US 20140079836A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0079836A1 McDaniel (43) Pub. Date: Mar. 20, 2014
(54) METHODS AND COMPOSITIONS FOR (52) U.S. Cl. ALTERING HEALTH, WELLBEING AND CPC ...... A61K 36/74 (2013.01); A61 K3I/122 LIFESPAN (2013.01) USPC ...... 424/777; 514/690: 435/375; 506/16; (71) Applicant: LifeSpan Extension, LLC, Virginia 435/6.12 Beach, VA (US) (72) Inventor: David H. McDaniel, Virginia Beach, VA (57) ABSTRACT (US) Described herein are the results of comprehensive genetic (73) Assignee: LifeSpan Extension, LLC expression and other molecular analysis p the effect s anti oxidants on biological systems, including specifically differ (21) Appl. No.: 14/084,553 ent human cells. Based on these analyses, methods and com (22) Filed: Nov. 19, 2013 positions are described for modifying or influencing the lifespan of cells, tissues, organs, and organisms. In various Related U.S. Application Data embodiments, there are provided methods for modulating the activity of the gene maintenance process in order to influence (60) Continuation of application No. 13/898.307, filed on the length and/or structural integrity of the telomere in living May 20, 2013, which is a division of application No. cells, as well as methods for modulating the rate/efficiency of 12/629,040, filed on Dec. 1, 2009, now abandoned. the cellular respiration provided by the mitochondria, mito (60) Provisional application No. 61/118,945, filed on Dec. chondrial biogenesis, and maintenance of the mitochondrial 1, 2008. membrane potential. Exemplary lifespan altering compounds include natural and synthetic antioxidants, such as plant anti Publication Classification oxidant and polyphenol compounds derived from coffee cherry, tea, berry, and so forth, including but not limited to (51) Int. Cl. caffeic acid, chlorogenic acid, ferulic acid, quinic acid, proan A6 IK36/74 (2006.01) thocyanidins, ubiquinone, idebenone, or a synthetic form or A6 IK3I/122 (2006.01) derivatives thereof. Patent Application Publication Mar. 20, 2014 Sheet 1 of 23 US 2014/0079836A1
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Figure 9 Expression of VEGFA in human fibroblast 24 hours after exposure to coffee cherry 1.5
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Figure 10 Expression of HMOX1 in human fibroblast 24 hours after exposure to coffee cherry
Concentration of CoffeeCherry O.01% OOOO% OOOOOO1% Patent Application Publication Mar. 20, 2014 Sheet 10 of 23 US 2014/0079836A1
Figure 11 Expression of CCL41 in human skin fibroblasts 24 hours after exposure to chlorogenic acid or coffee cherry
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Figure 13 Expression of NOS2A in human skin fibroblasts 24 hours after exposure to chlorogenic acid or coffee cherry
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Figure 15 Expression of TERT in human skin fibroblasts 24 hours 5 after exposure to chlorogenic acid or coffee cherry
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Figure 17 Expression of F144 in human skin fibroblasts 24 hours after exposure to chlorogenicacid or coffee cherry
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Figure 19(a) Relative expression of select genes in human skin fibroblasts 24 hours after exposure to chlorogenic acid
& 0.000005%, Chlorogenic Acid -4 i.------&t.005% Chlorogenic Acid : 0.0005% Chlorogenic Acid Gene & 0.005%, Chlorogenic Acid
Figure 19(b) Relative expression of select genes in human skin fibroblasts 24 hours after exposureto chlorogenic acid
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Figure 19(c) Relative expression of select genes in human skin fibroblasts 24 hours after exposure to chlorogenic acid
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& 0.000001%CoffeeCherry : 0.0001%. CoffeeCherry & 0.02% CoffeeCherry Patent Application Publication Mar. 20, 2014 Sheet 16 of 23 US 2014/0079836A1
Figure 20(b) Relative expression of Select genes in human fibroblast 24 hours after exposure to coffee cherry
80.000003%CoffeeCherry 12 . .8.0.0001% coffeecherry & 0.01% CoffeeCherry Figure 20(c) Relative expression of select genes in human ... fibroblasts 24 hours after exposure to coffee cherry
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Figure 20(d) Relative expression of select genes in human fibroblasts 24 hoursafter exposure to coffee cherry
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Figure 20(f) Relative expression of select genes in human fibroblasts 24 hours after exposure to coffee cherry
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Figure 20(h) Relative expression of select genes in human fibroblasts 24 hours after exposure to coffee cherry |
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Figure 21(a) Relative expression of select genes in the mitochondrial pathyay it skin fibrobiasts 24 hours after exposure to chorogenic acid
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Patent Application Publication Mar. 20, 2014 Sheet 20 of 23 US 2014/0079836A1
Figure 21(b) &etative expression of select genes in the mitochot dria pathway in skir fibrobiasts 24 hours after exposiretty coffee cherry
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Figure 22(b) Relative expression of select genes in the DNA repair pathway in human skin fibroblasts 24 hours after exposure to chlorogenic acid
& O.OOOOO5% Chlorogenic Acid -2. 80.00005% Chlorogenic Acid : 0.0005% Chlorogenic Acid & 0.005% chlorogenic Acid Figure 23 Relative expression of select genes in the telomere maintenance pathway in humanskin fibroblasts 24 hours after exposureto coffeecherry 5.0 gr or
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Figure 24 Relative expression of PARP1-4 in human skin fibroblasts 24 hours after exposureto coffee cherry extract
& 0.000001%CoffeeCherry 0.001% CoffeeCherry & 0.01%. Coffeecherry Figure 25 Relative expression of specific genes in human skin fibroblasts 24 hours after exposure to chlorogenic acid
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- Patent Application Publication Mar. 20, 2014 Sheet 23 of 23 US 2014/0079836A1
Figure 26 Relative expression of specific genes in human skin fibroblsts 24 hours after exposure to chlorogenic acid 12.0 :------: & O.8000005% Chlorogenic Acid 3.0 : 0.00005% Chlorogenit Acid x 0.0005% Chorogenic Acid & 0.0005% Chorogenic Acid 80 ------
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METHODS AND COMPOSITIONS FOR which plays an essential role in protecting these regions, but ALTERING HEALTH, WELLBEING, AND which may also be associated with cancer. Thus the ability to LIFESPAN modulate telomerase activity provides the opportunity to alter health both positively and negatively. CROSS REFERENCE TO RELATED 0005 One way to extend the lifespan of a living cell—and APPLICATION by extension possibly the organ, tissue or entire organism is 0001. This application is a continuation of co-pending to repair damage in addition to preventing damage. The genes which control the cellular repair mechanisms, if activated or U.S. application Ser. No. 13/898.307, filed May 20, 2013, enhanced in the proper way, may effectively extend the which is a divisional of U.S. application Ser. No. 12/629,040. lifespan of a cell. This may take several forms: extending the filed Dec. 1, 2009, and which claims the benefit of the earlier lifespan of a cell which is damaged or injured by properly filing date of U.S. Provisional Application No. 61/118,945, repairing that damage and/or by causing the cell to live longer filed Dec. 1, 2008. The entire content of these previously filed or replicate itself longer than it would have occurred natu applications is incorporated herein by reference. rally. FIELD 0006 Mammalian mitochondria are organelles that pro duce more than 90% of cellular ATP under aerobic conditions 0002. Described herein are methods and compositions for through a process called oxidative phosphorylation. Mito altering mitochondrial biogenesis and/or mitochondrial chondria are also involved in fatty acid metabolism, hormone maintenance, respiratory efficiency, DNA maintenance, production, ketone body production, apoptosis, and Ca" DNA repair, gene expression, and/or gene function, for homeostasis. Mitochondria contain, interalia, the TCA cycle instance in order to (in various embodiments) increase, (also known as the Kreb cycle), enzymes involved in heme extend, or shorten the lifespan and/or retard or increase rate of biosynthesis and the electron transport chain (OXPHOS sys senescence of a cell, tissue, organ, and/or organism. In tem). Due to the large flux of redox reactions necessary to example embodiments, this involves altering the maintenance maintain oxidative phosphorylation, the organelle is the site or function of telomeres and telomere structure, maintenance of production of reactive oxygen species (ROS), which in and control, cellular responses to oxidative stress and/or oxi controlled production have a signaling function, but in over dative DNA damage, and cellular response to environmental production are toxic and are believed to be the cause of many damage or disease or immune response or genetic alteration human diseases including, for example, Parkinson's disease of cells. and other neurodegenerative conditions, diabetes, and the aging process itself. BACKGROUND 0007. The OXPHOS system is composed of five large 0003 All living cells and organisms have a finite lifespan. multi-protein enzyme complexes, which collectively trans They live for a period of time and die. Cells and organisms form the reducing energy of NADH and FADH to ATP. have both a chronological age and a biological age. The NADH ubiquinone oxidoreductase (Complex I) contains 45 former is measured in days, months or years while the latter different subunits, and Succinate ubiquinone reductase (Com may be measured by a host of complex testing of biological plex II), ubiquinone-cytochrome c oxidoreductase (Complex functions including but not limited to: gene expression, pro III), cytochrome c oxidase (Complex IV) and the ATP syn tein production or metabolic pathways. The rate of aging may thase (Complex V) have 4, 11, 13 and 16 subunits respec also be measured, and an accelerated rate of aging may be tively. Although composed of five individual enzyme com considered premature aging, while a slower rate of aging plexes (each, an “OXPHOS complex” or “OXPHOS may extend lifespan. It is desirable to maximize the healthy enzyme’”) and containing a total of approximately 89 Subunit lifespan of cells and organisms and it is also desirable to proteins (each, an “OXPHOS protein'), the OXPHOS system extend the healthy lifespan by delaying the rate of aging and has traditionally been considered to function as a single unit. the onset of dysfunctional or disease states. Shortening the This single-unit concept has been Supported with evidence of lifespan and/or acceleratingapoptosis of unhealthy, diseased, structural associations between complexes, which associa damaged, or cancerous cells may also be desirable. tions are believed to enhance overall functional efficiency 0004 Oxidative stress is one of the primary causes of cell (Chen et al., J. Biol. Chem., 279:31761-31768, 2004; Ko et and organism dysfunction or disease and also accelerated or al., J. Biol. Chem., 278:12305-12309, 2003). premature aging and death. The ability to enhance in a favor 0008 Four of the OXPHOS enzyme complexes (Com able manner the ability of cells and organisms to resist or plexes I, III, IV andV) have a dual genetic origin. That is, they repair damage due to oxidative stress produced by environ are composed of both nuclear DNA-encoded proteins and mental injury, lifestyle choices as well as diseases and medi mtDNA-encoded proteins. Thus, 7 subunits of Complex I, 1 cal therapies may extend the healthy function and/or lifespan subunit of Complex III, 3 subunits of Complex IV and 2 and/or retard aging and senescence. Antioxidants have the subunits of Complex V are encoded by mtDNA. potential not only to neutralize reactive oxygen species, but 0009 Mitochondria contain their own DNA (mtDNA) also may provide vital anti-aging benefits by affecting various which is prokaryote-like. In mammals, this DNA is a 16 kb other key cellular mechanisms. One Such example is the double-stranded circular DNA encoding 13 different telomere (and/or telomere unit and associated proteins and polypeptides, all involved in oxidative phosphorylation, structural configurations) which are special chromatin struc along with 2 rRNAs and 22 tRNAS. mtDNA lacks protective tures at the end of chromosomes. Telomeres are coated by histones and has minimal repair mechanisms, which leads to DNA binding proteins, including TRF1 and TRF2 and asso a relatively high mutation rate that is further enhanced by the ciated proteins, TIN2, TPP1, POT1, Tankyrase 1, and Rap1 proximity of the DNA to the OXPHOS system, the site of Premature or accelerated telomere shortening may produce production of ROS. Accumulation of mutations and deletions premature aging and death. Telomerase is a DNA polymerase in mtDNA occurs throughout life in humans and becomes US 2014/0079836A1 Mar. 20, 2014
