1 23 Ift122
1427240 at Lgx only 815 1013 1033 1520 1 24 1 27 1 86 Dock6
1427241 at Lgx only 610 547 387 384 -1 12 -1 58 -1 59 Papolg
1427260 a at Res & Lgx 2978 2624 2355 1823 -1 13 -1 26 -1 63 Tpm3
1427266 at All 1 181 848 784 732 -1 39 -1 51 -1 61 Pbrmi
1427296 at Res & Lgx 4758 5054 3684 3677 1 06 -1 29 -1 29 BC010304
1427312 at Lgx only 12654 14384 15939 18697 1 14 1 26 1 48 Cmya5
1427314 at Res & Lgx 2995 2439 2188 1689 -1 23 -1 37 -1 77 Tmed7
1427319 at CR & Lgx 694 468 551 427 -1 48 -1 26 -1 63 A230046K03Rιk
1427342 at Res & Lgx 802 683 597 499 -1 18 -1 35 -1 61 Fastkdi
1427395 a at Lgx only 74 37 30 33 -2 00 -2 51 -2 27 Aldh1a3
1427418 a at Lgx only 1864 1555 1250 796 -1 20 -1 49 -2 34 H if 1 a
1427432 a at Lgx only 2372 2225 1569 1292 -1 07 -1 51 -1 84 SfrsiO
1427447 a at Res & Lgx 1080 1415 1561 1854 1 31 1 44 1 72 Triobp
1427490 at Lgx only 356 348 274 236 -1 02 -1 30 -1 51 Abcb7
1427529 at Res only 151 90 67 82 -1 68 -2 25 -1 84 Fzd9
1427555 at Lgx only 35 64 76 96 1 85 2 18 2 76 MII2
1427557 at Lgx only 180 201 256 281 1 11 1 42 1 56 AIg 12
1427604 a at Lgx only 871 1023 1176 1356 1 17 1 35 1 56 Atp9a
1427661 a at Res & Lgx 382 474 594 597 1 24 1 56 1 56 Tssc4
1427689 a at Lgx only 1261 1266 1376 1652 1 00 1 09 1 31 Tnιp1
1427720 a at Lgx only 2747 2851 3040 3415 1 04 1 1 1 1 24 Rrp1
1427728 at CR only 34 97 56 57 2 88 1 65 1 68 Chrng
1427735 a at CR & Res 8359 12700 12493 12108 1 52 1 49 1 45 Actai
1427873 at Lgx only 17 54 56 110 3 19 3 31 6 50 Defcr15
1427874 at Lgx only 1399 1574 1815 1899 1 13 1 30 1 36 Zfp313
1427876 at Lgx only 1630 1676 1431 1226 1 03 -1 14 -1 33 Zc3h15
1427886 at Lgx only 969 755 581 374 -1 28 -1 67 -2 59 Pom121
1427888 a at CR & Lgx 5357 6838 6596 7987 1 28 1 23 1 49 Spna2
1427894 at Lgx only 472 396 538 682 -1 19 1 14 1 44 Vasn
1427898 at Lgx only 822 714 616 515 -1 15 -1 33 -1 60 Rnf6
1427901 at Lgx only 3381 3126 2782 2128 -1 08 -1 22 -1 59 Mrps18c
1427903 at Res & Lgx 5849 5774 4732 4224 -1 01 -1 24 -1 38 Phpti
1427913 at Lgx only 2402 2230 1819 1452 -1 08 -1 32 -1 65 Rwddi
1427918 a at Lgx only 7544 7921 8074 9445 1 05 1 07 1 25 Rhoq
1427929 a at Lgx only 610 746 507 763 1 22 -1 20 1 25 Pdxk
1427943 at Lgx only 6234 5441 5157 3929 -1 15 -1 21 -1 59 Acyp2
1427947 at CR only 493 623 501 547 1 26 1 02 1 1 1 BC028440
1427955 a at Lgx only 6984 7396 5948 4819 1 06 -1 17 -1 45 Deb1
1427971 at Lgx only 957 802 781 608 -1 19 -1 22 -1 57 Cdc73
Figure imgf000077_0001
Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1428382 at Lgx only 964 1 129 1473 1531 1 17 1 53 1 59 Smarcc2
1428391 at Lgx only 267 295 348 432 1 10 1 30 1 62 Rab3ιl1
1428395 at Lgx only 487 508 647 727 1 04 1 33 1 49 Smurfi
1428405 at Lgx only 9641 10670 1 1324 12596 1 1 1 1 17 1 31 Hcfc1 r1
1428412 at Lgx only 2036 2091 2325 2629 1 03 1 14 1 29 Tm9sf3
1428421 a at Lgx only 2913 2410 2394 2019 -1 21 -1 22 -1 44 Glod4
1428423 at Lgx only 1714 1643 1657 1494 -1 04 -1 03 -1 15 Pcgf3
1428427 at CR only 92 36 39 101 -2 53 -2 37 1 10 Fbxl2
1428431 at Res & Lgx 1390 1453 1755 1963 1 04 1 26 1 41 2310047A01 Rιk
1428436 at Res & Lgx 1165 1047 815 839 -1 11 -1 43 -1 39 Lsm14a
1428440 at Lgx only 8819 10649 10766 12943 1 21 1 22 1 47 Slc25a12
1428441 at Lgx only 2992 2477 1975 1655 -1 21 -1 51 -1 81 Cιsd2
1428443 a at Lgx only 732 902 816 1006 1 23 1 11 1 37 Rap 1 gap
1428465 at Lgx only 6696 7010 5690 4717 1 05 -1 18 -1 42 Tmem147
1428468 at All 950 768 805 688 -1 24 -1 18 -1 38 31 10043021 Rik
1428476 a at Lgx only 441 575 672 824 1 30 1 52 1 87 Elac2
1428494 a at Lgx only 3176 3065 2853 2679 -1 04 -1 11 -1 19 Polr2ι
1428495 at Res & Lgx 2251 1796 1512 1278 -1 25 -1 49 -1 76 2410003K15Rιk
1428503 a at Lgx only 2042 1913 1596 1490 -1 07 -1 28 -1 37 Nkirasi
1428505 at Lgx only 4503 4374 3908 2889 -1 03 -1 15 -1 56 Ccdc90b
1428507 at Lgx only 1620 1610 1433 1331 -1 01 -1 13 -1 22 Hdhd2
1428508 at Lgx only 415 458 547 572 1 10 1 32 1 38 Tbc1 d2b
1428510 at Lgx only 331 473 485 525 1 43 1 46 1 58 Lphni
1428515 at Lgx only 845 790 736 569 -1 07 -1 15 -1 48 2410012H22Rιk
1428519 at Lgx only 3006 3200 2041 1928 1 06 -1 47 -1 56 2610528E23Rιk
1428540 at Lgx only 162 219 253 370 1 35 1 56 2 28 3321401 G04Rιk
1428544 at Res & Lgx 1458 1331 1205 1043 -1 10 -1 21 -1 40 0610007L01 Rιk
1428549 at Lgx only 404 587 708 973 1 45 1 75 241 Ccdc3
1428551 at All 518 361 300 223 -1 44 -1 72 -2 32 Trmti 1
1428552 at Res & Lgx 2497 2020 1544 1257 -1 24 -1 62 -1 99 2610001J05Rιk
1428554 a at Lgx only 2768 2661 2626 2233 -1 04 -1 05 -1 24 1810035L17Rιk
1428564 at Lgx only 417 459 555 738 1 10 1 33 1 77 Zfp579
1428580 at Lgx only 1441 1418 1326 1067 -1 02 -1 09 -1 35 Blvra
1428585 at Lgx only 720 831 840 1309 1 15 1 17 1 82 Actni
1428587 at Res & Lgx 2171 2060 1437 1374 -1 05 -1 51 -1 58 Tmem41 b
1428589 at Lgx only 7082 6392 5436 4990 -1 11 -1 30 -1 42 Mrpl41
1428593 at Res only 436 336 287 244 -1 30 -1 52 -1 79 1700029F09Rιk
1428594 at Lgx only 1864 1998 1599 1255 1 07 -1 17 -1 48 GarnM
1428598 at All 663 946 941 976 1 43 1 42 1 47 Tbc1 d7
1428613 at Res only 548 583 786 754 1 06 1 43 1 38 Ld hd
1428615 at CR only 1077 892 1088 1 153 -1 21 1 01 1 07 P2ry5
1428617 at Res & Lgx 1207 1018 969 859 -1 19 -1 25 -1 41 Hcfc2
1428626 at Lgx only 750 726 685 550 -1 03 -1 09 -1 36 Lysmd2
1428651 at CR & Lgx 5284 4538 5141 4573 -1 16 -1 03 -1 16 KIM24
1428652 at Res & Lgx 341 312 224 199 -1 09 -1 53 -1 72 0610010F05Rιk
1428668 at Lgx only 921 942 1096 1235 1 02 1 19 1 34 Acbd3
Figure imgf000079_0001
Figure imgf000080_0001
Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1429570 at Res only 126 63 36 102 -2 00 -3 45 -1 24 MIkI
1429621 at Lgx only 839 1 173 1240 1343 1 40 1 48 1 60 Cand2
1429648 at Lgx only 637 552 475 461 -1 15 -1 34 -1 38 Slc35a3
1429681 a at Lgx only 21171 22951 25817 28204 1 08 1 22 1 33 Gpsn2
1429698 at Res & Lgx 163 155 82 89 -1 05 -1 99 -1 83 Mterf
1429710 at Res & Lgx 839 763 558 459 -1 10 -1 51 -1 83 Styx
1429723 at Res & Lgx 2893 2604 1673 1495 -1 11 -1 73 -1 94 6330409N04Rιk
1429764 at CR only 2988 2220 3256 2552 -1 35 1 09 -1 17 1500005K14Rιk
1429771 at Lgx only 927 823 776 635 -1 13 -1 19 -1 46 31 10073H01 Rιk
1429783 at Res & Lgx 13193 13213 10275 9645 1 00 -1 28 -1 37 Pdhm5
1429819 at Lgx only 393 378 337 275 -1 04 -1 17 -1 43 Nmnatl
1429836 at CR only 142 251 216 269 1 78 1 53 1 90 Ugcgl2
1429860 at CR only 43 117 154 113 2 69 3 56 2 61 LOC677447
1429888 a at Lgx only 4996 5288 5443 6090 1 06 1 09 1 22 Hspb2
1429915 at Lgx only 56 63 79 116 1 12 1 42 2 08 4930426L09Rιk
1429918 at CR only 433 297 409 416 -1 46 -1 06 -1 04 Arhgap20
1429961 at CR & Lgx 308 198 313 200 -1 55 1 02 -1 54 1700021 C14Rιk
1429990 at Lgx only 1 14 138 174 186 1 21 1 52 1 63 Hyal4
1430000 at CR only 71 24 62 56 -2 92 -1 16 -1 27 B2301 17O15Rιk
1430045 at CR & Res 416 259 269 292 -1 61 -1 55 -1 42 Tsnax
1430078 a at Lgx only 284 264 259 154 -1 07 -1 09 -1 85 Ogg1
1430089 at Lgx only 355 291 227 142 -1 22 -1 57 -2 50 5830469G19Rιk
1430095 at Lgx only 90 95 135 240 1 06 1 50 2 66 D930020B18Rιk
1430123 a at Lgx only 13843 13323 16691 18357 -1 04 1 21 1 33 Akr1a4
1430137 at Res only 148 166 220 222 1 12 1 49 1 51 LOC100043489
1430170 at Res only 162 131 69 89 -1 24 -2 35 -1 82 Bbs10
1430224 at Res only 101 97 238 112 -1 03 2 36 1 12 Wfdc3
1430253 at Lgx only 1 14 159 173 252 1 40 1 52 2 22 2900006B1 1 Rιk
1430292 a at Res & Lgx 3466 3219 2398 2125 -1 08 -1 45 -1 63 1810030N24Rιk
1430309 at Res & Lgx 1747 1890 2535 2577 1 08 1 45 1 47 Nipbl
1430378 at Res & Lgx 205 160 103 72 -1 28 -2 00 -2 85 2900011 G08Rιk
1430388 a at Lgx only 1818 2657 2347 2718 1 46 1 29 1 49 Sulf2
1430474 a at Res & Lgx 8337 8097 5357 5222 -1 03 -1 56 -1 60 Mtch2
1430518 at Lgx only 85 28 55 17 -3 02 -1 56 -5 03 5430402E10Rιk
1430519 a at Lgx only 939 787 784 667 -1 19 -1 20 -1 41 Cnot7
1430527 a at Res & Lgx 2831 2873 3201 3440 1 01 1 13 1 22 Rnf167
1430544 at Lgx only 259 358 390 468 1 39 1 51 1 81 5830404H04Rιk
1430656 a at Res & Lgx 3948 3401 2556 2175 -1 16 -1 54 -1 82 Asnsdi
1430676 at CR & Lgx 26 92 89 122 3 52 3 39 4 65 Col19a1
1430685 at Res only 17 41 35 64 2 38 2 07 3 78 6330503C03Rιk
1430736 at Lgx only 176 135 173 48 -1 31 -1 02 -3 70 903041 1 M 15Rιk
1430768 at Lgx only 206 134 121 101 -1 54 -1 70 -2 03 9530018H14Rιk
1430770 at Res & Lgx 56 28 13 12 -1 97 -4 28 -4 68 3110080E11 Rιk
1430781 at Lgx only 164 131 1 18 78 -1 26 -1 39 -2 11 Ak7
1430799 at Res & Lgx 68 35 14 18 -1 91 -4 91 -3 76 5830432E09Rιk
1430818 at CR only 37 121 1 18 66 3 27 3 20 1 78 Tmc1 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1430835 at CR only 1 12 29 98 107 -3 85 -1 14 -1 05 Ccdc125
1430883 at CR only 103 36 71 54 -2 83 -1 44 -1 89 4933402C05Rιk
1430910 at CR only 173 44 108 126 -3 91 -1 60 -1 37 4930544L04Rιk
1430999 a at Res & Lgx 840 643 415 329 -1 31 -2 02 -2 55 Scoc
1431043 at Lgx only 596 596 738 893 -1 00 1 24 1 50 Kbtbd5
1431255 at CR only 604 385 610 572 -1 57 1 01 -1 06 Calr3
1431287 at CR only 71 140 68 62 1 97 -1 05 -1 15 Pcm1
1431293 a at Res & Lgx 2705 1856 1662 1 146 -1 46 -1 63 -2 36 Cldndi
1431302 a at CR & Lgx 4139 2946 3637 2685 -1 41 -1 14 -1 54 Nudt7
1431322 at Lgx only 704 836 854 982 1 19 1 21 1 40 Igsf3
1431415 a at Lgx only 681 643 595 493 -1 06 -1 14 -1 38 TbpM
1431428 a at Lgx only 1082 1202 1304 1403 1 11 1 21 1 30 Nosip
1431429 a at Lgx only 858 839 1095 1 129 -1 02 1 28 1 32 Arl4a
1431473 at Lgx only 18 7 25 81 -2 53 1 36 4 43 5330423111 Rik
1431498 at Res only 94 120 187 171 1 28 1 99 1 82 9530097N15Rιk
1431551 at Lgx only 62 105 104 164 1 69 1 68 2 64 2610028D06Rιk
1431561 a at Res only 485 676 690 684 1 39 1 42 1 41 Dhx34
1431587 at Res only 70 55 24 53 -1 28 -2 90 -1 32 Ccdc7
1431610 at Res only 49 101 141 107 2 05 2 87 2 18 5330439A09Rιk
1431618 a at CR & Res 325 230 221 179 -1 41 -1 47 -1 82 D14Ertd581e
1431619 a at Lgx only 3465 3627 4405 4627 1 05 1 27 1 34 Dtnbpi
1431679 at Res only 49 89 165 104 1 80 3 35 2 11 2510042H12Rιk
1431746 a at Lgx only 3679 3526 2924 2509 -1 04 -1 26 -1 47 Ubeic
1431785 at Lgx only 175 194 145 90 1 11 -1 20 -1 94 Rnaset2a
1431796 at Lgx only 31 106 75 91 3 40 2 40 2 93 2810430111 Rιk
1431804 a at Res & Lgx 690 548 337 279 -1 26 -2 04 -2 47 Sp3
1431822 a at Res & Lgx 3622 3320 2534 2374 -1 09 -1 43 -1 53 Azι2
1431827 a at Lgx only 896 797 717 590 -1 12 -1 25 -1 52 Tlk2
1431853 at CR only 92 40 75 92 -2 33 -1 22 1 00 4933413C19Rιk
1431893 a at Res & Lgx 638 758 379 436 1 19 -1 68 -1 46 Pdss 1
1431900 a at CR only 32 90 31 85 2 79 -1 06 2 64 Foxa3
1431934 at Lgx only 72 109 81 157 1 51 1 13 2 18 4930505O20Rιk
1431986 at Res only 197 193 296 133 -1 02 1 50 -1 48 4933421A08Rιk
1431998 at CR only 69 14 61 59 -4 95 -1 14 -1 16 4930432L08Rιk
1432000 a at Lgx only 342 378 448 482 1 11 1 31 1 41 Dedd
1432016 a at CR only 31956 37162 31250 32250 1 16 -1 02 1 01 Idh3a
1432057 a at Lgx only 662 743 770 91 1 1 12 1 16 1 38 Prdm5
1432073 at Res & Lgx 3205 3584 2249 2107 1 12 -1 43 -1 52 1700113l22Rιk
1432122 at Lgx only 128 89 121 44 -1 44 -1 05 -2 91 Lrrc44
1432158 a at Res & Lgx 2091 1924 1569 1489 -1 09 -1 33 -1 40 Trappc2
1432207 a at Lgx only 454 448 459 350 -1 01 1 01 -1 30 Toe1
1432248 at Lgx only 31 74 52 105 2 36 1 66 3 36 5430402P08Rιk
1432271 a at Res & Lgx 1856 1636 1172 967 -1 13 -1 58 -1 92 Dcun1 d5
1432348 at Lgx only 59 61 97 183 1 03 1 63 3 09 4930524O07Rιk
1432369 at CR & Res 60 139 152 155 2 30 2 52 2 57 3010027C24Rιk
1432420 a at Lgx only 1929 1605 1516 1 137 -1 20 -1 27 -1 70 2310002L09Rιk
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1435548 at Lgx only 726 703 596 466 -1 03 -1 22 -1 56 Mrs2l
1435549 at Lgx only 434 440 504 549 1 01 1 16 1 26 Trpm4
1435554 at Res & Lgx 1663 1372 1080 922 -1 21 -1 54 -1 80 Tmcc3
1435556 at Lgx only 530 463 373 373 -1 15 -1 42 -1 42 Zfp597
1435589 at Res & Lgx 510 650 831 11 14 1 27 1 63 2 18 Ccdc85b
1435641 at Res & Lgx 437 400 308 300 -1 09 -1 42 -1 45 9530018IO7Rιk
1435655 at Lgx only 248 223 189 131 -1 11 -1 31 -1 89 Snora65
1435674 at Lgx only 361 478 368 510 1 33 1 02 1 41 Rhobtb2
1435679 at Lgx only 1778 1866 1603 1228 1 05 -1 11 -1 45 Optn
1435693 at CR only 348 448 320 452 1 29 -1 09 1 30 Mall
1435695 a at Lgx only 876 756 620 449 -1 16 -1 41 -1 95 A030007L17Rιk
1435743 at Res & Lgx 987 808 671 634 -1 22 -1 47 -1 56 Klhl23
1435754 at Res & Lgx 1015 818 741 714 -1 24 -1 37 -1 42 Zyg1 1 b
1435768 at Lgx only 919 909 725 632 -1 01 -1 27 -1 46 Arιd4b
1435774 at Lgx only 487 394 360 292 -1 24 -1 35 -1 67 AV024533
1435777 at Lgx only 1189 995 853 715 -1 19 -1 39 -1 66 E030018N11 Rιk
1435782 at Lgx only 166 244 291 295 1 47 1 75 1 78 LOC668206
1435808 at Res & Lgx 579 704 924 1015 1 22 1 60 1 75 A230051 G13Rιk
1435813 at Lgx only 2373 3193 3181 3356 1 35 1 34 1 41 Mypn
1435864 a at Res & Lgx 2251 2148 1526 141 1 -1 05 -1 48 -1 59 1810063B05Rιk
1435874 at Lgx only 1005 1045 939 727 1 04 -1 07 -1 38 Prkab2
1435900 at Res & Lgx 775 768 643 518 -1 01 -1 20 -1 49 Zbtb43
1435912 at Lgx only 670 587 579 475 -1 14 -1 16 -1 41 Ubxd7
1435947 at Lgx only 647 572 495 456 -1 13 -1 31 -1 42 2810455D13Rιk
1436014 a at CR only 449 349 494 518 -1 28 1 10 1 16 Rusd
1436026 at Lgx only 476 418 620 736 -1 14 1 30 1 54 Zfp703
1436033 at Lgx only 879 984 1031 1238 1 12 1 17 1 41 BC031353
1436041 at CR & Lgx 1831 1425 1788 1414 -1 28 -1 02 -1 29 LOC100046086
1436045 at Lgx only 381 321 282 216 -1 19 -1 35 -1 76 TsgalO
1436059 at Res & Lgx 254 366 397 446 1 44 1 56 1 75 Rfx 1
1436075 at Lgx only 280 256 422 622 -1 09 1 51 2 22 Sfrp5
1436081 a at Lgx only 1053 1205 1255 1434 1 14 1 19 1 36 Zfp414
14361 12 at Res only 158 196 325 246 1 24 2 06 1 55 AM 18078
14361 13 a at Res only 2773 2845 2148 2176 1 03 -1 29 -1 27 St13
1436121 a at Lgx only 1870 1812 1483 1 122 -1 03 -1 26 -1 67 Nsmcel
1436122 at Res only 242 299 396 310 1 24 1 64 1 28 Zfp667
1436157 at Lgx only 1826 1496 1478 1265 -1 22 -1 24 -1 44 Ccari
1436188 a at CR only 7033 11508 7677 9091 1 64 1 09 1 29 Ndrg4
1436208 at CR & Lgx 551 716 663 794 1 30 1 20 1 44 Asb1
1436214 at Lgx only 567 465 395 293 -1 22 -1 44 -1 93 11 10028C15Rιk
1436215 at Lgx only 642 508 549 433 -1 26 -1 17 -1 48 lpmk
1436233 at Lgx only 1039 871 997 736 -1 19 -1 04 -1 41 Btnl9
1436240 at Lgx only 382 383 308 256 1 00 -1 24 -1 49 Sost
1436243 at Lgx only 2945 3472 2166 1917 1 18 -1 36 -1 54 Frmd5
1436275 at Lgx only 5589 6926 4236 4096 1 24 -1 32 -1 36 Kcnιp2
1436299 at Res & Lgx 1490 1306 11 13 877 -1 14 -1 34 -1 70 GIs Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1436310 at Lgx only 199 301 245 313 1 51 1 23 1 57 Gemιn5
1436332 at Lgx only 32378 38509 38078 46348 1 19 1 18 1 43 Hspb6
1436339 at Lgx only 6840 6976 10138 9417 1 02 1 48 1 38 1810058l24Rιk
1436342 a at Lgx only 1693 1473 1337 1170 -1 15 -1 27 -1 45 D19Ertd721e
1436367 at Lgx only 3325 3749 4038 4164 1 13 1 21 1 25 C130094E24
1436377 at Res only 595 716 830 810 1 20 1 40 1 36 Gpr137
1436408 at Lgx only 88 45 76 29 -1 95 -1 16 -3 02 Rprml
1436425 at Lgx only 182 121 125 126 -1 50 -1 45 -1 45 Ankrd38
1436446 at Res & Lgx 675 568 441 416 -1 19 -1 53 -1 62 2310007011Rιk
1436505 at CR only 722 511 816 739 -1 41 1 13 1 02 Ppig
1436511 at Res & Lgx 675 634 510 460 -1 06 -1 32 -1 47 BC031781
1436521 at Lgx only 995 1371 1104 1496 1 38 1 11 1 50 Slc36a2