physiologically relevant where they affect sufficient number lose their function or die. A cell normally has a finite lifespan of copies of the mtDNA to alter oxidative phosphorylation. determined by the number of cell divisions which are pos 0010 Unlike the nuclear genome, which is present in two sible. The Hayflick Limit theory discusses one view of copies, mtDNA is present in thousands of copies in mamma lifespan limitations. An organ may be repopulated with cells liancells, all of which are used in translation of gene products to regenerate itself from the stem cell population but the stem made within the organelle on bacterial-like ribosomes. Thus, and progenitor cells themselves have a finite lifespan. The inheritance and penetrance of mtDNA mutations is not Men ability to extend the lifespan of differentiated cells and/or delian, but rather depends on the relative amount (%) of stem and progenitor cells lies at the heart of extending wild-type and mutant mtDNA molecules per cell. The normal lifespan of an organism. state is 100% wild-type mtDNA or wild-type homoplasmy. A mutation in mtDNA can also be homoplasmic (present in all SUMMARY mtDNA molecules of a cell) in which case it is likely to have 0014 Provided herein are methods and compositions that a functional and possibly pathogenic effect. The presence of can be employed to increase telomerase activity, and/or a mixture of mutant and wild-type mtDNA molecules in an modulate the activity of other telomere maintenance genes so individual cell is referred to as heteroplasmy. Because normal as to repair, maintain or lengthen telomere structure to cells have an excess capacity of mtDNA and mtDNA-en lengthen the lifespan of healthy cells. Decreasing telomerase coded proteins, heteroplasmic mutant mtDNA are believed to activity in cancer cells, thus making cancer cells mortal and cause an altered functional (or pathogenic) phenotype if the healthy cells longer lasting if not immortal is another method mutant mtDNAS are present at levels exceeding some thresh to increase longevity. This disclosure describes methods of old value, usually 70-90%. An additional consequence of increasing or decreasing telomerase activity in healthy and heteroplasmy is the development of altered functions of mito stressed cells using antioxidant(s) that modulate gene activity chondria within a single cell, between cells and between and/or proteins which influence, regulate, and/or control tissues (Wallace, Science, 283: 1482-1488, 1999; Chinnery telomerase activity, the maintenance of the telomere unit and and Turnbull, Mol. Med. Today, 6:425-432, 2000). associated components, or telomere length. 0011 Transient ischemia (anoxia) results in the local pro 00.15 Exemplary compounds and compositions useful in duction of extremely high levels of ROS which can cause long the methods described herein include natural and synthetic term damage to mitochondria. Ironically, it is the Sudden antioxidants, such as plant antioxidant compounds derived re-supply of oxygen to the ischemic tissue during reperfusion from coffee cherry (e.g., including one or a mixture of caffeic that is believed to be the proximate cause of elevated ROS acid, chlorogenic acid, ferulic acid, quinic acid and proantho production. In the initial phase of transient ischemia, oxygen cyanidins or derivatives thereof); plant antioxidant com is scarce but tissue demands for ATP remain high, resulting in pounds derived from and plant antioxidant compounds continued functioning of the electron transport chain except derived from any of the plants listed herein. In another illus for the terminal reduction of oxygen to water by Complex IV. trative embodiment, the lifespan or health enhancing com Therefore, reduced electron acceptors “upstream” of Com pound is synthetic/bioengineered idebenone or an ester or plex IV accumulate to abnormally high levels. Upon resupply derivative thereof. In certain embodiments, if the modulating of oxygen, these excess reduced carriers react directly (inap compound is a naturally occurring compound, it may not be in propriately) with oxygen to generate highly toxic partially a form that is naturally occurring, for instance it may be a reduced oxygen species (Pitkanen and Robinson, J. Clin. synthetic form or an analog or derivative of the naturally Invest., 98:345-351, 1996; Genova et al., FEBS Lett., 505: occurring form. 364-368, 2001), which are capable of protein, lipid and DNA 0016. Importantly, embodiments of the methods and com modifying reactions. The resulting oxidative damage would positions described herein provide aspects of healthy longev be expected to occur mainly inside the mitochondrion, ity—that is, extended life span (of cells, tissues, organs, and/ because such radicals are so reactive that they are short lived or organisms) that is healthy and of high relative quality. and cannot diffuse far before finding a target for reaction. 0017 Thus, in various embodiments there are provided Accordingly, OXPHOS proteins and mtDNA are likely to be methods for modulating: the rate/efficiency of cellular respi the cellular molecules most affected by such oxidative stress. ration provided by mitochondria, the total number of mito The resulting defects in mtDNA and OXPHOS proteins may chondria per cell (mitochondrial biogenesis), and mitochon result in continued increased production of ROS, which may drial membrane potential. Also provided herein are methods also lead to a damaging positive feedback loop. for modulating the activity of the gene maintenance process, 0012. Oxidative stress is one of the primary causes of cell for instance for maintaining (or repairing) the length and/or and organism dysfunction or disease and also accelerated or structural integrity of the telomere in living cells. premature aging and death. Mitochondrial function or dys 0018. Also provided herein are methods for extending the function, biogenesis, death and regenesis also play a vital role lifespan of living cells, tissues, organs or organisms. In in the aging process. The ability to enhance in a favorable another embodiment a method of shortening the lifespan of manner the ability of cells and organisms to resist or repair diseased, unhealthy or cancerous cells is described. damage due to oxidative stress produced by environmental 0019 Presented herein are compositions and methods for injury, lifestyle choices as well as diseases and medical thera administering the life-span and/or health modulating com pies may extend the healthy function and/or lifespan and/or pound so that it contacts the living cells(s), thereby increasing retard aging and Senescence. (or in Some embodiments, decreasing) the lifespan or health 0013 The ability to extend or prolong lifespan (both of the cell, the tissue in which the cell is present, and/or the healthy and less healthy) lies in the ability to extend the organ or organism in which the cell is present. lifespan of cells, both differentiated specialized cells and also 0020. A cell may be contacted with a modulating com undifferentiated stem and progenitor cells so that cell lifespan pound alone or in combination with other modulating com is longer or so that new cells replace Senescent cells which pounds or synergistic non-modulating compounds which US 2014/0079836A1 Mar. 20, 2014
may enhance delivery to the contact cell or which may indi 0035 FIG. 12 is a graph illustrating the relative change in rectly enhance the modulating effect by altering a related expression of DDC in human fibroblasts 24 hours after expo cellular process which then facilitates the activity of the Sure to chlorogenic acid or coffee cherry. modulating compound. 0036 FIG. 13 is a graph illustrating the relative change in 0021 Embodiments described herein utilizes (conven expression of NOS2A in human fibroblasts 24 hours after tional and novel)antioxidant compounds to directly modulate exposure to chlorogenic acid or coffee cherry. the gene expression of genes/proteins and complexes vital to 0037 FIG. 14 is a graph illustrating the relative change in the maintenance of telomere length. expression of SIRT1 in human fibroblasts 24 hours after 0022. The foregoing and other objects, features, and exposure to chlorogenic acid or coffee cherry. advantages of the invention will become more apparent from 0038 FIG. 15 is a graph illustrating the relative change in the following detailed description, which proceeds with ref expression of TERT in human fibroblasts 24 hours after expo erence to the accompanying figures. Sure to chlorogenic acid or coffee cherry. 0039 FIG. 16 is a graph illustrating the relative change in BRIEF DESCRIPTION OF THE DRAWINGS expression of PTGS2 in human fibroblasts 24 hours after 0023 FIG. 1 shows a pictorial representation of the telo exposure to chlorogenic acid or coffee cherry. some/shelterin complex and telomere structure (Multani et 0040 FIG. 17 is a graph illustrating the relative change in al., J Cell Sci 120:713-721, 2007). (A) The telomere folds expression of IFI44 in human fibroblasts 24 hours after expo back onto itself to form a double-stranded t-loop and a single Sure to chlorogenic acid or coffee cherry. stranded D-loop. This complex protects telomeres at the G2 0041 FIG. 18 is a graph illustrating the relative change in phase of the cell cycle from inappropriate NHEJ- and HR expression of SIRT1, SIRT2, SIRT3, and SIRT4 in human mediated processing of telomeric DNA. The six-component fibroblasts 24 hours after exposure to different levels of coffee telosome/shelterin is shown schematically on the t-loop, with cherry. POT1 interacting with the D-loop. (B) During DNA replica 0042 FIGS. 19(a), 19(b), and 19(c) is a set of three graphs tion, the presence of WRN at the replication fork is postulated illustrating the relative change in relative expression of select to enable the replication complex to efficiently replicate telo genes (custom Array 2) in human skin fibroblasts 24 hours meric DNA. (C) The presence of WRN at telomeres may after exposure to chlorogenic acid. facilitate unwinding of the D-loop, enabling telomerase to 0043 FIG.20(a) through 200h) is a set of graphs illustrat extend telomeres. The linear 3' overhang is probably pro ing the relative change in expression of select genes (custom tected by POT1. Array 2) in human fibroblasts 24 hours after exposure to 0024 FIG. 2A-2D shows biosynthetic relationships coffee cherry. among stress-induced phenylpropanoids (derived from infor 0044 FIG. 21 is a pair of graphs illustrating the relative mation in Dixon et al., The Plant Cell 7:1085-1097, 1995). expression of genes in the mitochondrial pathway in skin 0025 FIG. 3 shows a diagrammatic representation of a fibroblasts 24 hours after exposure to (a) chlorogenic acid or coffee cherry fruit. (b) coffee cherry. 0026 FIG. 4 shows representative effects of environmen 0045 FIG. 22 is a pair of graphs illustrating the relative tal injury that lead to premature aging. expression of select genes in the DNA repair pathway in skin 0027 FIG. 5 shows a representation of various agents of fibroblasts 24 hours after exposure to (a) coffee cherry or (b) premature aging and the multiple pathologies they can gen chlorogenic acid. erate. 0046 FIG. 23 is a graph illustrating the relative expression 0028 FIG. 6 shows a pictorial representation of represen of select genes in the telomere maintenance pathway in skin tative mechanisms of action of premature aging in skin. fibroblasts 24 hours after exposure to coffee cherry. 0029 FIG. 7 is a graph showing the average expression 0047 FIG.24 is a graph illustrating the relative expression values of three separate PCR primer assays for five longevity of PARP genes in human skin fibroblasts 24 hours after expo genes (TPP1, TERF1, TERF2, TINF2, and) in cultured sure to coffee cherry. human skin fibroblasts 24 hours post exposure to the listed 0048 FIG.25 is a graph illustrating the relative expression antioxidant compounds (green tea, idebenone, or coffee of specific genes in human skin fibroblasts 24 hours after cherry extract). exposure to chlorogenic acid which demonstrate a classic bell 0030 FIG. 8 is a graph showing the change in the number shaped pattern for dose response that indicates a single direc of mitochondria in human cardiac myocytes in response to tional change and then return to baseline after a peak expres COFFEEBERRYR) treatment at 24 and 48 hours. As indi sion level. As the doses increase, the gene response either cated, five serial dilutions of COFFEEBERRYR) were used. increases or decreases until a peak expression level is 0031 FIG. 9 is a graph illustrating the relative change in reached. Beyond that dosage any increases in concentration expression of VEGFA in human fibroblasts 24 hours after of the compound gives "diminishing returns' or a lessening of exposure to coffee cherry. the effect. This effect is either an upregulation or a downregu 0032 FIG. 10 is a graph illustrating the relative change in lation, not bidirectional. expression of HMOX1 in human fibroblasts 24 hours after 0049 FIG. 26 is a graph illustrating the relative expression exposure to coffee cherry. of specific genes in human skin fibroblasts 24 hours after 0033 For FIGS. 11-17, treatment 1=0.00001% Cof exposure to chlorogenic acid which demonstrate a classic bell feecherry, 0.0000005% Chlorogenic Acid; 2=0.0001% Cof shaped pattern for dose response that begins as a negative feecherry, 0.00005% Chlorogenic Acid; and 3=0.01% Cof expression value and as the dosage increases it passes through feecherry, 0.005% Chlorogenic Acid. the Zero expression value and has an positive expression value 0034 FIG. 11 is a graph illustrating the relative change in until a threshold dose is reached and then returns to the other expression of CCL4L1 in human fibroblasts 24 hours after side of the axis similar to the starting dose. This is the first exposure to chlorogenic acid or coffee cherry. type of bi-directional dose response noted. US 2014/0079836A1 Mar. 20, 2014
0050 FIG.27 is a graph illustrating the relative expression ways and that they are characterized RAD50 and RAD51, of specific genes in human skin fibroblasts 24 hours after genes that encode proteins essential for double stranded DNA exposure to chlorogenic acid which demonstrate a classic bell break repair. Break Induced Replication (BIR) can then shaped pattern for dose response that begins as a positive lengthen the telomere by the above described processes. This expression value and as the dosage increases it passes through genomic instability, leads to breaks in the double stranded the Zero expression value and has an negative expression DNA which must be repaired (this repair mechanism has been value until a threshold dose is reached and then returns to the shown to be inhibited by KU70). Inhumancells these specific other side of the axis similar to the starting dose. This is the genetic requirements are not known, however numerous stud second type of bi-directional dose response noted. ies demonstrated that human chromosome termini are subject to enhanced levels of recombination, as demonstrated in the DETAILED DESCRIPTION yeast ALT pathway studies. A study in human cells demon 0051 Telomeres are structures at the end of chromosomes strated that the action of telomerase can effectively inhibit the that undergo shortening with cell division; they are considera alternate recombination pathway by maintaining genomic biological clock of sorts for how many cycles of cell replica stability. Cells relying on the recombination based pathway tion may occur (FIG.1). They are protective structures similar for telomere maintenance were forced to express telomerase. to the plastic cap on the end of shoelaces which prevent them These cells never demonstrated the shortened telomeres from unraveling. With each cell division these telomere struc required for initiation of recombination, due to the expressed tures shorten, and this shortening accompanies aging. Even telomerase preventing such an occurrence. Other alternate tually after the telomere shortens to a certain level the cell can pathways or mechanisms may also exist. no longer divide, its metabolism slows down, it ages and 0057. A recent study has also demonstrated that over eventually dies. expression of TERT, the catalytic subunit of telomerase pro 0052. After birth, telomerase activity is diminished; but in tects fibroblasts against oxidative stress. When the cell is embryonic stem and progenitor cells, telomerase is activated under oxidative stress, as Supposed in the free radical theory and maintainstelomere length and cellular immortality. How of aging, the mitochondrial membrane loses potential, and ever, the level of telomerase activity is low or absent in the mtDNA is damaged as the ion levels increase. TERT func majority of stem and progenitor cells regardless of their pro tions primarily to maintain the length of the telomere, but in liferative capacity. cells under chronic oxidative stress, cells overexpressing 0053 Thus, even in stem and progenitor cells, except for TERT lose telomere length at only a slightly lesser rate than embryonal stem and progenitor cells and cancer stem and similarly stressed, non expressing cells. It has also been dem progenitor cells, telomere shortening occurs during replica onstrated in the same study that TERT is (reversibly) released tive ageing, possibly at a slower rate than that in normal from the nucleus in a dose/time dependant fashion, where it somatic cells. This telomere limit prevents cell survival after co localizes with the mitochondria. In these TERT overex extensive proliferation and thereby inhibits malignant trans pressing cells, mtDNA is protected, mitochondrial membrane formation or survival, but in combination with certain other potentials are higher, and concentrations of free radicals are gene expression changes (such as deficient expression of the lower which indicates better mitochondrial function/viability p53 tumor Suppressor) then it may facilitate tumor formation and decreased damage. or expansion. 0058. The activity of the enzyme telomerase is the best 0054 Telomere shortening not only accompanies normal understood mechanism for maintaining the length of the aging, but dysfunction of the telomere unit is associated with telomere unit or structure. Modulating the activity of telom Some premature aging syndromes and various diseases erase is one method for extending the lifespan of living cells. including aplastic anemia and many other diseases. While normally repressed in human somatic cells it may be 0055 Telomerase is a reverse transcriptase repair enzyme activated by certain repair mechanisms, certain agents and which can replace lost telomere DNA structure. Typically the also in tumor progression or transformation. activity of telomerase is low, but it is a critical factor in 0059 Agents which can be utilized to modulate or alter the maintaining telomere length. The activation of telomerase gene expression of this telomerase complex or its subunits may rejuvenate cells and thus tissues, organs or organisms can play a vital role in extending (or shortening) the lifespan and the modulation of telomerase activity has many applica of living cells. Suchagents which extend the lifespan of living tions in medicine and for extending lifespan. cells may be administered in many forms and may be used to 0056 Alternate, telomerase independent, recombination treat disease as well as to maintain and promote health. These based pathways are also a method by which cellular lifespan living cells may range from human or animal cells to plants can be lengthened. In this method of telomere maintenance, and any other living cell. Understanding the telomerase com originally discovered in telomerase defective yeast Strain S. plex is critical then to selecting agents to modulate the activity cerevisiae EST1 genetic recombination of break induced of telomerase. replication adds G rich telomeric repeats to the end of, or a 0060. One subunit of interest is Telomerase Reverse Tran break induced replication occurs between a critically (but still scriptase (TERT) which is a catalytic subunit; additional viable, i.e. retaining the repeat segments) short telomere and genes of interest are listed below. TERT gene expression then another portion of the telomere, essentially “lengthening the is controlled by Sp1 and c-myc transcription factors (genes telomere unit. The fact that these critically short telomeres are which interestingly are frequently altered in human tumors). so recombinogenic, has caused speculation that the telomeres 0061 The gene designated SP1 (or Transcription Factor either: 1) become more recombinogenic in response to the SP1, Specificity Protein 1), when overexpressed in humans, absence of telomeres, 2) critically short telomeres trigger has been shown to induce apoptosis. The apoptotic pathways recombination events, or 3) critically short telomeres are involved required the binding of SP1 to the DNA (via a zinc preferred substrates for specific types of recombination. finger domain) and were generally cell type specific. The SP1 Recent evidence Suggests that there are 2 recombination path regulated apoptosis involved alteration (downregulation) of US 2014/0079836A1 Mar. 20, 2014
BCLXL and BAX, no other caspases or BCL2 related genes become rigid and more permeable eventually leading to cyto were affected. It is involved in gene expression in the early plasmic ion leakage and cell apoptosis. development of an organism and when bound regulates tran 0065. The mitochondrial theory of aging states that the Scription. mitochondria (the organelles in the cell that create energy 0062. The gene designated as cMyc codes for a protein through the electron transport system and ATP synthase), lose that binds to the DNA of other genes and modulates the function due to damage and changes to the mitochondrial activity or transcription. It is estimated that cMyc transcrip DNA which codes for the proteins of the electron transport tion factor regulates about 15% of all genes. Induction of system. (This is similar to the Free radical theory of aging, but cMyc promotes cell proliferation/transformation by binding/ with a broader scope looking at the genetics, bioenergetics activating growth promoting genes. When cMyc is overex and membrane potential of the cell: not just the Free Radical chemistry). This decreased function allows for inability to pressed or mutated the DNA binding doesn’t occur correctly process the free radicals created during the cellular respira and cancer can result. cMyc is activated via many pathways tion process and causes further oxidative damage (membrane (WNT, SHH, and EGF to name a few) and modifies the permeability, loss of membrane potential and triggering of expression of many target genes resulting in a diverse number programmed cell death by leakage of cytochromes into the of biological effects.cMyc has also demonstrated direct acti cytoplasm) and a shortening of the cells lifespan. The mito vation of telomerase by inducing expression of TERT. It has chondrial DNA is without protection from oxidative stress, so been discovered that along with transcription, cMyc can (the most common damaging agent is the 8OHdG 8-hy affect cell growth, differentiation, stem cell self renewal and droxy-2'-deoxyguanosine formed by oxidized guanine apoptosis. It is often found to be upregulated in many cancers. bases) and much more Susceptible to deletions/damage. As 0063. The mitochondria function as the primary producer biomechanically described above, these mutations accumu of chemical cellular energy through the production of adenos late during normal aging, the most frequent of which being ine triphosphate (ATP) via the electron transport system. By the 4,977 base pair deletion known as the common deletion the transfer of electrons down a gradient ATP is formed for (significantly increased in photoaged skin). When human use in powering the cell. Additionally the mitochondria func fibroblasts are chronically exposed to UVA irradiation, there tion in other aspects of cellular regulation, for example, the was a time/dose dependent generation of the common dele mitochondrial membrane potential (the amount of potential tion caused by singlet oxygen generation (free radical forma to move ions across the membrane to facilitate energy pro tion). This generation of the common deletion was dimin duction) is a key regulator of apoptosis, or programmed cell ished in the presence of singlet oxygen quenchers death. The proton pump capacity of the membrane aids in (antioxidants) and duplicated in non-irradiated cells by ther reduction of compounds leading to energy production, and mochemical means (deuterium oxide enhanced production of also helping regulate oxidative stress caused by free radicals. singlet oxygen) indicating a clear role in free radical modu The mitochondria can also produce oxidative stress if, in the lated formation of mitochondrial deletions and prevention of process of cellular respiration, the electrons are not trans same via antioxidants. ferred from Complex I to Complex III. In essence, a back log 0.066 Mitochondrial deletions have been shown to be is formed and the generation of free radicals is the result. The either causative agents or co-factors in many aging disorders. ubiquinone, and synthetic idebenone