1436537 at CR only 624 518 554 573 -1 20 -1 13 -1 09 Zfp629
1436538 at Res & Lgx 425 320 201 116 -1 33 -2 12 -3 68 Ankrd37
1436546 at Res & Lgx 1262 1105 994 881 -1 14 -1 27 -1 43 Lιx11
1436547 at CR only 380 285 282 352 -1 33 -1 34 -1 08 Dgke
1436562 at Lgx only 579 545 498 363 -1 06 -1 16 -1 59 Ddx58
1436584 at Res & Lgx 825 721 541 545 -1 15 -1 53 -1 51 Spry2
1436594 at Lgx only 180 124 152 103 -1 45 -1 18 -1 74 Zfp719
1436609 a at Lgx only 4537 4960 5297 5655 1 09 1 17 1 25 Lrpapi
1436650 at CR & Lgx 729 1049 944 1072 1 44 1 30 1 47 Filipi
1436665 a at Lgx only 3944 5547 5624 7673 1 41 1 43 1 95 Ltbp4
1436739 at Lgx only 2627 2725 2561 2047 1 04 -1 03 -1 28 Agtiia
1436747 at Lgx only 2419 2508 2836 3129 1 04 1 17 1 29 1110014K08Rιk
1436797 a at Lgx only 1519 1814 1790 2289 1 19 1 18 1 51 Surf4
1436809 a at All 1520 2518 2464 2994 1 66 1 62 1 97 Spιn1
1436817 at Lgx only 1182 1030 929 752 -1 15 -1 27 -1 57 Exoc5
1436842 at Res & Lgx 3911 3696 2482 2369 -1 06 -1 58 -1 65 B230380D07Rιk
1436844 at Lgx only 536 431 451 371 -1 24 -1 19 -1 44 AW046287
1436865 at CR only 308 173 311 209 -1 78 1 01 -1 47 Slc26a11
1436867 at CR only 23230 28014 23888 25409 1 21 1 03 1 09 SrI
1436883 at Lgx only 558 425 421 399 -1 31 -1 32 -1 40 Mbtps2
1436918 at Lgx only 857 823 801 562 -1 04 -1 07 -1 53 LOC100044376
1436947 a at Lgx only 4903 4267 3399 2703 -1 15 -1 44 -1 81 TxnM
1436984 at Lgx only 752 734 574 497 -1 02 -1 31 -1 51 Abι2
1436985 at Res & Lgx 1800 1660 1487 1422 -1 08 -1 21 -1 27 Zfp644
1436999 at Res only 619 512 464 480 -1 21 -1 33 -1 29 5033414K04Rιk
1437026 at Lgx only 472 457 287 194 -1 03 -1 65 -2 44 BC057893
1437069 at Lgx only 1632 1471 1282 972 -1 11 -1 27 -1 68 Osbplδ
1437077 at Res & Lgx 2086 1987 1493 1649 -1 05 -1 40 -1 26 Dcun1d2
1437092 at Res & Lgx 535 440 282 286 -1 22 -1 90 -1 87 LOC100048376
1437111 at Lgx only 474 441 430 320 -1 08 -1 10 -1 48 Zc3h12c
1437136 at Res only 60 130 240 141 2 17 4 00 2 35 5830436119Rιk
1437143 a at All 4498 3397 2547 1894 -1 32 -1 77 -2 38 Txndd
1437148 at CR & Lgx 7652 9004 8835 9409 1 18 1 15 1 23 Arpc2
1437149 at Lgx only 2370 2826 3182 3347 1 19 1 34 1 41 Slc6a6 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1437151 at Lgx only 1757 1972 2085 2237 1 12 1 19 1 27 Usp22
1437216 at Lgx only 363 289 295 210 -1 25 -1 23 -1 73 Ccdc88a
1437236 a at Lgx only 449 348 371 302 -1 29 -1 21 -1 49 Zfp110
1437241 at CR only 760 1025 755 926 1 35 -1 01 1 22 KIf11
1437283 at Lgx only 376 462 480 662 1 23 1 28 1 76 Tnpo2
1437287 at Res & Lgx 2217 1647 1611 1014 -1 35 -1 38 -2 19 1110020G09Rιk
1437290 at Lgx only 1027 946 711 722 -1 09 -1 44 -1 42 lmpadi
1437354 at Res & Lgx 1278 1069 925 811 -1 20 -1 38 -1 58 C230091 D08Rιk
1437382 at Lgx only 1211 1154 1069 922 -1 05 -1 13 -1 31 Acvr2a
1437394 at Lgx only 343 383 446 567 1 12 1 30 1 65 Centg2
1437397 at Res & Lgx 277 210 181 160 -1 32 -1 53 -1 73 PrIr
1437398 a at All 781 1052 1289 1566 1 35 1 65 201 Aldh9a1
1437403 at Lgx only 41 70 69 98 1 72 1 69 241 Samd5
1437405 a at Lgx only 5956 7647 8743 11879 1 28 1 47 1 99 Igfbp4
1437426 at Lgx only 275 200 273 162 -1 37 -1 01 -1 70 Wac
1437432 a at Res only 220 153 98 165 -1 44 -225 -1 33 Trim12
1437442 at Res & Lgx 1223 963 774 621 -1 27 -1 58 -1 97 Pcdh7
1437449 at Lgx only 727 595 602 560 -1 22 -1 21 -1 30 Rsadi
1437482 at Res & Lgx 7128 6639 4396 3270 -1 07 -1 62 -2 18 Srd5a2l2
1437484 at Res & Lgx 321 372 527 541 1 16 1 64 1 68 Zbtb5
1437513 a at Lgx only 5779 5896 6781 7125 1 02 1 17 1 23 Serind
1437533 at Res & Lgx 2000 1894 1502 1237 -1 06 -1 33 -1 62 Bιrc4
1437537 at Res & Lgx 249 339 340 398 1 36 1 36 1 60 Casp9
1437704 at Res & Lgx 194 157 376 378 -1 23 1 93 1 94 290002401ORik
1437729 at Lgx only 1474 1094 793 412 -1 35 -1 86 -3 58 Rpl27a
1437740 at Lgx only 763 1122 1005 1343 1 47 1 32 1 76 Plekhm2
1437741 at Lgx only 2327 2390 1778 1560 1 03 -1 31 -1 49 Rab21
1437785 at Lgx only 474 397 463 337 -1 19 -1 03 -1 41 Adamtsθ
1437869 at Res & Lgx 14570 13664 11826 11731 -1 07 -1 23 -1 24 3222402P14Rιk
1437875 at Lgx only 2753 2607 2485 2181 -1 06 -1 11 -1 26 Bιcd2
1437900 at Lgx only 566 564 505 437 -1 00 -1 12 -1 30 4930523C07Rιk
1437917 at Lgx only 370 334 293 223 -1 11 -1 26 -1 65 D530037H12Rιk
1438024 at Res & Lgx 3610 3226 2484 2237 -1 12 -1 45 -1 61 Ccdc90a
1438026 at Lgx only 623 498 498 393 -1 25 -1 25 -1 58 Zfp560
1438045 at Lgx only 737 678 702 495 -1 09 -1 05 -1 49 Eea1
1438047 at Res & Lgx 1053 1176 1296 1256 1 12 1 23 1 19 Zfp384
1438062 at CR only 588 372 440 434 -1 58 -1 34 -1 36 4832420A03Rιk
1438077 at Lgx only 18 38 17 62 2 11 -1 09 3 42 Nlrp4a
1438097 at Lgx only 611 574 490 391 -1 06 -1 25 -1 56 Rab20
1438169 a at Lgx only 747 494 424 347 -1 51 -1 76 -2 15 Frmd4b
1438195 at Lgx only 2915 3104 2321 2279 1 06 -1 26 -1 28 Gpdil
1438208 at Res only 286 277 424 361 -1 03 1 48 1 26 Taok2
1438213 at Lgx only 15 30 39 79 2 00 2 61 5 30 A830018L16Rιk
1438229 at Res only 843 887 609 701 1 05 -1 38 -1 20 Pggtib
1438238 at Lgx only 231 217 198 106 -1 07 -1 17 -2 18 2010315B03Rιk
1438241 at Lgx only 525 593 734 837 1 13 1 40 1 59 Rgma
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1448163 at Lgx only 798 1050 981 1240 1 32 1 23 1 55 Gnpdal
1448167 at Lgx only 684 808 806 1 130 1 18 1 18 1 65 IfngM
1448174 at Lgx only 1875 1928 2181 2232 1 03 1 16 1 19 CuM
1448181 at CR only 1549 221 1 1745 1742 1 43 1 13 1 12 KIf 15
1448185 at Lgx only 5495 7055 5636 8302 1 28 1 03 1 51 Herpudi
1448188 at Lgx only 5526 6650 8466 12288 1 20 1 53 2 22 Ucp2
1448189 a at Lgx only 2277 2853 3180 3351 1 25 1 40 1 47 Flu
1448196 at Res & Lgx 3368 2651 2008 1537 -1 27 -1 68 -2 19 Mat2b
1448198 a at Lgx only 38229 43229 40539 48367 1 13 1 06 1 27 Ndufbδ
1448199 at Res only 679 631 507 543 -1 08 -1 34 -1 25 AnkrdiO
1448206 at Lgx only 6419 7041 7577 10844 1 10 1 18 1 69 Psma2
1448208 at Lgx only 632 474 499 348 -1 34 -1 27 -1 82 Smadi
1448209 a at Lgx only 246 316 363 502 1 29 1 48 2 04 Slc22a17
1448212 at Lgx only 1004 973 746 707 -1 03 -1 35 -1 42 Tnfsf5ιp1
1448221 at CR only 4042 4996 4086 4247 1 24 1 01 1 05 Bati a
1448224 at Lgx only 1460 1359 11 17 995 -1 07 -1 31 -1 47 Tf am
1448225 at Lgx only 498 691 692 796 1 39 1 39 1 60 Gpaai
1448240 at Lgx only 1199 1319 1410 1668 1 10 1 18 1 39 Mbtpsi
1448242 at Lgx only 1162 1336 1617 1835 1 15 1 39 1 58 Sec61 a1
1448244 at Lgx only 1573 1505 1192 813 -1 05 -1 32 -1 93 Lyplai
1448252 a at Lgx only 7588 7012 6014 5451 -1 08 -1 26 -1 39 Eef1 b2
1448258 a at Lgx only 6413 6297 5305 4246 -1 02 -1 21 -1 51 Spcsi
1448269 a at Res & Lgx 613 517 374 350 -1 19 -1 64 -1 75 KIhM 3
1448276 at Lgx only 1808 2156 1862 2730 1 19 1 03 1 51 Tspan4
1448284 a at Lgx only 37586 40364 44938 56730 1 07 1 20 1 51 Ndufd
1448287 at Lgx only 4436 4040 3639 2830 -1 10 -1 22 -1 57 Rpo1-3
1448304 a at Res & Lgx 3053 2867 2183 1770 -1 06 -1 40 -1 72 Rab6
1448313 at Lgx only 2609 2807 3061 3302 1 08 1 17 1 27 Tpp1
1448325 at Res & Lgx 757 893 11 12 1080 1 18 1 47 1 43 Myd1 16
1448327 at Lgx only 30728 36837 37776 43889 1 20 1 23 1 43 Actn2
1448330 at Res & Lgx 7365 8521 9720 11395 1 16 1 32 1 55 Gstmi
1448336 at Lgx only 1711 1622 1611 1433 -1 05 -1 06 -1 19 Drg1
1448339 at Res & Lgx 4104 3995 2913 2634 -1 03 -1 41 -1 56 Tmem30a
1448341 a at Lgx only 321 317 503 653 -1 01 1 57 2 04 Stxbp2
1448345 at Lgx only 1081 1303 1204 1435 1 21 1 1 1 1 33 Tomm34
1448346 at CR only 2920 2207 2287 2450 -1 32 -1 28 -1 19 CfM
1448351 at Lgx only 1035 1248 1300 1665 1 21 1 26 1 61 Coroi b
1448356 at Lgx only 2083 1903 1705 1572 -1 09 -1 22 -1 32 Ube2d2
1448362 at Lgx only 1772 2097 2189 2519 1 18 1 24 1 42 Dnajc7
1448363 at Res & Lgx 2486 2740 3232 3785 1 10 1 30 1 52 Yap1
1448365 at Res & Lgx 1790 1723 1184 1227 -1 04 -1 51 -1 46 Exosc7
1448379 at Res only 647 563 413 518 -1 15 -1 57 -1 25 Pot 1a
1448380 at Res & Lgx 1020 1208 1439 1659 1 19 1 41 1 63 Lgals3bp
1448388 a at Lgx only 2505 2405 1802 1 158 -1 04 -1 39 -2 16 11 10002B05Rιk
1448402 at Lgx only 1415 1869 2287 2869 1 32 1 62 2 03 TInI
1448412 a at Lgx only 3472 4076 4244 4721 1 17 1 22 1 36 Tsc22d4 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1448415 a at Lgx only 387 438 593 653 1 13 1 53 1 69 Sema3b
1448416 at Lgx only 16601 20394 19345 31156 1 23 1 17 1 88 Mgp
1448417 at Lgx only 1000 1181 1301 1299 1 18 1 30 1 30 Nm1I
1448429 at Res & Lgx 3339 3198 2421 1974 -1 04 -1 38 -1 69 Gyg
1448430 a at CR & Lgx 8516 10720 9639 10638 1 26 1 13 1 25 Naca
1448432 at Lgx only 369 514 544 661 1 40 1 48 1 79 Plcdi
1448438 at Lgx only 2204 1814 1971 1577 -1 22 -1 12 -1 40 Derl2
1448463 at Lgx only 4645 4072 3480 3091 -1 14 -1 33 -1 50 4933434E20Rιk
1448467 a at Lgx only 1218 1447 1612 1817 1 19 1 32 1 49 EhbpiH
1448476 at CR & Lgx 5612 6609 6127 7217 1 18 1 09 1 29 Nap1l4
1448480 at Res & Lgx 1000 728 556 360 -1 37 -1 80 -2 78 Nιp7
1448484 at Res & Lgx 5744 5110 3530 3419 -1 12 -1 63 -1 68 Amd1
1448488 at CR & Lgx 831 1036 919 1013 1 25 1 11 1 22 Mrps5
1448492 a at Res & Lgx 4733 4441 3842 3020 -1 07 -1 23 -1 57 Psmd12
1448493 at CR & Lgx 9202 6905 8442 6228 -1 33 -1 09 -1 48 Paιp2
1448495 at Lgx only 723 911 890 1046 1 26 1 23 1 45 Tsta3
1448498 at Lgx only 793 1061 951 1228 1 34 1 20 1 55 Rps6ka4
1448505 at Res & Lgx 1738 1400 1033 860 -1 24 -1 68 -202 C1d
1448508 at Lgx only 237 284 322 442 1 20 1 36 1 86 Traf3ιp2
1448517 at Lgx only 2047 2144 1562 1376 1 05 -1 31 -1 49 Tιmm22
1448527 at Lgx only 1955 1753 1345 1338 -1 12 -1 45 -1 46 PdcdiO
1448533 at Res & Lgx 3076 2967 2613 2263 -1 04 -1 18 -1 36 Tbcb
1448535 at Lgx only 306 250 237 154 -1 22 -1 29 -1 98 Elp4
1448536 at Lgx only 4579 4812 3261 2727 1 05 -1 40 -1 68 Lsm3
1448537 at Res & Lgx 2417 2026 1762 1591 -1 19 -1 37 -1 52 Ttc1
1448543 at Res & Lgx 1715 1494 908 822 -1 15 -1 89 -2 09 Slmo2
1448548 at Lgx only 1328 1385 1288 1631 1 04 -1 03 1 23 Tulp4
1448549 a at Res only 1179 1068 850 1106 -1 10 -1 39 -1 07 Dpagti
1448559 at Lgx only 5131 5547 6227 7143 1 08 1 21 1 39 Floti
1448564 at Res only 511 609 713 573 1 19 1 40 1 12 Cιb1
1448565 at Lgx only 2831 2386 2740 1942 -1 19 -1 03 -1 46 Ppp1r11
1448567 at Lgx only 590 668 763 806 1 13 1 29 1 37 Tmem115
1448568 a at CR only 688 524 629 533 -1 31 -1 09 -1 29 Slc20a1
1448570 at Lgx only 1666 1576 1258 948 -1 06 -1 32 -1 76 Gmfb
1448579 at Lgx only 2689 2976 3392 3692 1 11 1 26 1 37 GIgI
1448585 at Res only 751 809 578 655 1 08 -1 30 -1 15 Gtf2h4
1448591 at CR only 1010 720 1099 736 -1 40 1 09 -1 37 Ctss
1448613 at Lgx only 1206 1308 1426 1969 1 08 1 18 1 63 Ecm1
1448615 at Res & Lgx 769 858 1028 1113 1 12 1 34 1 45 Ccs
1448621 a at Res & Lgx 7092 7555 5213 5291 1 07 -1 36 -1 34 Smpdi
1448623 at Lgx only 1699 1287 1178 807 -1 32 -1 44 -2 11 Tmem123
1448625 at Res & Lgx 1612 1957 2268 2558 1 21 1 41 1 59 Golga2
1448637 at Lgx only 1033 1248 1507 1924 1 21 1 46 1 86 Med25
1448638 at Res only 68 79 140 150 1 17 206 221 Mtbp
1448644 at Lgx only 1286 1471 1597 1684 1 14 1 24 1 31 Pef1
1448645 at Lgx only 2239 2328 1993 1844 1 04 -1 12 -1 21 Msl31
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1450203 at Lgx only 5141 5970 4705 4331 1 16 -1 09 -1 19 Smydi
1450308 a at Res & Lgx 373 389 657 764 1 04 1 76 2 05 Xm1
1450355 a at Res only 432 329 314 351 -1 31 -1 38 -1 23 Capg
1450361 at CR only 129 52 132 65 -247 1 02 -1 99 Propi
1450376 at Lgx only 2003 2127 2276 2577 1 06 1 14 1 29 Mxι1
1450377 at Lgx only 165 219 246 321 1 33 1 49 1 94 LOC640441
1450395 at Lgx only 1580 1719 1822 2403 1 09 1 15 1 52 Slc22a5
1450405 at Res & Lgx 725 643 354 320 -1 13 -2 05 -2 27 Mrpl19
1450409 a at Lgx only 1748 1899 2253 2570 1 09 1 29 1 47 4930570C03Rιk
1450415 at CR only 56 17 56 44 -3 33 -1 01 -1 29 Pde6a
1450424 a at CR only 188 296 252 253 1 58 1 34 1 34 Il18bp
1450431 a at Res & Lgx 11524 12897 16998 18939 1 12 1 48 1 64 Nedd4
1450435 at Lgx only 277 284 353 397 1 03 1 27 1 44 L1cam
1450449 a at Lgx only 3983 4570 4434 5830 1 15 1 11 1 46 2900002H16Rιk
1450490 at Lgx only 417 575 570 646 1 38 1 37 1 55 Kcna7
1450519 a at Lgx only 4324 5666 5682 6518 1 31 1 31 1 51 Prkaca
1450531 at CR only 102 24 54 78 -4 30 -1 90 -1 30 H2-BI
1450584 at Res only 25 69 107 61 2 76 4 32 2 45 Hoxd11
1450623 at Lgx only 3269 3884 4094 4247 1 19 1 25 1 30 Gnb2
1450627 at Res & Lgx 10383 11098 8410 8645 1 07 -1 23 -1 20 Ank
1450649 at Lgx only 1489 966 883 760 -1 54 -1 69 -1 96 Gng10
1450650 at Lgx only 987 1096 1732 1739 1 11 1 76 1 76 Myo10
1450662 at Lgx only 584 632 750 830 1 08 1 28 1 42 Teski
1450664 at CR & Lgx 1309 969 977 814 -1 35 -1 34 -1 61 Gabpa
1450670 at Lgx only 884 1083 1043 1276 1 22 1 18 1 44 Dbh
1450672 a at Lgx only 892 966 977 1154 1 08 1 10 1 29 Trexi
1450678 at CR only 384 292 365 338 -1 32 -1 05 -1 14 Itgb2
1450690 at Lgx only 1537 1178 953 774 -1 30 -1 61 -1 98 Ranbp2
1450691 at Res only 621 793 919 894 1 28 1 48 1 44 Caskιn2
1450700 at Res & Lgx 1859 1642 1543 1404 -1 13 -1 20 -1 32 Cdc42ep3
1450706 a at Lgx only 2959 2743 2744 2114 -1 08 -1 08 -1 40 ArI3
1450714 at Res & Lgx 2451 2021 1702 1453 -1 21 -1 44 -1 69 Azιn1
1450729 at Lgx only 637 561 625 508 -1 14 -1 02 -1 25 Hs2st1
1450735 at Res & Lgx 2114 2019 1724 1463 -1 05 -1 23 -1 45 Pno1
1450738 at Lgx only 938 886 1089 1210 -1 06 1 16 1 29 Kιf21a
1450740 a at Res & Lgx 4235 4039 3546 3025 -1 05 -1 19 -1 40 Maprei
1450744 at Lgx only 488 355 297 213 -1 38 -1 65 -2 29 EII2
1450759 at Res only 852 857 1146 1021 1 01 1 35 1 20 Bmp6
1450791 at Lgx only 15042 15107 14450 8913 1 00 -1 04 -1 69 Nppb
1450798 at Lgx only 819 1305 1266 1651 1 59 1 54 201 Tnxb
1450801 at Res & Lgx 37 93 146 227 2 53 3 98 6 20 Adam21
1450816 at Res only 384 341 301 362 -1 13 -1 27 -1 06 Polg2
1450839 at Res only 1123 1002 829 1015 -1 12 -1 35 -1 11 D0H4S114
1450840 a at Lgx only 50799 56471 65223 75285 1 11 1 28 1 48 Rpl39
1450842 a at CR & Lgx 2062 1704 1758 1347 -1 21 -1 17 -1 53 Cenpa
1450857 a at Lgx only 2960 3430 3914 4745 1 16 1 32 1 60 CoI1a2 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1450866 a at Lgx only 3015 3253 3652 3857 1 08 1 21 1 28 Mrpl17
1450878 at Lgx only 2049 1928 1635 1368 -1 06 -1 25 -1 50 Sn
1450879 at Lgx only 610 710 716 993 1 16 1 17 1 63 Atp9b
1450883 a at CR only 31708 23985 32717 30704 -1 32 1 03 -1 03 Cd 36
1450890 a at Lgx only 948 726 729 585 -1 31 -1 30 -1 62 Abi 1
1450891 at Lgx only 5718 5089 4197 3750 -1 12 -1 36 -1 53 Srp19
1450894 a at Res & Lgx 2906 3585 3746 3815 1 23 1 29 1 31 Ap2m1
1450897 at Lgx only 1688 1363 1427 1 165 -1 24 -1 18 -1 45 Arhgapδ
1450903 at Lgx only 1020 958 834 627 -1 06 -1 22 -1 63 Rad23b
1450927 at Lgx only 514 591 685 712 1 15 1 33 1 39 Lztri
1450934 at Res & Lgx 22353 18933 16228 15963 -1 18 -1 38 -1 40 Eιf4a2
1450948 a at Lgx only 827 736 541 458 -1 12 -1 53 -1 81 MrpM
1450953 at Res & Lgx 2794 2661 2247 2084 -1 05 -1 24 -1 34 Cιao1
1450957 a at Lgx only 14361 13299 20795 20648 -1 08 1 45 1 44 Sqstml
1450958 at Lgx only 5954 4641 4404 4138 -1 28 -1 35 -1 44 Tm4sf1
1450965 at Res & Lgx 1995 2296 2538 3135 1 15 1 27 1 57 Tex261
1450966 at Lgx only 927 836 863 679 -1 11 -1 07 -1 37 Crot
1450968 at Lgx only 39927 41899 42555 55205 1 05 1 07 1 38 Uqcrfsi
1450970 at Lgx only 25949 32719 31690 36332 1 26 1 22 1 40 Got1
1450971 at Res & Lgx 551 827 965 1069 1 50 1 75 1 94 Gadd45b
1450974 at Res & Lgx 776 1077 1180 1212 1 39 1 52 1 56 Tιmp4