compounds and deriva In Parkinson's disease clear evidence of a high burden of tives serve to alleviate this potential backlog by serving as mitochondrial deletions in the neurons of the Substantia nigra electron carriers/transfer agents facilitating the transition in aged individuals has been demonstrated. Mitochondrial from Complex I and down the respiratory chain. deletions and defects have also been identified in heart dis 0064. The free radical theory of aging postulates that ease, Alzheimer's disease, fatigue syndromes and many aging and related degenerative conditions are caused by the genetic disorders. Increased mtDNA deletions have been negative effects of free radicals (highly reactive molecules or reported in multiple cell types, fibroblasts, retinal pigment atoms that have an unpaired electron in an outer orbital that is epithelial cells, and neurons. Specifically of interest is the not contributing to molecular bonding “free”) on cells and ratio of “clean mtDNA to the “common deletion', which has tissues. The free radicals are formed as byproducts of cata been shown to increase with the aging of the cell, the addition lyzing molecular oxygen inside the cell with oxidative of oxidative stress and in cancerous cells. The ability of enzymes, and also in the connective tissues by traces of met antioxidants to modulate the production of these deletions has als like iron, cobalt and manganese. The most common free been demonstrated to be dose dependant (lower dosages are radicals are the Superoxide ion (O), the hydroxyl radical effective in reducing the formation of the common deletion (OH), and lipid peroxyl radicals (LOO). The hydroxyl radical while conversely higher doses have been shown to be inef is highly reactive (short halflife) and covalent cross linking is fective, and in fact are thought to act as electron donors and the most common effect. This cross linking can also perpetu facilitate the production of ROS). ate the cycle by creating more free radicals. Superoxide ions 0067. This theory has been supported through experimen are even more reactive and found mainly in the cell cytoplasm tal evidence which Suggests, among other things, that there (less often the nucleus). Hydrogen peroxide molecules are are morphological differences between mitochondria in more stable and capable of passing through cellular and young and old cells, membrane potentials in older mitochon nuclear membranes and forming hydroxyl radicals with met dria are decreased, the activity level of cytochrome oxidase als. Proteins in direct contact with hydrogen peroxide mol (COX) present in old muscle cells old cells is diminished, and ecules can also be damaged and hydrogen peroxide is one of that mtDNA deletions, point mutations and other changes to the most common methods for putting cells under oxidative the structure of mitochondrial DNA increase with age. stress. Lipid peroxidation of polyunsaturated fatty acids is a 0068. The rate of mutation in mitochondrial DNA is as contributing factor to food becoming rancid. In living animal much as ten times higher than the rate of mutation in nuclear cells lipid membranes that have undergone peroxidation DNA. This may be due to the limited ability to repair DNA in US 2014/0079836A1 Mar. 20, 2014 mitochondria. Mitochondrial DNA is particularly susceptible may speed up the aging process. So while there are many to damage from free oxygen radicals generated during the theories of aging, it is probable that the processes involved in production of ATP within the electron transport chain. ROS many of these theories play Some role in the process of aging are produced in part when electrons get stalled on complex I and also accelerated or premature aging. Foremost among or III and thus bypassing complex I with the electron transport these appear to be the role of oxidative stress and ROS and is one mechanism to reduce ROS. Mitochondrial DNA is mitochondrial DNA changes (both secondary to oxidative attached to the mitochondrial inner membrane which is a stress as well as DNA changes independent of oxidative Source of oxygen radicals and also it lacks protective histones stress) as well as inflammation. All three of these processes making the innate repair ability more limited. This accumu may be impacted in a favorable way by various antioxidants lated damage can make it difficult to copy the DNA, or pro which interact with these processes. Different antioxidants duce deletions and mutations in the DNA. This damage over have different mechanisms of action and there are different the years not only creates reduced function, but also prema pathways involved in ROS and oxidative stress damage. Thus ture aging and in Some cases disease states. While the mito the use of antioxidants to prevent premature aging and to act chondria have some ability to repair DNA, the importance of as an antidote to oxidative stress is well documented if not the ability to protect, defend or repair mitochondrial DNA and fully understood. function can be appreciated. 0073 However, while preventing the premature aging of a 0069. Another pathway for increasing lifespan is to cell, organ, tissue or organism generally speaking is one way increase mitochondrial respiration either directly or by to extend the lifespan, these processes are primarily methods increasing the total overall number of mitochondria. Increas to prevent premature shortening of the lifespan. To use ing the NAD/NADH ratio is produced with an increase in humans as an example, humans are all exposed to various mitochondrial respiration which can be associated with the factors including but not limited to those just described which activation of PGC-1alpha which can induce mitochondrial if we were to counteract those would effectively extend our biogenesis which increases mitochondrial numbers. Thus lifespan, but this is primarily by preventing damage rather increasing the number of mitochondria by modulating the than repairing damage that shortens lifespan. activity of PGC1-a is a target for lifespan altering/modulating 0074 To truly extend the lifespan of a living cell—and by compounds. extension the organ, tissue or entire organism—it is beneficial 0070. It is well known that oxidative stress produced by to repair damage in addition to preventing damage. The genes free radicals or reactive oxygen species (ROS) can produce a which control the cellular repair mechanisms, if activated or wide range of cellular damage which if not perfectly repaired enhanced in the proper way, may effectively extend the results in cellular damage, injury, aging or apoptosis. Many lifespan of a cell. This may take two forms: extending the Small imperfectly repaired injuries accumulate over time to lifespan of a cell which is damaged or injured by properly degrade the vital functions of cells, tissues, organs and ulti repairing that damage and also by causing the cell to live or mately the entire organism. This damage may occur in any of replicate itself longer than it would have occurred naturally. the cellular components, but of particular interest are the 0075 Cancer cells may accomplish the latter by a process mitochondria which are the power plants of living cells and termed immortalization and this may also be created in the which provide energy for cellular activities but which also laboratory in research conditions. Some view cancer as a control a process called apoptosis or programmed cell death. form of aging as the cellular repair mechanisms have either The mitochondria also possess their own unique DNA sepa not fully repaired damage or they have failed to kill a cell rate from the cell's nuclear DNA. Unlike nuclear DNA, the which is damaged beyond repair. One example is a type of mitochondrial DNA has a more limited ability to repair DNA skin cancer which is produced by injury from UVB light. UV damage. Also mitochondria can actually create their own free light is known to produce changes in DNA termed thymine radicals as part of normal cellular functioning. Thus mito dimers whereby there is cross linking which occurs within the chondria are particularly Susceptible to damage from oxida DNA and this defect or mutation produces basal cell carci tive stress and the cellular damage can profoundly affect the noma of the skin—a very common type of skin cancer caused function of the entire cell. This may result in decreased by sunlight. There are also DNA repairenzymes which if they lifespan of the cells and ultimately the entire organism. perform their job properly will repair this damage thus pre 0071. In general, increasing the chronological lifespan is venting the skin cancer from developing. In the case of basal related to protection from oxidative stress, minimizing DNA cell skin cancer, Sun light exposure leads to the formation of mutations within the mitochondria, and increasing resistance thymine dimers, a form of DNA damage and when the DNA to heat shock. It has been demonstrated that increased large repair does not remove this UV-induced damage, not all scale mitochondrial DNA mutations termed deletions pro crosslinks are excised. There is, therefore, cumulative DNA duced by exposure to a toxic chemical (such as ethidium damage leading to mutations. Apart from the mutagenesis, bromide) decreases various vital mitochondrial functions Sunlight depresses the local immune system, possibly including oxygen consumption which is required to produce decreasing immune Surveillance for new tumor cells. This cellular energy or ATP. When an antioxidant was provided risk is primarily based on UV exposure and the degree of Some of these changes were prevented or minimized Suggest protective pigment in the skin, but there is also a rare syn ing that increased environmentally induced ROS production drome termed basal-cell nevus syndrome, or Gorlin’s Syn leads to altered mitochondrial gene or protein expression drome in which much less UV exposure is required because which may be diminished by the protective effects of certain there is defective DNA repair. The cause of this syndrome is antioxidants. Chronic oxidative stress leads to premature a mutation in the PTCH1 tumor-suppressor gene at chromo aging. Some 9q22.3, which inhibits the hedgehog signaling pathway 0072 Some studies have shown that inducing changes in ultimately leading to production of the cancer. While basal the mitochondrial DNA without oxidative stress also accel cell carcinoma of the skin is not a fatal form of cancer it does erates the aging process of cells. Chronic inflammation also illustrate the role of environmental damage in producing can US 2014/0079836A1 Mar. 20, 2014
cer and also the role of genetic inheritance in making some for the primary energy metabolism of food). Resveratrol is the individuals more or less likely to have this problem as well as most potent activator of these sirtuin compounds. affecting the age of onset and severity of the cancer. 