1450994 at Lgx only 2773 2136 2256 1821 -1 30 -1 23 -1 52 Rocki
1451002 at Lgx only 54266 62734 61354 75692 1 16 1 13 1 39 Aco2
1451006 at Lgx only 1143 1483 1634 1746 1 30 1 43 1 53 Xdh
1451010 at Lgx only 656 786 728 858 1 20 1 11 1 31 NoM 1
1451017 at Lgx only 1586 1728 1871 2173 1 09 1 18 1 37 Ergιc3
1451019 at Lgx only 1338 1281 1678 2014 -1 04 1 25 1 51 Ctsf
1451022 at Lgx only 545 506 713 852 -1 08 1 31 1 56 Lrp6
1451025 at Lgx only 2443 2161 1929 1347 -1 13 -1 27 -1 81 ArM
1451050 at Lgx only 3147 2948 2847 2290 -1 07 -1 11 -1 37 Nt5c3
1451051 a at Lgx only 837 1040 1137 1455 1 24 1 36 1 74 ScyM
1451067 at Lgx only 3259 4112 4448 4416 1 26 1 37 1 36 Sgta
1451070 at Lgx only 2031 2545 2472 3265 1 25 1 22 1 61 Gdι1
1451074 at Res & Lgx 4210 3824 3180 2816 -1 10 -1 32 -1 49 Rnf13
1451096 at Lgx only 29178 32946 32150 36689 1 13 1 10 1 26 Ndufs2
1451099 at Lgx only 535 685 718 838 1 28 1 34 1 57 Mbc2
1451104 a at CR & Lgx 1056 1430 1492 1790 1 35 1 41 1 69 Snrp70
14511 18 a at Lgx only 790 918 855 1042 1 16 1 08 1 32 2410018C17Rιk
14511 19 a at Lgx only 2418 2685 3222 3877 1 11 1 33 1 60 FbInI
1451121 a at Res & Lgx 11 12 1325 1636 1783 1 19 1 47 1 60 Gltscr2
1451126 at Lgx only 4627 4444 5283 5589 -1 04 1 14 1 21 Maf1
1451134 a at Res & Lgx 1207 953 612 601 -1 27 -1 97 -2 01 Tm2d2
1451144 at All 1442 1150 986 886 -1 25 -1 46 -1 63 Bxdc2
1451159 at Res & Lgx 4282 5561 6247 8339 1 30 1 46 1 95 Arhgef12
1451168 a at Res & Lgx 4732 5809 6829 7722 1 23 1 44 1 63 Arhgdia
1451177 at Res & Lgx 6681 6017 4465 4080 -1 11 -1 50 -1 64 Dnajb4 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1451187 at Lgx only 4203 4110 3516 3225 -1 02 -1 20 -1 30 0610037P05Rιk
1451204 at Res only 2057 1633 1312 1410 -1 26 -1 57 -1 46 Scara5
1451217 a at Lgx only 6459 5713 5401 4388 -1 13 -1 20 -1 47 lmmpil
1451219 at Lgx only 915 111 738 662 -1 18 -1 24 -1 38 OrmdH
1451223 a at Lgx only 2697 2588 2232 1739 -1 04 -1 21 -1 55 Btf3l4
1451225 at Lgx only 6229 6428 6944 7210 1 03 1 11 1 16 Ptpn11
1451226 at Lgx only 1149 1523 1685 2053 1 32 1 47 1 79 Pex6
1451232 at Lgx only 6475 6003 4860 4346 -1 08 -1 33 -1 49 Cd151
1451244 a at Lgx only 1155 1068 902 915 -1 08 -1 28 -1 26 Zfp422
1451245 at Lgx only 1838 1659 1561 1417 -1 11 -1 18 -1 30 Lrrc3b
1451248 at Res & Lgx 1477 1443 1184 1086 -1 02 -1 25 -1 36 Prmt7
1451254 at Res & Lgx 1081 1005 719 681 -1 08 -1 50 -1 59 lkbkap
1451269 at Lgx only 1025 970 886 831 -1 06 -1 16 -1 23 Pdzd11
1451272 a at Res & Lgx 1518 1300 972 856 -1 17 -1 56 -1 77 Ube2f
1451274 at Lgx only 18853 24930 23212 26074 1 32 1 23 1 38 Ogdh
1451281 at Res only 182 134 126 176 -1 36 -1 44 -1 03 Zscan12
1451284 at Lgx only 824 1042 937 1011 1 26 1 14 1 23 Yιpf3
1451285 at Lgx only 1415 1654 1921 2284 1 17 1 36 1 61 Fus
1451290 at Lgx only 19210 20375 21862 26463 1 06 1 14 1 38 Map1lc3a
1451291 at CR only 985 1221 1225 1080 1 24 1 24 1 10 Obfc2b
1451293 at CR only 334 573 472 317 1 71 1 41 -1 05 Rrp9
1451295 a at Res & Lgx 645 582 1919 1477 -1 11 2 98 2 29 Chd4
1451297 at Res only 17 25 80 44 1 45 466 2 55 GuIo
1451298 at Lgx only 536 562 684 976 1 05 1 28 1 82 Plekhh3
1451312 at Lgx only 31762 35863 37350 45671 1 13 1 18 1 44 Ndufs7
1451316 a at Res & Lgx 5859 5369 3411 3242 -1 09 -1 72 -1 81 Picalm
1451343 at Lgx only 832 752 752 595 -1 11 -1 11 -1 40 Vps36
1451344 at Lgx only 284 314 447 534 1 11 1 57 1 88 Tmem119
1451349 at Lgx only 129 111 75 50 -1 16 -1 73 -2 56 BC020077
1451364 at Res & Lgx 1068 1040 814 549 -1 03 -1 31 -1 94 Polr3gl
1451369 at Lgx only 1208 1284 1405 1759 1 06 1 16 1 46 Commd5
1451381 at Lgx only 3059 2545 2352 2042 -1 20 -1 30 -1 50 1810020D17Rιk
1451382 at Res & Lgx 247 447 470 562 1 81 1 90 2 28 Chad
1451388 a at Lgx only 1122 1092 1224 1351 -1 03 1 09 1 20 Atp11b
1451405 at Lgx only 3973 4289 5228 5041 1 08 1 32 1 27 Pcca
1451415 at CR only 1529 2052 1641 1570 1 34 1 07 1 03 1810011O10Rιk
1451420 at Res & Lgx 2878 2478 2242 2325 -1 16 -1 28 -1 24 Ccdc47
1451427 a at Lgx only 1907 2012 2224 2426 1 05 1 17 1 27 Egfl7
1451448 a at Lgx only 1765 1548 1206 984 -1 14 -1 46 -1 79 1110005A03Rιk
1451453 at Lgx only 246 248 265 336 1 01 1 08 1 36 Dapk2
1451455 at Res only 273 220 478 374 -1 24 1 75 1 37 Thnsl2
1451462 a at Lgx only 1261 1287 1200 958 1 02 -1 05 -1 32 Ifnar2
1451465 at Lgx only 1407 1531 1643 1908 1 09 1 17 1 36 Ubl7
1451471 at Lgx only 421 391 360 290 -1 08 -1 17 -1 45 Ears2
1451488 at Lgx only 7929 9254 9001 10760 1 17 1 14 1 36 1110028A07Rιk
1451502 at CR only 160 305 224 282 1 90 1 40 1 76 Pla2g10 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1451508 at Lgx only 1347 1314 1107 988 -1 02 -1 22 -1 36 Larp2
1451523 a at Lgx only 1126 879 1016 827 -1 28 -1 11 -1 36 Mιf4gd
1451538 at Lgx only 135 123 153 242 -1 10 1 13 1 78 Sox9
1451553 at Lgx only 713 650 619 510 -1 10 -1 15 -1 40 Art5
1451561 at Res & Lgx 450 622 809 978 1 38 1 80 2 18 Prr12
1451583 a at Res & Lgx 69 119 158 261 1 72 2 29 3 78 BC025076
1451604 a at Lgx only 658 716 707 849 1 09 1 07 1 29 Acvrl 1
1451622 at Lgx only 2006 2187 2476 2643 1 09 1 23 1 32 Lmbrdi
1451663 a at Lgx only 743 755 847 912 1 02 1 14 1 23 Trιm3
1451665 a at Res & Lgx 5435 4923 4504 4166 -1 10 -1 21 -1 30 Ap4s1
1451674 at Lgx only 1 14 131 180 195 1 15 1 57 1 70 SId 2a5
1451678 at Res & Lgx 2482 2409 1553 1507 -1 03 -1 60 -1 65 Narf
1451700 a at Res & Lgx 2076 2271 2807 2740 1 09 1 35 1 32 11 10007L15Rιk
1451728 at Lgx only 792 823 931 1 122 1 04 1 17 1 42 Wdr13
1451741 a at Lgx only 732 614 585 518 -1 19 -1 25 -1 41 Cdk7
1451742 a at Lgx only 14534 14261 12397 11426 -1 02 -1 17 -1 27 Ugp2
1451782 a at Lgx only 2095 2186 2027 2552 1 04 -1 03 1 22 Slc29a1
1451789 a at CR only 651 1037 851 972 1 59 1 31 1 49 Ryk
1451803 a at Lgx only 6764 8556 8339 9104 1 26 1 23 1 35 Vegfb
1451820 at Lgx only 131 214 241 536 1 64 1 84 4 10 Dirasi
1451839 a at CR only 890 1444 1029 1071 1 62 1 16 1 20 Pde7a
1451854 a at Lgx only 383 572 575 694 1 49 1 50 1 81 Shroom3
1451857 a at Lgx only 194 239 319 373 1 23 1 64 1 92 Notum
ENSMUSG-
1451883 at Res & Lgx 401 497 932 825 1 24 2 32 2 0600000074670
1451902 at Lgx only 572 427 446 344 -1 34 -1 28 -1 66 Zfp758
1451911 a at Lgx only 784 928 1055 1218 1 18 1 35 1 55 Ace
1451974 at Lgx only 3494 3897 3875 4394 1 12 1 1 1 1 26 Osbpl2
1451984 at Res & Lgx 1996 1833 1480 1574 -1 09 -1 35 -1 27 HnrpuM
1452012 a at Lgx only 688 561 589 433 -1 23 -1 17 -1 59 Exosd
1452024 a at Lgx only 972 977 1305 1295 1 00 1 34 1 33 Ldb1
1452043 at Lgx only 638 593 728 510 -1 07 1 14 -1 25 231001 1J03Rιk
1452047 at Res & Lgx 2343 2094 1524 1383 -1 12 -1 54 -1 69 Cacybp
1452057 at Lgx only 1040 1024 1176 1469 -1 02 1 13 1 41 Actri b
1452058 a at Res & Lgx 8691 8360 5764 5728 -1 04 -1 51 -1 52 Rnf11
1452072 at Res & Lgx 1185 937 765 822 -1 27 -1 55 -1 44 Mycti
1452080 a at Lgx only 1213 1057 971 775 -1 15 -1 25 -1 57 Dcun1 d1
1452088 at Res only 153 231 81 127 1 51 -1 89 -1 21 Zbed3
1452091 a at CR & Lgx 852 1 127 962 1 169 1 32 1 13 1 37 Rbm28
14521 10 at Lgx only 459 361 351 327 -1 27 -1 31 -1 41 Mtrr
1452130 at Res & Lgx 1764 1802 966 869 1 02 -1 83 -2 03 Txndc14
1452140 at Res & Lgx 867 934 1137 1294 1 08 1 31 1 49 Tbd d20
1452141 a at Lgx only 25419 26171 28265 32953 1 03 1 1 1 1 30 Seppi
1452143 at All 21772 24259 24942 27902 1 1 1 1 15 1 28 Spnb2
1452145 at Lgx only 524 698 748 1076 1 33 1 43 2 06 H6pd
1452152 at Lgx only 2710 2333 2165 1785 -1 16 -1 25 -1 52 Clinti
1452155 a at Res & Lgx 3205 3897 4271 4549 1 22 1 33 1 42 Ddx17 Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1452156 a at Lgx only 4703 5715 5941 7648 1 22 1 26 1 63 Nisch
1452159 at Res & Lgx 1379 1311 1056 977 -1 05 -1 31 -1 41 2310001A20Rιk
1452173 at Lgx only 26826 30831 34012 38315 1 15 1 27 1 43 Hadha
1452174 at Res & Lgx 500 634 880 1000 1 27 1 76 2 00 Srebf2
1452202 at Lgx only 1135 1145 1336 1475 1 01 1 18 1 30 Pde2a
1452203 at All 2414 1686 1491 1035 -1 43 -1 62 -2 33 Obfc2a
1452208 at Lgx only 485 620 638 696 1 28 1 31 1 43 Prdm4
1452213 at Res & Lgx 1320 1264 1036 845 -1 04 -1 27 -1 56 Tex2
1452214 at Lgx only 1513 1185 1126 850 -1 28 -1 34 -1 78 Skil
1452221 a at Lgx only 1063 1125 1281 1393 1 06 1 20 1 31 Cxxd
1452222 at Lgx only 1656 1643 1732 2090 -1 01 1 05 1 26 Utrn
1452225 at Lgx only 1335 1061 1047 1000 -1 26 -1 28 -1 33 2010106G01Rιk
1452250 a at Lgx only 2260 2785 2667 3592 1 23 1 18 1 59 Col6a2
1452262 at Lgx only 708 642 550 557 -1 10 -1 29 -1 27 Grpel2
1452286 at Res & Lgx 6581 4659 2979 2444 -1 41 -2 21 -2 69 Slaιn2
1452291 at Lgx only 564 461 566 360 -1 22 1 00 -1 57 Centdi
1452292 at Lgx only 1094 1334 1435 1847 1 22 1 31 1 69 Ap2b1
1452296 at Lgx only 237 266 412 579 1 12 1 74 2 45 Sht3
1452308 a at Lgx only 4905 5839 6466 6497 1 19 1 32 1 32 Atp1a2
1452309 at Lgx only 721 839 763 1132 1 16 1 06 1 57 CgnM
1452318 a at Res & Lgx 536 385 278 289 -1 39 -1 93 -1 86 Hspalb
1452319 at CR only 283 194 251 220 -1 46 -1 13 -1 28 Zfp82
1452327 at Lgx only 904 1190 1305 1397 1 32 1 44 1 55 Iqsed
1452329 at Res & Lgx 265 311 451 486 1 17 1 71 1 84 Plekhni
1452330 a at Lgx only 2664 2991 3519 4501 1 12 1 32 1 69 Mxraδ
1452333 at Res & Lgx 475 512 815 822 1 08 1 71 1 73 Smarca2
1452335 at Res & Lgx 485 467 382 319 -1 04 -1 27 -1 52 Mfsdδ
1452339 at Lgx only 320 425 415 535 1 33 1 30 1 67 Adamts7
1452374 at Lgx only 756 605 602 553 -1 25 -1 26 -1 37 Zfp322a
1452375 at Lgx only 1059 1280 1336 1441 1 21 1 26 1 36 Aldh4a1
1452395 at Res & Lgx 550 421 386 295 -1 31 -1 42 -1 87 Med19
1452398 at Lgx only 1052 998 837 640 -1 05 -1 26 -1 64 Plcei
1452401 at Lgx only 1940 1839 1632 1573 -1 05 -1 19 -1 23 Wtap
1452411 at Lgx only 261 364 308 474 1 40 1 18 1 82 Lire1
1452432 at Lgx only 2342 2445 2339 1802 1 04 -1 00 -1 30 Tfpi
1452446 a at Lgx only 1260 1260 1436 1569 1 00 1 14 1 25 Tmub2
1452462 a at Lgx only 342 469 538 591 1 37 1 58 1 73 Banp
1452469 a at Res & Lgx 1833 2687 2690 3184 1 47 1 47 1 74 Smtn
1452472 at Lgx only 128 142 189 242 1 11 1 47 1 89 Rtp3
1452499 a at Res only 705 659 530 573 -1 07 -1 33 -1 23 Kιf2a
ENSMUSG-
1452502 at Lgx only 190 121 131 88 -1 56 -1 45 -2 1500000050599
1452509 at CR only 114 33 80 89 -344 -1 44 -1 29 Usp9y
1452587 at Lgx only 3962 3990 3352 3268 1 01 -1 18 -1 21 Actr2
1452596 at Lgx only 4654 3854 3014 2105 -1 21 -1 54 -221 Polr2k
1452601 a at Lgx only 978 969 1221 1242 -1 01 1 25 1 27 Acbd6
1452607 at Lgx only 978 917 707 587 -1 07 -1 38 -1 67 2610030H06Rιk
Figure imgf000104_0001
Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1453058 at Lgx only 174 168 136 55 -1 04 -1 28 -3 18 Wdr5b
1453059 at Lgx only 8551 8336 7741 7324 -1 03 -1 10 -1 17 2310046A06Rιk
1453062 at CR only 223 400 295 433 1 80 1 33 1 95 A930026l22Rιk
1453097 a at Lgx only 1577 1678 1749 2148 1 06 1 1 1 1 36 Ubtf
14531 19 at Res & Lgx 559 481 365 290 -1 16 -1 53 -1 92 Otudi
1453129 a at Lgx only 557 617 773 837 1 11 1 39 1 50 Rgs12
1453137 at Lgx only 1023 1022 916 767 -1 00 -1 12 -1 33 Fbxo30
1453149 at Lgx only 290 191 219 11 1 -1 52 -1 33 -2 61 Slc25a32
1453154 at CR only 79 24 84 44 -3 27 1 06 -1 78 1700029M20Rιk
1453155 at Lgx only 2675 2692 2995 3076 1 01 1 12 1 15 Tmem50a
1453160 at Lgx only 738 596 734 458 -1 24 -1 01 -1 61 Med 13
1453180 at Res only 782 784 635 639 1 00 -1 23 -1 23 6530404N21 Rιk
1453187 at All 605 410 365 248 -1 48 -1 66 -2 44 Ocιad2
1453191 at Lgx only 374 503 391 738 1 34 1 04 1 97 Col27a1
1453196 a at Res & Lgx 254 340 389 41 1 1 34 1 53 1 62 Oasl2
1453206 at Res & Lgx 890 1 145 1255 1633 1 29 1 41 1 84 Acad9
1453212 at Lgx only 319 237 226 141 -1 34 -1 41 -2 27 Zfp383
1453224 at Res & Lgx 809 940 1461 1631 1 16 1 81 2 02 Zfand5
1453257 at Lgx only 853 753 660 637 -1 13 -1 29 -1 34 Agpat5
1453271 at Res & Lgx 1296 1098 939 900 -1 18 -1 38 -1 44 Phf14
1453296 at CR only 1 13 32 90 134 -3 51 -1 25 1 19 Tmem103
1453312 at Lgx only 790 818 632 503 1 04 -1 25 -1 57 Iqwdi
1453377 at Res & Lgx 606 603 406 399 -1 00 -1 49 -1 52 Sh2d4a
1453391 at CR only 28 75 84 70 2 64 2 97 2 47 Speer7-ps1
1453399 at Res & Lgx 607 726 1159 1232 1 20 1 91 2 03 Ccnt2
1453412 a at CR only 2089 271 1 2591 2801 1 30 1 24 1 34 Sec14l1
1453486 a at Lgx only 1455 1240 1142 1034 -1 17 -1 27 -1 41 Scube2
14534g4 at Res only 98 79 44 66 -1 23 -2 21 -1 49 4921513H07Rιk
1453502 at Lgx only 77 79 94 27 1 02 1 22 -2 82 2210408121 Rik
1453552 at CR & Lgx 1567 1069 1454 980 -1 47 -1 08 -1 60 2310014F07Rιk
1453572 a at Lgx only 2411 2727 2793 3149 1 13 1 16 1 31 Plp2
1453592 at Lgx only 3846 3055 3329 2431 -1 26 -1 16 -1 58 Lrrc39
1453673 at Lgx only 76 65 94 172 -1 18 1 24 2 26 LOC100046982
1453728 a at Lgx only 3261 3568 2905 2627 1 09 -1 12 -1 24 Mrps17
1453729 a at Lgx only 41216 43763 46983 55549 1 06 1 14 1 35 Rpl37
1453731 a at Lgx only 3325 2887 2681 2049 -1 15 -1 24 -1 62 Tmem77
1453739 at Res & Lgx 450 425 316 266 -1 06 -1 42 -1 69 Tmem126b
1453740 a at Lgx only 2063 231 1 2547 2926 1 12 1 24 1 42 Ccnl2
1453761 at Res only 74 144 179 199 1 95 243 2 71 Phf6
1453795 at Lgx only 1896 2038 2175 2434 1 08 1 15 1 28 Fahd2a
1453804 a at Lgx only 804 758 637 604 -1 06 -1 26 -1 33 Orc4l
1453821 at Res only 642 596 1039 923 -1 08 1 62 1 44 N6amt1
1453850 at Lgx only 43 35 37 131 -1 24 -1 19 3 01 1500002101 Rik
1453851 a at Lgx only 352 496 457 640 1 41 1 30 1 82 Gadd45g
1453865 a at Res & Lgx 869 969 1199 1 124 1 12 1 38 1 29 Otud5
1453866 a at Lgx only 43 74 75 126 1 71 1 73 2 91 Xk
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res 4X Entrez Info
1455482 at All 516 724 797 1018 1 40 1 54 1 97 Ap2a2
1455491 at Res & Lgx 1099 1208 811 846 1 10 -1 36 -1 30 Hnrph3
1455506 at Lgx only 5998 5890 8637 9404 -1 02 1 44 1 57 Slc25a34
1455508 at Lgx only 354 367 312 217 1 04 -1 14 -1 63 A530082C1 1 Rιk
1455538 at Lgx only 942 726 609 472 -1 30 -1 55 -2 00 6330403M23Rιk
1455585 at Lgx only 601 526 486 404 -1 14 -1 24 -1 49 Rnf168
1455587 at Res only 11 16 20 21 1 39 1 75 1 82 BC030183
1455588 at Res & Lgx 1065 816 578 556 -1 30 -1 84 -1 91 Lyrm4
1455655 a at Res & Lgx 1506 11 15 925 831 -1 35 -1 63 -1 81 Tardbp
1455688 at Lgx only 951 817 745 702 -1 16 -1 28 -1 35 Ddr2
1455689 at Lgx only 270 358 391 488 1 33 1 45 1 81 Fzd10
1455700 at Res & Lgx 1328 1 183 811 664 -1 12 -1 64 -2 00 Mterfd3
1455702 at Lgx only 584 734 962 803 1 26 1 65 1 37 Wdr22
1455733 at Res & Lgx 135 216 228 227 1 60 1 69 1 68 Taok3
1455734 at CR & Lgx 1289 881 11 18 921 -1 46 -1 15 -1 40 Crbn
1455741 a at Res & Lgx 1206 1346 1718 2034 1 12 1 42 1 69 Ece1
1455750 at Res & Lgx 1072 1041 733 717 -1 03 -1 46 -1 50 A230067G21 Rιk
1455757 at Res & Lgx 522 526 371 305 1 01 -1 41 -1 71 D3Ertd254e
1455794 at Lgx only 566 625 809 1034 1 10 1 43 1 83 Smtnl2
1455832 a at Res only 420 352 325 287 -1 19 -1 29 -1 46 Umps
1455854 a at Res & Lgx 470 513 771 829 1 09 1 64 1 76 Ssh1
1455870 at Lgx only 2176 2652 2908 3503 1 22 1 34 1 61 Akap2
1455873 a at Lgx only 271 383 402 483 1 41 1 48 1 78 Vps18
1455884 at Lgx only 508 747 821 851 1 47 1 62 1 67 Dpp9
1455914 at Lgx only 168 138 101 93 -1 22 -1 66 -1 81 AI987944
1455915 at CR only 191 132 150 138 -1 44 -1 27 -1 38 Galnt4
1455922 at Lgx only 1213 1256 1440 1560 1 04 1 19 1 29 Rab3gap1
1455936 a at CR & Lgx 1540 2056 1827 2200 1 33 1 19 1 43 Rbpms
1455944 at Lgx only 153 127 219 289 -1 21 1 43 1 88 Zfp516
1455945 at Lgx only 192 166 136 117 -1 16 -1 41 -1 65 Zfp817