0080. The technology described herein is different from 0076. If one thinks then of other cancers which destroy use of resveratrol and sirtuin modulation because the current cellular or organ function thus limiting healthy lifespan or technology utilizes antioxidant compounds to directly modu which lead to a fatal outcome producing a shortened lifespan, late the gene expression of genes/proteins and complexes one may better appreciate that while there are many factors Vital to the maintenance of telomere length and/or mitochon which lead to shortened lifespan there are also many means to drial membrane stability/free radical elimination, whereas diminish or avoid these factors, and that the ability to repair the sirtuins and resveratrol modify the energy metabolism of the damage is vital to achieving and optimal healthy lifespan. the cell and boost the “anti-stress' response. Additionally, the antioxidant idebenone may help reduce ROS activity in mito 0077. The ability to extend or prolong lifespan (both chondria by helping electrons in the electron transport system healthy and less healthy) lies in the ability to extend the bypass Complex I (where most of the ROS is formed) and lifespan of cells, both differentiated specialized cells and also donate the electrons into Complex III. undifferentiated stem and progenitor cells so that cell lifespan I0081 Emerging evidence suggests that microRNA is longer or so that new cells replace Senescent cells which (miRNA) may play a regulatory role in both aging and cancer. lose their function or die. A cell normally has a finite lifespan miRNAs appear to influence such systems as cell cycle, DNA determined by the number of cell divisions which are pos repair, oxidative stress responses and apoptosis and have been sible. The Hayflick Limit theory discusses one view of shown to be abnormally expressed later in life. In view of this, lifespan limitations. An organ may be repopulated with cells also provided herein are methods of altering the expression of to regenerate itself from the stem cell population but the stem one or more of the life-span influencing genes identified and progenitor cells themselves have a finite lifespan. The herein. ability to extend the lifespan of differentiated cells and/or I0082. There is value in extending the lifespan of a cell stem and progenitor cells lies at the heart of extending whether in vitro or in vivo. Protecting the cell against stress or lifespan of an organism. oxidative stress responses or DNA damage and controlling 0078. The ability to repair cellular or DNA damage pro the cell cycle or apoptosis or stem cell activity can potentially duced by environmental or genetic factors may extend the extend the life of the cell. lifespan of a cell. The ability to extend the natural lifespan of such a cell will also extend the lifespan of a cell. Such a cell I. Abbreviations may be a differentiated cell or a stem cell. On a broader scale 0.083 ANT ADP/ATP translocase either or both of these events may lead to extending the lifespan of an entire organ or organism provided that some CoA Coenzyme A other intervening factor limits or shortens the life of the organ or organism. An exception is that extending the life of a I0084 Complex I NADH ubiquinone oxidoreductase cancerous cell or stem cell may instead shorten the life of the Complex II Succinate ubiquinone reductase organ or organism and is thus undesirable. Making cancer Complex III ubiquinone-cytochrome c oxidoreductase cells mortal while making healthy cells if not immortal at Complex IV (or COX) cytochrome c oxidase least longer lasting is an important concept as longevity and Complex V (F1/F0 ATPase) ATP synthase tumor Suppression are in Some ways opposite goals. Telom erase is a critical enzyme in determining cell lifespan and its IF1 Inhibitor of F1/FOATPase activation enables cells to overcome senescence, but also I0085 LC-MS/MS liquid chromatography mass spectrom allows cancer cells to proliferate. Activity of telomerase then etry/mass spectrometry becomes a vital issue to consider in extending or shortening M F1FO mitochondrial F1/FO ATPase the lifespan of living cells. mAb monoclonal antibody 007.9 The discovery of sirtuins (cellular enzymes that MALDI-TOF matrix assisted laser desorption/ionization increase DNA repair and the production of antioxidants) and time-of-flight the SIRT pathway able to increase the lifespan of yeast cells mtDNA mitochondrial DNA with no decrease in the replicative capacity was another NADH nicotinamide adenine dinucleotide breakthrough in understanding aging. Sirtuins and the SIRT ORAC oxygen radical absorbance capacity pathway are thought to be regulated via changes in the intra OD optical density cellular NAD/NADH ratio and the related energy metabolism of the mitochondria. The SIRT pathway is involved in the OMIM Online Mendelian Inheritance in Man caloric restriction process, although the mechanism is poorly I0086 OXPHOS oxidative phosphorylation understood, it is thought primarily to revolve around the PDH pyruvate dehydrogenase complex lowered instance of glycolysis that CR creates. Three of the PMSF phenylmethylsulfonyl fluoride seven mammalian sirtuins (SIRT3, 4 and 5) are targeted to mitochondria. SIRT1 itself also regulates mitochondrial ROS reactive oxygen species activity. The activity of SIRT3 has been most clearly II. Terms described and it functions in the mitochondria as an activator of special enzymes that spontaneously form NADPH the key 0087. Unless otherwise noted, technical terms are used component need for the regeneration of cellular anti stress according to conventional usage. Definitions of common systems (this alternate energy production pool explains why terms in molecular biology may be found in Benjamin Lewin, the stressful cellular event of caloric restriction seems to Genes V, published by Oxford University Press, 1994 (ISBN enhance cell longevity; by creating NADPH without the need 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of US 2014/0079836A1 Mar. 20, 2014
Molecular Biology, published by Blackwell Science Ltd., instance signal intensity). In some examples of computer 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), readable array formats, the individual spots on the array Sur Molecular Biology and Biotechnology: a Comprehensive face will be arranged regularly, for instance in a Cartesian grid Desk Reference, published by VCH Publishers, Inc., 1995 pattern, that can be correlated to address information by a (ISBN 1-56081-569-8). computer. 0088. In order to facilitate review of the various embodi 0097. The sample application spot (or feature) on an array ments of the invention, the following explanations of specific may assume many different shapes. Thus, though the term terms are provided: “spot' is used herein, it refers generally to a localized deposit 0089 Addressable: Capable of being reliably and consis of nucleic acid or other biomolecule, and is not limited to a tently located and identified, as in an addressable location on round or Substantially round region. For instance, Substan an array. tially square regions of application can be used with arrays, as 0090 Antioxidant: A molecule or atom capable of slowing can be regions that are substantially rectangular (such as a slot or preventing transfer of electrons from one molecule? atom to blot-type application), or triangular, oval, irregular, and so another (oxidizing agent). forth. The shape of the array substrate itself is also immate 0091 Antisense, Sense, and Antigene: Double-stranded rial, though it is usually substantially flat and may be rectan DNA (dsDNA) has two strands, a 5'->3' strand, referred to as gular or Square in general shape. the plus strand, and a 3'->5' strand (the reverse complement), 0.098 Binding or interaction: An association between two referred to as the minus strand. Because RNA polymerase Substances or molecules, such as the hybridization of one adds nucleic acids in a 5'->3' direction, the minus strand of the nucleic acid molecule to another (or itself). Disclosed arrays DNA serves as the template for the RNA during transcription. are used to detect binding of, in some embodiments, a labeled Thus, the RNA formed will have a sequence complementary nucleic acid molecule (target) to an immobilized nucleic acid to the minus Strand and identical to the plus strand (except that molecule (probe) in one or more features of the array. A U is substituted for T). labeled target molecule “binds to a nucleic acid molecule in 0092 Antisense molecules are molecules that are specifi a spot on an array if, after incubation of the (labeled) target cally hybridizable or specifically complementary to either molecule (usually in Solution or Suspension) with or on the RNA or the plus strand of DNA. Sense molecules are mol array for a period of time (usually 5 minutes or more, for ecules that are specifically hybridizable or specifically instance 10 minutes, 20 minutes, 30 minutes, 60 minutes, 90 complementary to the minus Strand of DNA. Antigene mol minutes, 120 minutes or more, for instance over night or even ecules are either antisense or sense molecules directed to a 24 hours), a detectable amount of that molecule associates dsDNA target. with a nucleic acid feature of the array to such an extent that 0093. Apoptosis: The process by which cells are pro it is not removed by being washed with a relatively low grammed to die or lose viability. Commonly triggered by stringency buffer (e.g., higher salt (such as 3xSSC or higher), cytochrome leakage from the mitochondria and accompanied room temperature washes). Washing can be carried out, for by signaling cascades (caspases and other proteins) resulting instance, at room temperature, but other temperatures (either in: decreased mitochondrial and energy potential via the elec higher or lower) also can be used. Targets will bind probe tron transport system, an build up of reactive oxygen species nucleic acid molecules within different features on the array and free radical and loss of membrane integrity. to different extents, based at least on sequence homology, and 0094. Array: An arrangement of molecules, particularly the term “bind’ encompasses both relatively weak and rela biological macromolecules (such as polypeptides or nucleic tively strong interactions. Thus, some binding will persist acids) or biological samples (such as tissue sections) in after the array is washed in a more stringent buffer (e.g., lower addressable locations on a Substrate, usually a flat Substrate salt (such as about 0.5 to about 1.5xSSC), 55-65°C. washes). Such as a membrane, plate or slide. The array may be regular (0099. Where the probe and target molecules are both (arranged in uniform rows and columns, for instance) or nucleic acids, binding of the test or reference molecule to a irregular. The number of addressable locations on the array feature on the array can be discussed in terms of the specific can vary, for example from a few (Such as three) to more than complementarity between the probe and the target nucleic 50, 100, 200, 500, 1000, 10,000, or more. A “microarray” is acids. Also contemplated herein are protein-based arrays, an array that is miniaturized to Such an extent that it benefits where the probe molecules are or comprise proteins or pep from microscopic examination for evaluation. tides, and/or where the target molecules are or comprise pro 0095 Within an array, each arrayed molecule (e.g., oligo teins or peptides. nucleotide) or sample (more generally, a “feature' of the 0100 Biological Sample: Any sample that may be array) is addressable, in that its location can be reliably and obtained directly or indirectly from an organism, including consistently determined within the at least two dimensions on whole blood, plasma, serum, tears, mucus, saliva, urine, pleu the array Surface. Thus, in ordered arrays the location of each ral fluid, spinal fluid, gastric fluid, Sweat, semen, vaginal feature is usually assigned to a sample at the time when it is secretion, sputum, fluid from ulcers and/or other Surface spotted onto or otherwise applied to the array Surface, and a eruptions, blisters, abscesses, tissues, cells (such as, fibro key may be provided in order to correlate each location with blasts, peripheral blood mononuclear cells, or muscle cells), the appropriate feature. organelles (such as mitochondria), organs, and/or extracts of 0096. Often, ordered arrays are arranged in a symmetrical tissues, cells (such as, fibroblasts, peripheral blood mono grid pattern, but samples could be arranged in other patterns nuclear cells, or muscle cells), organelles (such as mitochon (e.g., in radially distributed lines, spiral lines, or ordered dria) or organs. An "organism' includes, without limitation, clusters). Arrays are computer readable, in that a computer plants, animals, or microbes. The term “animal' includes can be programmed to correlate a particular address on the Vertebrate or invertebrate animals, such as mammals (for array with information (such as identification of the arrayed example, humans), insects (for example, Drosophila melano sample and hybridization or binding data, including for gaster), nematodes (for example, Caenorhabditis elegans), US 2014/0079836A1 Mar. 20, 2014
and fish (for example, Danio rerio, aka, Zebrafish). A biologi more generally, free radical) modulatory capabilities, and can cal sample may also be a laboratory research sample Such as have non-steroidal anti-inflammatory effects as well. These a cell culture Supernatant. The sample is collected or obtained polyphenols and procyanidins are commonly extracted from using methods well known to those skilled in the art. the bean by fermenting, drying and grinding the cocoa seeds. 0101 Caffeic Acid (3-(3,4-Dihydroxyphenyl 3,4-Dihy droxy-cinnamic acid trans-Caffeate 3,4-Dihydroxy-trans 0110 Coffee Cherry Fruit of the coffee tree Coffea rubi cinnamate) 2-propenoic acid (E)-3-(3,4-dihydroxyphenyl)- aceae. The pulp, husk (FIG. 3) (to a lesser degree) and muci 2-propenoic acid 3,4-Dihydroxybenzeneacrylicacid): lage of the whole coffee cherry contain high levels of Formally known as carbolic acid, this phenolic (crystalline polyphenols antioxidants if kept in a nonfermented State and acid compound derived from aromatic hydrocarbons) com preserved. The extract of the coffee cherry is generally pro pound can be extracted from the coffee cherry and has been duced by being contacted with a solvent and will include the shown to be anti-carcinogenic, anti-inflammatory and have nutrients. Further processing of the extract (or “tea') can antioxidant properties with a chemical structure similar to allow for the purification of various aspects of the coffee cinnamic acid. It is soluble in water and alcohol. Methods for cherry. One commercial producer of a coffee cherry extract is the isolation and characterization of caffeic acid are well VDF FutureGeuticals, Inc. (Momence, Ill.; marketed as COF known in the art; in addition, this compound is commercially FEEBERRYR); a significant portion of their preparation is available. chlorogenic acid, with the other coffee acids, proanthocyani 0102 Carnosine: A natural amino acid with strong anti dins, etc. making up the remainder of active ingredients. By oxidant properties (it helps bind and flush ionic metals from way of example, coffee cherry extract can be prepared as the system). Carnosine has been shown to extend the lifespan described previously (see, e.g., U.S. publication no. 2007/ of fibroblast cells treated with the amino acid in culture up to 028.1048 and other patent documents cited therein; U.S. Pub 10 divisions past the Hayflick limit of non-treated cells. Car nosine also helps prevent the cross linking of protein and lications No. 2006/0210689, 2006/0263508, and 2009/ DNA molecules and preventing cell damage. 0175973; and PCT publications no. WO 2004/098320, WO 0103 Catechin 3 gallate (CG): A minor polyphenolic con 2004/0983.03, WO 2006/022764 and WO 2004/098320). stituent of green tea having antioxidant properties. 0111 Isolation of the coffee acids, including caffeic, chlo 0104 cDNA: A DNA molecule lacking internal, non-cod rogenic, quinic and ferulic acids, as well as proanthocyani ing segments (e.g., introns) and regulatory sequences that dins via (for instance) ion exchange columns and Sodium determine transcription. By way of example, cDNA may be acetate solutions will yield purified antioxidant components. synthesized in the laboratory by reverse transcription from The greatest amounts of antioxidants are found in the green messenger RNA extracted from cells. coffee cherries with ripe coffee cherries having somewhat 0105 Cell Proliferation: The process by which there is an less. Polyphenols constitute a substantial portion of the active increase in the number of cells as a result of cell growth and ingredients in coffee cherry extract; these polyphenols division (mitotic cell division). include chlorogenic acid, caffeine, caffeic acid, ferulic acid, 0106 Cell Senescence: The process of cellular aging and quinic acid, and so forth. Representative analyses of different loss of cell function and viability (death). coffee cherry extracts are shown, for instance, in Table 2 of 0107 Chalcone: An aromatic ketone (chemical compound U.S. Publication Mo. 2007/028.1048. containing a carbonyl C=O group) intermediate in the bio synthesis of flavonoids that forms the central core of many biologically important compounds and has been shown to be O OH able to block Voltage dependant potassium channels. OH 0108 Chlorogenic Acid (-3-(3,4-Dihydroxyphenyl)-1- oXo-2-propenyloxy)-1,4,5-trihydroxycyclohexanecarboxy HO OH lic acid): A family of esters formed between certain trans cinnamic acids and quinic acid (most common individual OH chlorogenic acid formed from caffeic and quinic acids) and a QUINIC ACID major phenolic compound found in coffee and the cherry OH thereof. Chlorogenic acid has been shown to be effective in 21 reducing free radicals (antioxidant ability) and inhibitory to HO O the tumor formation process. Methods for the isolation and O O OH characterization of chlorogenic acid are well known in the art; in addition, this compound is commercially available. HO 0109 Cocoa Bean: A fatty seed from the cacao tree; it contains substantial levels of polyphenols as well as levels of HO procyanidins. A cacao pod has a rough leathery rind about OH which varies in thickness dependent on species is filled with CHLOROGENIC ACID Sweet, mucilaginous pulp that encases 30 to 50 large beans OH that are fairly soft and pinkish or purplish in color. It is these beans, containing cocoa butter and cocoa solids (the dried O rs Solids produce cocoa powder and the combination of the two N 21 OH creates chocolate in its many incarnations based on the amount of cocoa Solids present. Inside the bean and pod itself OH are the polyphenolic and procyanidin compounds. These CAFFEIC ACID compounds have antioxidant anti cancer, nitric oxide (and US 2014/0079836A1 Mar. 20, 2014 10
-continued and second substrate molecules typically are, but need not be, HCO O proteins, carbohydrates, lipids, or Small co-factors. 0117 Oxidation and/or reduction can be detected by any OH / OH method known in the art. In some examples, a detectable change in a physical property of the oxidized and/or reduced Substrate molecule(s) is measured; for example, a change in FERULIC ACID optical density (OD) at some defined wavelength. In particu lar examples, ODao can be used to monitor the ratio of NAD/NADH redox (such as, in assays of Complex I activity), 0112 DNA (deoxyribonucleic acid): DNA is a long chain or ODoo can be used to monitor reduction of 2,6-dichlo polymer that contains the genetic material of most living rophenolindophenol (such as, in assays for Complex II activ organisms (the genes of some viruses are made of ribonucleic ity), or ODsso can be used to monitor oxidation of cytochrome acid (RNA)). The repeating units in DNA polymers are four c (II) (Such as, in assays for Complex IV activity) (see, e.g., different nucleotides, each of which includes one of the four Birch-Machin and Turnbull, Meth. Cell Biol. 65: 97-117, bases (adenine, guanine, cytosine and thymine) bound to a 2001). In other examples, oxidation and/or reduction can be deoxyribose Sugar to which a phosphate group is attached. detected by monitoring a change in the properties of a pros Triplets of nucleotides (referred to as codons) code for each thetic group in the oxidoreductase enzyme; for example, the amino acid in a polypeptide, or for a stop signal. The term ratio of ODos/OD can be used to monitor heme aa3 of "codon’ is also used for the corresponding (and complemen Complex IV (see, e.g., Rickwood et al., in Mitochondria. A tary) sequences of three nucleotides in the mRNA into which Practical Approach, ed. by Darley-Usmar et al., Oxford:IRL the DNA sequence is transcribed. Press, 1987). In still other examples, oxidation and/or reduc 0113. Enriched: The term “enriched” means that the con tion can be detected by coupling the oxidation or reduction centration of a material is at least about 2, 5, 10, 100, or 1000 reaction of interest to another more easily monitored redox times its natural concentration (for example), advantageously reaction, Such as oxidation or reduction of a chromogenic at least 0.01% by weight. Enriched preparations of about (Birch-Machin and Turnbull, Meth. Cell Biol. 65: 97-117, 0.5%, 1%. 5%, 10%, and 20% by weight are also contem 2001) or fluorogenic (Molecular Probes, Inc.) substrate. plated. 0118 “Reductase activity” is the ability of an enzyme to 0114 Enzymatic Activity: A detectable (and usually quan reduce (add electrons to) a Substrate molecule, which typi tifiable) characteristic of at least one function of an enzyme cally is, but need not be, a protein, a carbohydrate, a lipid or (such as, an OXPHOS enzyme), often monitored over time or a small co-factor. The reducing equivalents are obtained by in comparison to a standard curve. Methods are well knownto the enzyme from some other molecule which is thereby oxi those of ordinary skill in the art, for detecting, determining, dized either contemporaneously with, or at Some time prior monitoring, and/or quantifying various enzymatic activities. to, the reductase enzyme? substrate reaction. Reductase activ Also well known are ways of using enzymatic activity assays ity can be measured using various assays known to those of to assess the ability of compounds (for instance, test com ordinary skill in the art. For example, assays for activity of pounds) to affect the function of the enzyme, for instance, as Complex II can follow reduction of the oxidized substrate an inhibitor or enhancer. 2,6-dichlorophenolindophenol by monitoring changes in 0115 For instance, ATPase activity” is usually contem OD600 (Birch-Machin & Turnbull, Meth. Cell Biology, plated as the ability to detectably hydrolyze ATP ATPase 65:97-117, 2001). activity can be measured using various assays known to those 0119 “Oxidase activity” is the ability of an enzyme to of ordinary skill in the art, including those assays provided oxidize (remove protons and electrons, or reducing equiva herein, for instance, in Example 2. In some examples, ATPase lents, from) a Substrate molecule, which typically is, but need activity is measured in Solution by detecting (quantitatively or not be, a carbohydrate, a lipid or a small co-factor. The reduc qualitatively) free phosphate released by enzyme activity ing equivalents are typically transferred by the enzyme to (such as, Complex V activity). Methods of detecting free some other molecule which is thereby reduced either contem phosphate are known and include, for example, both colori poraneously with, or at Some time after, the oxidase enzyme? metric and fluorescent techniques (see, e.g., Aggeler et al., J. Substrate reaction. Oxidase activity can be measured using Biol. Chem., 277:33906-33912, 2002). In other examples, various assays known to those of ordinary skill in the art. For ATPase activity of an immobilized enzyme (for instance, instance, Complex IV oxidase activity can be detected by Complex V immunocaptured on a dipstick) is detected, for observing the oxidation of cytochrome c by measuring ODsso example, by fluorescent techniques (for example, fluores (Birch-Machin and Turnbull, Meth. Cell Biol., 65: 97-117, cence-based assays for free phosphate as provided by 2001). Molecular Probes, Inc., or by direct application of tissue I0120 Epigallocatechin gallate (EGCG): The most abun based histochemical techniques (see, e.g., Bancroft and dant of the antioxidant catechins found in green tea. It is an Stevens. Theory and Practice of Histological Techniques, 4th ester of epigallocatechol and gallic acid. edition, London:Churchill-Livinstone, 1996) or slight modi I0121 Epicatechin gallate (ECG): A polyphenol found in fications thereof, for example to account for the physical green tea and having antioxidant properties. handling differences of tissue sections as compared to dip 0.122 Ester: A class of chemical compound that consists of Sticks. an acid that has at least one —OH (hydroxyl) group replaced 0116 “Oxidoreductase activity” is the ability of an by an —O-alkyl (alkoxy) group. enzyme to reversibly oxidize (remove protons and electrons, I0123 Ferulic Acid ((E)-3-(4-hydroxy-3-methoxy-phenyl) or reducing equivalents from) a first Substrate molecule and prop-2-enoic acid): A compound serving as a precursor for contemporaneously reduce (add protons and electrons, or other aromatic compounds, it is found most commonly in the reducing equivalents to) a second Substrate molecule. First plant cell walls where it associates with dihydroferulic acid, US 2014/0079836A1 Mar. 20, 2014 to facilitate the crosslinking of lignin and polysaccharides goals would improve the length of the lifespan or make the conferring rigidity to the cell wall. It can be found in coffee remaining lifespan duration healthier. Modulating cell func cherry, has antioxidant activity and is biologically synthe tion to achieve one or more of these goals is then a means of sized by methylation of caffeic acid. Methods for the isolation producing a state termed healthy longevity. The modulation and characterization offerulic acid are well known in the art; of cell activity to accomplish this may involve in some in addition, this compound is commercially available. instances modulating to kill cells prematurely and in a man 0.124 Free Radical: Any atom or molecule having a single, ner diminish the cells health to the point of cell death in order unpaired electron in an outer shell. to remove unhealthy cells which may harm the tissue, organ 0125 FOXO1, 3 and 4 (Forkhead Box O1A, O3A, and or organism or even which may stimulate the creation and O4A): Activation of serine threonine kinase which inactivates replacement of the unhealthy or sub-optimally healthy cell(s) apoptotic machinery. Overexpression causes growth Suppres with new cells via cell division of healthy cells, biogenesis of sion in a of cell lines variety. new cells or replacement of cells via stem cells or autologous 012.6 Gallocatechin gallate (GCG): A member of antioxi transplant or allograft or other types of transplanted cells dant polyphenols found in green tea. including genetically engineered cells for transplantation. 