1456046 at Lgx only 3587 3017 2786 2266 -1 19 -1 29 -1 58 Cd93
1456058 at Lgx only 807 745 690 598 -1 08 -1 17 -1 35 Rbm27
1456059 at Lgx only 4664 4475 3594 3214 -1 04 -1 30 -1 45 Psmd11
1456061 at Res & Lgx 740 574 371 258 -1 29 -1 99 -2 87 Gimapδ
1456065 at CR only 128 49 171 119 -2 60 1 33 -1 07 U bash 3a
1456092 at Lgx only 86 52 58 26 -1 67 -1 49 -3 35 Kctd7
1456099 at Res only 128 174 254 207 1 35 1 98 1 61 D930017J03Rιk
1456161 at Lgx only 403 283 297 279 -1 42 -1 36 -1 44 0610040B10Rιk
1456169 at Res only 128 171 178 144 1 34 1 39 1 13 EG226654
1456210 at Lgx only 150 178 101 68 1 18 -1 48 -2 21 5430407P10Rιk
1456241 a at Lgx only 3667 3705 4635 4984 1 01 1 26 1 36 1810073N04Rιk
1456257 at Res only 495 565 327 409 1 14 -1 51 -1 21 C130065N10Rιk
1456315 a at Res & Lgx 5639 5549 3253 2716 -1 02 -1 73 -2 08 Ptpla
1456398 at Lgx only 1951 1675 1544 1204 -1 16 -1 26 -1 62 Tug1
1456487 at Lgx only 270 438 425 592 1 62 1 57 2 19 Adcyi
1456599 at CR only 106 165 1 18 109 1 55 1 1 1 1 03 Nxt2
Figure imgf000110_0001
Table 3
CO CR RES LGX FC FC FC
Probe Set ID Treatment mean mean mean mean CR Res Lcjx Entrez Info
1459363 at Lgx only 312 405 407 506 1 30 1 31 1 62 Atxn2
1459578 at Res only 54 61 155 88 1 12 2 85 1 62 AA407175
1460033 at CR only 74 180 125 105 2 42 1 69 1 42 C030002C1 1 Rιk
1460053 at CR only 242 144 218 221 -1 68 -1 1 1 -1 10 Smyd4
14601 13 at CR only 104 187 144 134 1 80 1 38 1 29 B930093H17Rιk
1460165 at Res & Lgx 9064 9043 7129 6522 -1 00 -1 27 -1 39 Pppi ca
1460167 at Lgx only 988 1 120 1063 1253 1 13 1 08 1 27 Aldh7a1
1460169 a at Lgx only 2453 3136 3555 4351 1 28 1 45 1 77 Pctki
1460177 at Res & Lgx 1301 1575 1640 1671 1 21 1 26 1 28 Cndp2
1460184 at Lgx only 36182 38955 42606 49147 1 08 1 18 1 36 Hadh
1460189 at Res & Lgx 4600 3988 3323 2755 -1 15 -1 38 -1 67 Wdr23
1460194 at Lgx only 12145 13386 14000 14613 1 10 1 15 1 20 Phyh
1460196 at Lgx only 1079 1068 1158 1385 -1 01 1 07 1 28 Cbr1
1460210 at Lgx only 683 848 775 1026 1 24 1 13 1 50 Pkd1
1460214 at CR only 27 97 92 108 3 67 3 46 4 07 Pcp4
1460216 at Lgx only 5275 5712 6310 7377 1 08 1 20 1 40 Acad s
1460230 at Lgx only 106 114 120 192 1 07 1 13 1 81 Syn2
1460239 at Lgx only 3477 3308 3484 3037 -1 05 1 00 -1 15 Tspan13
1460251 at Lgx only 464 467 407 262 1 01 -1 14 -1 77 Fas
1460254 at Res & Lgx 1563 1408 1168 924 -1 11 -1 34 -1 69 1810049H13Rιk
1460271 at CR only 157 66 1 18 125 -2 38 -1 33 -1 26 Trem3
1460276 a at Lgx only 822 886 967 1097 1 08 1 18 1 33 Gpr175
1460321 at CR only 129 52 1 17 105 -248 -1 10 -1 23 Cntn4
1460326 at Res & Lgx 1395 1325 1867 1690 -1 05 1 34 1 21 Pιk3ca
1460328 at Res & Lgx 619 628 880 862 1 01 1 42 1 39 Brd3
1460329 at Res only 713 637 458 504 -1 12 -1 56 -1 42 LOC675709
1460330 at Lgx only 1601 1521 1413 1219 -1 05 -1 13 -1 31 Anxa3
1460331 at Res & Lgx 5861 5156 4527 4030 -1 14 -1 29 -1 45 Tm9sf2
1460336 at Lgx only 2689 2686 4293 5218 -1 00 1 60 1 94 Ppargd a
1460337 at CR only 4383 5948 3663 4114 1 36 -1 20 -1 07 Sh3kbp1
1460344 at Lgx only 1002 1085 1313 1398 1 08 1 31 1 40 2310033F14Rιk
1460396 at Lgx only 483 621 790 817 1 29 1 63 1 69 Ddx54
1460409 at Lgx only 1345 1292 1713 2001 -1 04 1 27 1 49 Cpti a
1460412 at CR only 58 20 45 57 -2 84 -1 30 -1 03 1600015H20Rιk
1460420 a at Lgx only 634 820 934 1 142 1 29 1 47 1 80 Egfr
1460428 at Lgx only 919 958 1120 1 123 1 04 1 22 1 22 Ankrd13a
1460432 a at Lgx only 12140 9937 10609 8730 -1 22 -1 14 -1 39 Eιf3e
1460433 at Lgx only 662 763 813 1034 1 15 1 23 1 56 Entpd6
1460435 at Lgx only 573 713 699 935 1 25 1 22 1 63 1500002O20Rιk
1460444 at Lgx only 329 323 411 491 -1 02 1 25 1 49 Arrbi
1460500 at Lgx only 313 176 158 98 -1 78 -1 98 -3 20 5033421 C21 Rιk
1460510 a at Res & Lgx 3505 3184 2702 2908 -1 10 -1 30 -1 21 CoqiOb
1460539 at Res only 68 139 141 153 2 04 2 07 2 25 4933404K13Rιk
1460547 a at Res & Lgx 4912 3450 2670 1733 -1 42 -1 84 -2 84 Hnrpk
1460552 at Lgx only 2039 2109 2286 2886 1 03 1 12 1 42 Ascc3l1
1460557 at Lgx only 1577 1866 1401 1297 1 18 -1 13 -1 22 Supv3l1
Figure imgf000112_0001
[00114] Several genes of particular interest showed expression patterns indicating that compounds of the present invention (Longevinex®) up-regulated survival/longevity genes or down-regulate genes whose expression enhances cellular damage to a greater extent than resveratrol:
(A) The sirtuin family of genes, and in particular Sirtuin 1, are thought to be critical mediators of extended lifespans (Boily, G. et al. (2008) "SirTl Regulates Energy Metabolism And Response To Caloric Restriction In Mice," PLoS ONE 3(3):el759; Huang, J. et al. (2008) "SIRTl Overexpression Antagonizes Cellular Senescence with Activated ERK/S6M Signaling in Human Diploid Fibroblasts " PLoS ONE 3(3):el710). Whereas mice receiving resveratrol showed only a 1.22 fold decrease in expression and mice subjected to a calorie restricted diet showed only a 1.12 fold reduction in Sirtuin 1 expression, expression of Sirtuin 1 was found to be decreased 1.71 fold in mice receiving Longevinex®.
(B) Pgc- lα (peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; ppargcla) is a transcriptional co-factor that controls energy metabolism and mitochondrial biogenesis; its expression is increased in skeletal muscle tissue upon long-term calorie restriction (Conley, K.E. et al. (2007) "Mitochondrial Dysfunction and Age," Curr. Opin. Clin. Nutr. Metab. Care. 10(6):688-692; Wu, Z. et al. (2007) "Targeting PGC-I Alpha To Control Energy Homeostasis," Expert Opin. Ther. Targets 11(10): 1329-1338). Whereas mice receiving resveratrol showed only a 1.6 fold increase in expression and mice subjected to a calorie restricted diet showed no increase in Pgc-lα expression, mice receiving Longevinex® showed a 1.94 fold increase in Pgc- 1 α expression.
(C) Uncoupling protein-3 is believed to be a target of Pgc-lα and to play a role in fatty acid metabolism; its expression is increased in cardiac tissue upon long-term calorie restriction (Bezaire, V. et al. (Epub 2007 Jan 3) "Uncoupling Protein-3: Clues 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(3):271-283). Whereas mice receiving resveratrol showed only a 2.02 fold increase in expression and mice subjected to a calorie restricted diet showed only a 1.8 fold increase in uncoupling protein-3 expression, mice receiving Longevinex® showed a 2.79 fold increase in uncoupling protein-3 expression.
(D) Pyruvate dehydrogenase kinase 4 coordinates fuel selection during fasting to promote fatty acid metabolism (Sugden, M.C. et al. (2006) "Mechanisms Underlying Regulation Of The Expression And Activities Of The Mammalian Pyruvate Dehydrogenase Kinases," Arch. Physiol. Biochem. 112(3): 139-149; Pilegaard, H. et al. (2004) " 'Transcriptional Regulation Of Pyruvate Dehydrogenase Kinase 4 In Skeletal Muscle During And After Exercise," Proc. Nutr. Soc. 63(2):221-226; Sugden, M.C. (2003) "PDK4: A factor in fatness?," Obes. Res. 11 (2): 167- 169). It is a target of Pgc-lα and is induced in multiple tissues by long-term calorie restriction. Whereas mice receiving resveratrol showed only a 2.78 fold increase in expression and mice subjected to a calorie restricted diet showed only a 1.48 fold increase in pyruvate dehydrogenase kinase 4 expression, mice receiving Longevinex® showed a 3.25 fold increase in pyruvate dehydrogenase kinase 4 expression.
[00115] Analysis of the genes up-regulated or down-regulated by a compound of the present invention (Longevinex®) revealed that oxidative phosphorylation genes, which are involved in mitochondrial ATP production, were markedly up-regulated (Table 4).
Figure imgf000114_0001
Example 4 Biochemical Pathways Affected by the Compositions of the Present Invention
[00116] Recent research has suggested that complex traits are emergent properties of molecular networks that are modulated by complex genetic loci and environmental factors. Chen, Y. et al. (Epub 2008 Mar 16) "Variations In DNA Elucidate Molecular Networks That Cause Disease," Nature 452(7186):429-435). [00117] Indeed, research within the last decade has revealed that most chronic illnesses such as cancer, cardiovascular and pulmonary diseases, neurological diseases, diabetes, and autoimmune diseases exhibit dysregulation of multiple cell signaling pathways (Harikumar, K.B. et al. (Epub February 15, 2008) "Resveratrol: A Multitargeted Agent For Age-Associated Chronic Diseases," Cell Cycle. 2008:7(8)). The compounds of the present invention were therefore evaluated for their effect on the expression of biochemical pathways and were found to affect the expression of genes involved in 220 biological processes (P < 0.05) by (Table 5).
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
[00118] Calorie restriction affected genes associated with 5% of these processes, administration of resveratrol affected genes associated with 10% of these processes. Compounds of the present invention (e.g., Longevinex®) were found to affect 85% of these processes. Administration of resveratrol to calorie restricted mice failed to affect any genes 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 the modulation of the genes of the oxidative phosphorylation pathway (GO:0006119) caused by calorie restriction (CR), resveratrol alone (Res), or the compositions of the present invention (LGX).
Figure imgf000120_0002
Figure imgf000121_0001
[00119] Table 7 shows the modulation of the genes of the glucose metabolism pathway (GO: 0006006) caused by calorie restriction (CR), resveratrol alone (Res), or the compositions of the present invention (LGX).
Figure imgf000121_0002
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
[00120] Table 8 shows the modulation of the genes of the tricarboxylic acid metabolism pathway (GO:0006099) caused by calorie restriction (CR), resveratrol alone (Res), or the compositions of the present invention (LGX).
Figure imgf000124_0002
[00121] Table 9 shows the modulation of the genes of the fatty acid metabolism pathway (GO:0006631) caused by calorie restriction (CR), resveratrol alone (Res), or the compositions of the present invention (LGX).
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
[00122] A study of the expression of 20,341 genes in cardiac tissue revealed that 2,829 genes exhibited statistically significant differences in expression (P<0.01). Of these, 7% (approximately 189 genes) exhibited altered expression in animals subjected only to calorie reduced diets; 8% (approximately 226 genes) exhibited altered expression in animals receiving only resveratrol; no additional genes exhibited altered expression in animals that received resveratrol and which were subjected to calorie reduced diets. In contrast, 61% of the 20,341 genes (approximately 1,729 genes) exhibited altered expression in animals receiving only compounds of the present invention (e.g., Longevinex®); an additional 2% of the genes (approximately 56 genes) exhibited altered expression in animals that had received compounds of the present invention (e.g., Longevinex®) and which had been subjected to calorie reduced diets; an additional 21% of the genes (approximately 594 genes) exhibited altered expression in animals that had received compounds of the present invention (e.g., Longevinex®) and resveratrol; an additional 1% of the genes (approximately 28 genes) exhibited altered expression in animals that had received compounds of the present invention (e.g., Longevinex®), resveratrol and which had been subjected to calorie reduced diets.
[00123] The above data demonstrates that compounds of the present invention (e.g., Longevinex®) were effective in modulating gene expression in hear tissue to an extent surpassing even that of calorie restriction. Similar effects have been observed in non-heart tissue. A study of the expression of 20,341 genes in brain tissue revealed that 3,572 genes exhibited statistically significant differences in expression (P<0.01). Of these, 124 genes exhibited altered expression in animals subjected only to calorie reduced diets; 424 genes exhibited altered expression in animals receiving only resveratrol; 10 genes exhibited altered expression in animals that received resveratrol and which were subjected to calorie reduced diets. In contrast, 2,560 genes exhibited altered expression in animals receiving only compounds of the present invention (e.g., Longevinex®); 19 additional genes exhibited altered expression in animals that had received compounds of the present invention (e.g., Longevinex®) and which had been subjected to calorie reduced diets; 430 additional genes exhibited altered expression in animals that had received compounds of the present invention (e.g., Longevinex®) and resveratrol; 5 additional genes exhibited altered expression in animals that had received compounds of the present invention (e.g., Longevinex®), resveratrol and which had been subjected to calorie reduced diets.
Example 4 Model Mechanism of Action of the Compositions of the Present Invention
[00124] The compounds of the present invention were thus found to greatly exceed the modulation of gene expression observed upon calorie restriction and to alter the expression of genes in key patyhways of lipid metabolism, glucose metabolism, oxidative phosphorylation, the Kreb's cycle, ATP synthesis and fatty acid β oxidation. In summary, the compounds of the present invention were found to have a greater specific activity than resveratrol alone, both in terms of the number of genes and the number of different biochemical pathways affected. The results are significant since calorie restriction (CR) is considered the unequivocal method of prolonging life in all forms of life. Generally, reduction of 50% of caloric intake doubles the lifespan of any organism. The above-described experiments demonstrate that the compositions of the present invention exert a more powerful influence over genome expression than resveratrol or CR, and marks the first time any technology has been shown to exceed the effects of CR. Furthermore, the compositions of the present invention were found to influence genome expression at an earlier stage of life than CR (which requires a life-long adherence to a CR diet to differentiate genes).
[00125] Without intending to be bound by any mechanism of action, the above results suggest that the compounds of the present invention act by enhancing the activity of the forkhead Foxol (daf-16, dFoxO) transcription factor (Figure 5). Studies in model organisms has shown that Foxol mediates lifespan expression by enhancing gene expression. Insulin/IGF- 1 signaling phosphorylates Foxol, thereby causing it to be excluded from the nucleus and downregulating its actions. The compounds of the present invention decrease insulin and IGF-I signaling thereby decreasing Foxol phosphorylation. Consistent with this model are the observations that the insulin receptor signaling pathway (e.g., GO:008286; genes Ide, Igfbp4, and Igfbpό) is affected by the compounds of the present invention. Expression of Foxol is increased by 1.75 fold. The compounds of the present invention mediate decreased glycolysis and increased gluconeogenesis (e.g., GO:0006006), enhanced Pgc-lα expression (thereby leading to stimulation of Pdk4 expression (e.g., a 1.94 fold increase in Ppargclα and a 3.25 fold increase in Pdk4), increased expression of lipid metabolism genes (e.g., a 2.79 fold increase in Ucp3, 1.49 fold increase in Cptla, and a 1.45 fold increase in Cptlb). Lipid and fatty acid metabolism genes GO:0006629 and GO:0006635 are uniquely affected by the compounds of the present invention. The compounds of the present invention thus exert a more pronounced favorable effect on key processes affected by calorie restriction and resveratrol (e.g. , chromatin remodeling, transcription from RNA polymerase II promoter, and the ubiquitin cycle. Genes GO:0006333 and GO:0006367 are uniquely affected by the compounds of the present invention; Gene GO:0006512 is affected by resveratrol and Longevinex®. Thus, in sum, a proposed mechanism of action is that the compositions of the present invention deliver resveratrol to cells, where it passes through cell walls, enters the cytoplasm, and facilitates the translocation of Foxol gene into the cell nucleus, which produces the longevity effects.
[00126] All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

Claims

What Is Claimed Is:
Claim 1. A resveratrol-containing composition that, upon administration to a recipient, modulates the concentration or activity, relative to resveratrol alone or calorie restriction, of the product of a survival/longevity gene or the product of a gene whose expression enhances cellular damage.
Claim 2. The resveratrol-containing composition of claim 1, wherein said modulation alters:
(A) oxidative phosphorylation;
(B) actin filament length or polymerization;
(C) intracellular transport;
(D) organelle biogenesis;
(E) insulin signaling;
(F) glycolysis;
(G) gluconeogenesis; or (H) fatty acid metabolism in said recipient.
Claim 3. The resveratrol-containing composition of any of claims 1 -2, wherein said survival/longevity gene product is Sirtuin 1 or the forkhead Foxo 1 transcription factor.
Claim 4. The resveratrol-containing composition of any of claims 1-3, wherein said gene whose expression enhances cellular damage encodes uncoupling protein 3 or pyruvate dehydrogenase kinase 4.
Claim 5. The resveratrol-containing composition of any of claims 1-4, wherein said composition comprises:
(a) trans-resveratrol; and
(b) a metal chelating agent; wherein said ?ra«,y-resveratrol is encapsulated to thereby substantially preserve the ability of said composition to modulate the concentration or activity of said product of said survival/longevity gene or said product of said gene whose expression enhances cellular damage, from loss due to exposure of said trαns- resveratrol to light or oxygen.
Claim 6. The resveratrol-containing composition of claim 5, wherein said metal chelating agent is nordihydroguaiaretic acid.
Claim 7. The resveratrol-containing composition of claim 5, wherein said metal chelating agent is phytic acid.
Claim 8. The resveratrol-containing composition of any of claims 5-7, wherein said composition additionally comprises quercetin.
Claim 9. The resveratrol-containing composition of any of claims 5-8, wherein said composition additionally comprises hyaluronic acid.
Claim 10. The resveratrol-containing composition of any of claims 5-9, wherein said composition additionally comprises vitamin D.
Claim 11. The resveratrol-containing composition of any of claims 5-10, wherein said encapsulation of resveratrol is a microencapsulation.
Claim 12. The use of a resveratrol-containing composition for the manufacture of a medicament for ameliorating a symptom associated with an existing disease of an individual or for preventing onset of said symptom in an individual prior to the occurrence of said disease in said individual, wherein said resveratrol-containing composition modulates the concentration or activity, relative to resveratrol alone or calorie restriction, of the product of a survival/longevity gene or the product of a gene whose expression enhances cellular damage, and wherein said disease is selected from the group consisting of: cardiovascular disease, cancer, macular degeneration, a disease associated with aging, and inflammation.
Claim 13. The use of the resveratrol-containing composition of claim 12, wherein said modulation alters:
(A) oxidative phosphorylation;
(B) actin filament length or polymerization;
(C) intracellular transport;
(D) organelle biogenesis;
(E) insulin signaling;
(F) glycolysis;
(G) gluconeogenesis; or (H) fatty acid metabolism in said individual.
Claim 14. The use of the resveratrol-containing composition of any of claims 12-13, wherein said survival/longevity gene product is Sirtuin 1 or the forkhead Foxo 1 transcription factor.
Claim 15. The use of the resveratrol-containing composition of any of claims 12-14, wherein said gene whose expression enhances cellular damage encodes uncoupling protein 3 or pyruvate dehydrogenase kinase 4.
Claim 16. The use of the resveratrol-containing composition of any of claims 12-15, wherein said composition comprises:
(a) trans-resveratrol; and
(b) a metal chelating agent; wherein said ?rα«,y-resveratrol is encapsulated to thereby substantially preserve the ability of said composition to modulate the concentration or activity of said product of said survival/longevity gene or said product of said gene whose expression enhances cellular damage, from loss due to exposure of said trαns- resveratrol to light or oxygen.
Claim 17. The use of the resveratrol-containing composition of any of claims 12-16, wherein said disease is cancer.
Claim 18. The use of the resveratrol-containing composition of any of claims 12-16, wherein said disease is a disease associated with aging.
Claim 19. The use of the resveratrol-containing composition of claim 18, wherein said disease associated with aging is a neurodegenerative disease.
Claim 20. The use of the resveratrol-containing composition of any of claims 12-19, wherein said composition additionally comprises quercetin, hyaluronic acid and/or vitamin D.
PCT/US2008/076707 2007-09-20 2008-09-17 Resveratrol-containing compositions for modulating gene product concentration or activity WO2009039195A1 (en)

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