0127 Gnetin H: A stilbene (a hydrocarbon with a trans The treatment of such cells with the process of this invention ethane double bond substituted with a phenyl group on both prior to or after transplantation is also envisioned as a means carbon atoms of the double bond) resveratrol derivative from to produce healthy longevity in these new cells. peony seeds having antioxidant properties and mimicking the 0131 High throughput genomics: Application of genomic effects of resveratrol. or genetic data or analysis techniques that use arrays, 0128 Golgi apparatus: A cell organelle involved in the microarrays or other genomic technologies to rapidly identify processing and packaging of proteins and lipids produced by large numbers of genes or proteins, or distinguish their struc and/or moved through a cell. ture, expression or function from normal or abnormal cells or 0129 Hayflick Limit: The number of times a cell can tissues, or from cells or tissues of Subjects with known or undergo mitosis before the telomeres are shortened to a criti unknown phenotype and/or genotype. cal length and the cell begins to senesce. Each mitosis event 0.132. Histone(s): Lysine and Arginine rich, basic proteins decreases the length of the telomere and pushes the “aging associated with DNA in eukaryotic chromosomes resembling cell towards senescence. This limit is thought to be a mecha “beads on a string. These proteins form the scaffold which nism through which the body can control cancerous cell the DNA wraps around to form the chromatin structure. growth; since the more times a cell undergoes mitosis the I0133 HPGD (Hydroxyprostaglandin Dehydrogenase): more chances for a problematic mutation or transcription Involved in many cellular processes specifically inflamma error to OCCur. tion. As an NAD dependant dehydrogenase, HPGD is the 0130 Healthy longevity: The concept of having entire primary prostaglandin degrading enzyme. organisms (as well as organs, tissues and individual cells) at I0134 HSPA1A (Heat Shock 70-KD Protein 1A): Ubiqui optimal genetic and functional health. While not limited to tous highly conserved protein involved in many functions. these issues, this means for example that the DNA is not HSPA1A is thought to be proliferative, when expressed, for significantly damaged or mutated and is in a state comparable cancer cells, involved in apoptosis and regulation of acute to the configuration that would occur in a natural healthy StreSS. infant or fetus. In other embodiments, the DNA has been 0.135 HSPA1B (Heat Shock 70-KD Protein 1B): A altered to be equivalent or better than that status through, e.g., 'stress' protein expressed in response to heat, oxidative dam repair or genetic engineering. Similarly, in some embodi age, free radicals and toxic metalions. Structurally and func ments the mitochondrial number and/or function and/or res tionally comparable to other heat shock proteins (varying in piratory efficiency are similarly optimal or Supra optimal. their inducibility due to stress) and involved in the cell Metabolic pathways and immune function also may be like response to damage/stress. wise optimized, and existing environmental damage may 0.136. HSPA1L (Heat Shock 70-KD Protein 1 L): A have been repaired. Intrinsic chronologic aging and/or oxida 'stress' protein expressed in response to heat, oxidative dam tive stress damage from normal cellular processes such as free age, free radicals and toxic metalions. Structurally and func radical damage within mitochondria have also been mitigated tionally comparable to other heat shock proteins (varying in or reversed or repaired or otherwise restored to a youthful their inducibility due to stress) and involved in the cell optimally functional status or a close approximation of the response to damage/stress. same. Unhealthy cells, including even cancerous cells, which 0.137 Human Cells: Cells obtained from a member of the have not been repaired, are eliminated via apoptosis or the species Homo sapiens. The cells can be obtained from any death of these cells has been modulated to be accelerated. Source, for example peripheral blood, urine, saliva, tissue Significantly gene expression patterns and pathways have biopsy, skin scrape, Surgical specimen, amniocentesis been reregulated, or reset or resignalled in Such a fashion as to samples and autopsy material. From these cells, biological optimize the function and health of the cells and by extension components such as genomic or mitochondrial DNA, mRNA the tissues, organs and organisms that these cells comprise. (from which one can make cDNA), RNA, and/or protein can One end result of at least one or perhaps more of these pro be isolated. cesses is that the cells achieve maximal longevity or lifespan 0.138 Hybridization: Nucleic acid molecules that are and/or function optimally or at maximal efficiency and effec complementary to each other hybridize by hydrogen bond tiveness for the duration of their lifespan. Understanding that ing, which includes Watson-Crick, Hoogsteen or reversed Such a process may not be undertaken until Substantial dam Hoogsteen hydrogen bonding between complementary age from aging, disease, diet, injury, environmental exposure, nucleotide units. For example, adenine and thymine are medication or medical therapy side effects, etc. it is under complementary nucleobases that pair through formation of stood that even a partial achievement of one or more of these hydrogen bonds. "Complementary refers to sequence US 2014/0079836A1 Mar. 20, 2014
complementarity between two nucleotide units. For example, bound forms or other derivatives. Examples of idebenone if a nucleotide unit at a certain position of an oligonucleotide derivatives include esters of idebenone where idebenone is is capable of hydrogen bonding with a nucleotide unit at the esterified using glycosaminoglycans (GAGS), and/or their same position of a DNA or RNA molecule, then the oligo salts, for example HA (hyaluronic acid) having a molecular nucleotides are complementary to each other at that position. weight of 1 to 1,000,000 and its salts of hyaluronidase inhibi The oligonucleotide and the DNA or RNA are complemen tors like inter-alpha-trypsin inhibitor. An example of a hydro tary to each other when a sufficient number of corresponding philic idebenone ester is idebenone sulphonic acid. positions in each molecule are occupied by nucleotide units 0144. IDH2 (Isocitrate Dehydrogenase 2): NADP depen which can hydrogen bond with each other. dant isocitrate dehydrogenase that is responsible for playing 0139 “Specifically hybridizable” and “complementary” a major role in mitochondrial redox balance and mitigating are terms that indicate a sufficient degree of complementarity damage by oxidative stress by providing NADPH for such that stable and specific binding occurs between the oli NADPH dependent antioxidant enzymes. gonucleotide and the DNA or RNA or PNA target. An oligo (0145 IFI44 (Interferon Induced Protein 44): Interferon nucleotide need not be 100% complementary to its target stimulated response element induced by interferon alpha and nucleic acid sequence to be specifically hybridizable. An bets, but not gamma possibly produced in response to viral oligonucleotide is specifically hybridizable when binding of induction. the oligonucleotide to the target DNA or RNA molecule 0146 In vitro amplification: Techniques that increase the interferes with the normal function of the target DNA or number of copies of a nucleic acid molecule in a sample or RNA, and there is a sufficient degree of complementarity to specimen. An example of in vitro amplification is the poly avoid non-specific binding of the oligonucleotide to non merase chain reaction, in which a biological sample collected target sequences under conditions in which specific binding is from a subject is contacted with a pair of oligonucleotide desired, for example under physiological conditions in the primers, under conditions that allow for the hybridization of case of in vivo assays, or under conditions in which the assays the primers to nucleic acid template in the sample. The prim are performed. ers are extended under Suitable conditions, dissociated from 0140 Hybridization conditions resulting in particular the template, and then re-annealed, extended, and dissociated degrees of stringency will vary depending upon the nature of to amplify the number of copies of the nucleic acid. the hybridization method of choice and the composition and 0147 The product of in vitro amplification may be char length of the hybridizing DNA used. Generally, the tempera acterized by electrophoresis, restriction endonuclease cleav ture of hybridization and the ionic strength (especially the Na age patterns, oligonucleotide hybridization or ligation, and/or concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybrid nucleic acid sequencing, using standard techniques. ization conditions required for attaining particular degrees of 0.148. Other examples of in vitro amplification techniques stringency are discussed by Sambrook et al. in Molecular include strand displacement amplification (see U.S. Pat. No. Cloning: A Laboratory Manual, Cold Spring Harbor Labo 5,744.311); transcription-free isothermal amplification (see ratory Press (1989), chapters 9 and 11, herein incorporated by U.S. Pat. No. 6,033,881); repair chain reaction amplification reference. (see WO 90/01069); ligase chain reaction amplification (see 0141 Idebenone (6-(10-hydroxydecyl)-2,3-dimethoxy-5- EP-A-320 308); gap filling ligase chain reaction amplifica methyl-1,4-benzoquin-one): Reference German patent docu tion (see U.S. Pat. No. 5,427.930); coupled ligase detection ment DE3049039, European patent 788793, and U.S. Pat. and PCR (see U.S. Pat. No. 6,027,889); and NASBATM RNA Nos. 4,436,753, 5,059,627, 5916925, application transcription-free amplification (see U.S. Pat. No. 20050152857 and WIPO 9907355 to describe the use of oral, 6,025,134). parenteral or percutaneous preparations of idebenone or 0149 Isolated: An "isolated biological component (such derivatives for the treatment of dementia, circulatory disease as a nucleic acid molecule, protein or organelle) has been induction of neural growth factors and resistance to Sunburn Substantially separated or purified away from other biological cell formation. Methods for the isolation and characterization components in the cell of the organism in which the compo of idebenone are well known in the art; in addition, this nent naturally occurs, i.e., other chromosomal and extra compound is broadly commercially available. Idebenone is a chromosomal DNA and RNA, proteins and organelles. synthetic molecule that does not occur in nature and mimics Nucleic acids and proteins that have been "isolated include the structure and function of ubiquinone and ubiquinol with nucleic acids and proteins purified by Standard purification similar results for Redox potential and free radical quenching methods. The term also embraces nucleic acids and proteins capabilities. prepared by recombinant expression in a host cell as well as 0142 Idebenone has also been shown via chemilumines chemically synthesized nucleic acids. cence to intercept the pro-oxidative effect of tocopherol oxi 0150. Keratinocyte: A cell type comprising 95% of the dation products occurring after 24 hours. In the measure epidermis and producer of the structural protein(s) keratin(s). ments of the lipid hydroperoxides generated as a result of the 0151 KL (Klotho): Reduced expression of KL is thought oxidation of lipids due to, for example, UV radiation or free to be a causative factor in many degenerative processes radical damage, idebenone was shown to have the highest including, arteriosclerosis, osteoporosis, skin atrophy and reduction of said products of the tested antioxidants (U.S. Pat. general aging. KL functions by converting members of the No. 6,756,045). Fibroblast Growth Factor/FGFR family and mediating trans 0143 Idebenone (chemical) derivative: Derivatives of ide epithelial calcium transport and metabolism. This effect on benone may also be suitable for use methods described cellular calcium levels may tie into apoptosis and membrane herein, including the maintenance of telomere length and potentials. increase in the longevity of cellular lifespan. Such derivatives 0152 KU70 (Thyroid Autoantigen, 70 kD: G22P1): Part may include the salts and/or esters of idebenone, protein of a cell cycle dependant (associated with the chromosomes US 2014/0079836A1 Mar. 20, 2014
in interphase and dissociated in prophase) dsDNA binding and shape of the nucleus. An intermediate filament thought to complex with a proposed role in DNA repair or transposition. be involved in Hutchinson-Guilford progeria syndrome. 0153 Label: Any molecule or composition bound to an 0159 Melanocyte: A pigment producing cell that provides analyte, analyte, detector reagent, analog or binding partner color to skin, hair and eyes. It is most commonly found in the that is detectable by spectroscopic, photochemical, biochemi bottom layer of the skins epidermis and mid-layer of the eye. cal, immunochemical, electrical, optical or chemical means. (0160 MDH1 (Soluble Malate Dehydrogenase 1): Cata Non-limiting examples of labels include enzymes, colloidal lyzes a reversible reaction in the citric acid cycle (L-malate-- gold particles, colored latex particles, radioactive isotopes, NAD to form NADH--Oxaloacetate). MDH1 is located on the enzyme Substrates, co-factors, ligands, chemiluminescent or same chromosome as IDH. fluorescent agents, haptens, protein-adsorbed silver particles, (0161. MDH2 (Mitochondrial Malate Dehydrogenase): protein-adsorbed iron particles, protein-adsorbed copper par Mitochondrial bound dehydrogenase. MDH2 catalyzes a ticles, protein-adsorbed selenium particles, protein-adsorbed reversible reaction in the citric acid cycle (L-malate--NAD to Sulphur particles, protein-adsorbed tellurium particles, pro form NADH--oxaloacetate). tein-adsorbed carbon particles, and protein-coupled dye sacs. (0162 ME1 (Malic Enzyme 1): NADP+-dependent enzyme Methods for labeling and guidance in the choice of labels link between the citric acid cycle and glycolytic pathway that appropriate for various purposes are discussed, e.g., in Sam catalyzes the reversible oxidative decarboxylation of malate brook et al., Molecular Cloning. A Laboratory Manual. to form pyruvate, CO and NADPH. CSHL, New York, 1989 and Ausubel et al., Current Protocols (0163 ME2 (Malic Enzyme 2): Mitochondrial enzyme in Molecular Biology, Greene Publ. Assoc. and Wiley-Inter determined by nuclear genes, similar to ME1. Sciences, 1998. The attachment of a compound (e.g., an anti (0164 ME3 (Malic Enzyme 3): NADP+-dependent mito body) to a label can be through covalent bonds, adsorption chondrial enzymes, catalyzes the oxidative decarboxylation processes, hydrophobic and/or electrostatic bonds, as in che of malate to pyruvate using NAD+ or NADP+ as cofactors. lates and the like, or combinations of these bonds and inter 0.165 Meristematic: The quality of being undifferentiated actions and/or may involve a linking group. or progenitor cell like, and can apply to both cells and tissues. 0154 Specific example detectable labels suitable for con 0166 Merkel cell: Large oval cells found in the epidermis jugating to antibodies, including antibodies used in high of vertebrates and associated with the sense of touch. throughput Screening systems, include radiolabels and other 0.167 Mitochondrion (mitochondria): The small, mem detectable molecules linked to the antibodies using various brane lined organelle providing most of the cells chemical chemical linking groups or bifunctional peptide linkers. A energy through the electron transport systems production of terminal hydroxyl can be esterified with inorganic acids, e.g., adenosine triphosphate. The mitochondria are also involved 'P phosphate, or ''C organic acids, or else esterified to in cell growth, cellular signaling, cell cycle regulation, apo provide linking groups to the label. Enzymes of interest as ptosis, and cellular differentiation. The loss of mitochondrial detectable labels will primarily be hydrolases, particularly membrane potentials/functions and deletions of the mtDNA esterases and glycosidases, or oxidoreductases, particularly are also thought to be key events in the aging of cells. peroxidases. Fluorescent compounds include fluorescein and 0168 Mitochondrial Biogenesis: The process by which its derivatives, rhodamine and its derivatives, dansyl, umbel new mitochondria are formed during the lifespan of the cell. liferone, and so forth. Chemiluminescers include luciferin, 0169 Mitochondrial Damage: any physical alteration in and 2,3-dihydrophthalazinediones (e.g., luminol), and the mitochondrial components, including mtDNA, proteins like. (such as, one or more OXPHOS proteins), or lipids, that alters 0155 Langerhans cell: (Dendritic) cells found in the epi mitochondrial function in a way that is detrimental to cell dermis and lymph nodes responsible for the capture, uptake physiology, growth or faithful replication. and processing of antigens and foreign particles in the skin. 0170 Mitochondrial Disorder: A disease resulting from 0156 Lifespan: The length of time a cell, tissue or organ altered mitochondrial function, caused by any alteration or ism remains viable. There are 2 components to this the Poten combination of alterations of mitochondrial components (for tial (or Inherent) Lifespan defined as the unaltered lifespan of instance, mitochondrial protein (Such as, one or more the cell or organism based solely on genetic factors and the OXPHOS proteins), mtDNA, or lipid) caused by genetic and/ Observed Lifespan defined as the length of time the cell or or environmental factors, including autotoxicity caused by organism will remain viable when all damaging (Oxidative normal cellular metabolic processes. “Late onset mitochon Stress, Poor Nutrition) stimuli are factored in. drial disorder” or “late onset disease” means such diseases as 0157 Liposome or liposomal: An aqueous compartment late onset diabetes (Diabetes Type I), Huntington's, Parkin or pocket, often microscopic, enclosed by a bimolecular Sons and Alzheimer's diseases, ALS (amyotrophic lateral phospholipid membrane; a lipid vesicle. Liposomes have sclerosis), Schizophrenia and the like, wherein the subject is been exploited to deliver compounds and compositions, for free of the disease in early life, but develops the disease during instance cells; when the liposome comes in contact with puberty or thereafter, sometimes as late as age 70 or 80. another membrane (e.g., a cell membrane), the two mem (0171 MTND5 (NADHDehydrogenase Subunit 5): 1 of 7 branes fuse and the encapsulated liposomal contents are of the mitochondrial subunits of the respiratory complex. released into the cell. This effectively transports the aqueous Complex I (of which subunit 5 is a part) accepts electrons contents trapped in the liposome across and into the contacted from NADH and transfers them to ubiquinone and uses the membrane-bound compartment (e.g., cell). Means of prepar energy released to drive protons across the inner mitochon ing liposomes are well known to those of skill in the art. See, drial membrane. e.g., Betageri et al., Liposome Drug Delivery Systems, Tech (0172 MTHD1 (Methylenetetrahydrofolate Dehydroge nomic Publishing Co., Inc., Lancaster, Pa. (1993). nase 1): Encodes trifunctional protein in eukaryotes that is 0158 LMNA (Lamin A/C): Gene that codes for structural involved in the conversion of 1-carbon derivatives into sub components of the protein network that determines the size strates for methionine and purine synthesis. US 2014/0079836A1 Mar. 20, 2014
(0173 MTHFD1L (Methylenetetrahydrofolate Dehydro 0185. NOXA1 (NADPH Oxidase Activator 1): Activates genase, NADP+Dependent 1 Like): Localized to the mito (more effective with NOX) the various NADPH oxidases that chondria and involved in THF (tetrahydrofolate) synthesis generate reactive oxygen species (ROS). therein. Involved in synthesis of purines and thymidylate: 0186 NOXO1 (NADPH Oxidase Organizer 1): Respon Supporting cellular methylation reactions. sible for targeting NOX activators to NOX and relocating (0174 MTHFR (5-10, Methylenetetrahydrofolate Reduc NOX to subcellular compartments. tase): Catalyzes the formation of a substrate for remethylating 0187 NRF1 (Nuclear Respiratory Factor 1): A transcrip methionine. tion factor that codes for respiratory Subunits and components (0175 NADK (NAD Kinase): Catalyzes formation of of the mitochondrial transcription and replication machinery, NADP which is reduced to act as an electron donor for various which allows for the transcription of mitochondrial DNA. biochemical reactions. 0188 NRF2 (Nuclear Factor Erythroid 2 Like 2) A gene (0176 NADSYN1 (NAD Synthetase 1): Responsible for coding for a family of leucine Zipper transcription factors the final step in the formation of NAD, a coenzyme in redox with some highly conserved regions with FOS and JUN. reactions, a Substrate for posttranslational modifications and Regulates transcription of SSAT gene and aids in protein a common cell signaling mechanism. interactions. 0177 NDUFA2, 3, 4, 4L2, 5, 6, 7, 9, 10 and 12 (NADH (0189 NQO1 (NAD(P)H Dehydrogenase Quinone 1): A Ubiquinone Oxidoreductase 1 alpha, Subcomplexes 2, 3, 4, two-electron reductase involved in the detoxification of 4L2, 5, 6, 7, 9, 10 and 12): Genes coding for the various quinones and protection against benzene metabolites. Subcomplexes that compose the first and largest complex in 0190. Nucleic acid: A deoxyribonucleotide or ribonucle the respiratory chain (Complex I). Complex I is responsible otide polymer in either single or double stranded form, and for NADH oxidation, ubiquinone reduction and proton ejec unless otherwise limited, encompassing known analogues of tion from the mitochondria. natural nucleotides that hybridize to nucleic acids in a manner (0178 NDUFB2, 3, 5, 6, 7, 8, and 9 (NADH-Ubiquinone similar to naturally occurring nucleotides. Oxidoreductase 1 beta, subcomplexes 2, 3, 5, 6, 7, 8 and 9): 0191) Nucleic acid array: An arrangement of nucleic acids Genes coding for the various Subcomplexes in the hydrophilic (such as DNA or RNA) in assigned locations on a matrix. Such region of the first and largest complex in the respiratory chain as that found in cDNA arrays, or oligonucleotide arrays. (Complex I). Complex I is responsible for NADH oxidation, 0.192 Nucleic acid molecules representing genes: Any ubiquinone reduction and proton ejection from the mitochon nucleic acid, for example DNA (intron or exon or both), dria. cDNA or RNA, of any length suitable for use as a primer (e.g., (0179 NDUFC2 (NADH-Ubiquinone Oxidoreductase 1 for in vitro amplification), probe or other indicator molecule, subunit c2): Gene coding for the subunit C2 of the first and and that is informative about the corresponding gene. largest complex in the respiratory chain (Complex I). Com 0193 Nucleotide: A grouping of a phosphate, a sugar and plex I is responsible for NADH oxidation, ubiquinone reduc a nitrogenous base that form the structures of RNA and DNA tion and proton ejection from the mitochondria. (the RNA or DNA is determined by which sugar, ribose or 0180 NDUFS2, 4, 5, 7, and 8 (NADH-Ubiquinone Oxi deoxyribose, is involved in the grouping). "Nucleotide' doreductase Fe—S Proteins 2, 4, 5, 7, and 8): Genes coding includes, but is not limited to, a monomer that includes a base for the iron sulfur protein (IP) fraction of the first and largest linked to a Sugar, Such as a pyrimidine, purine or synthetic complex in the respiratory chain (Complex I). Complex I is analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in responsible for NADH oxidation, ubiquinone reduction and a polynucleotide. A nucleotide sequence refers to the proton ejection from the mitochondria. sequence of bases in a polynucleotide. 0181 NDUFV2 and 3 (NADH-Ubiquinone Oxidoreduc 0194 Oligonucleotide: A linear single-stranded poly tase Flavoprotein 2 and 3): Genes coding for 24 kD fraction of nucleotide sequence ranging in length from 2 to about 5,000 the first and largest complex in the respiratory chain (Com bases, for example a polynucleotide (such as DNA or RNA) plex I). Complex I is responsible for NADH oxidation, which is at least 6 nucleotides, for example at least 10, 12, 15, ubiquinone reduction and proton ejection from the mitochon 18, 20, 25, 50, 100, 200, 1,000, or even 5,000 nucleotides dria. long. Oligonucleotides are often synthetic but can also be 0182 NFKB1 (Nuclear Factor Kappa B; Subunit 1): Gene produced from naturally occurring polynucleotides. that encodes for protein involved in the inflammatory process 0.195 An oligonucleotide analog refers to moieties that and responsible for the induction of many inflammatory pro function similarly to oligonucleotides but have non-naturally teins. Inhibition of NFKB1 has been shown to lead to delayed occurring portions. For example, oligonucleotide analogs can cell growth, apoptosis and inappropriate immune cell devel contain non-naturally occurring portions, such as altered opment. Sugar moieties or inter-Sugar linkages. Such as a phospho 0183 NHP2L1 (Non-Histone Chromosome Protein 2, S. rothioate oligodeoxynucleotide. Functional analogs of natu Cerevisiae, Homolog Like 1): Component of the spliceosome rally occurring polynucleotides can bind to RNA or DNA, complex required for activation of the complex. The spliceo and include PNA molecules. Such analog molecules may also Some is involved in removing introns from a transcribed bind to or interact with polypeptides or proteins. pre-RNA segment. 0196. Oxidative Stress: An imbalance within the cell, tis 0184) NOX1, 3, 4 and 5 (NADPH Oxidase 1, 3, 4 and 5): Sue or organism which results in a diminished ability to: Associated with the plasma membrane of multiple cell types, reduce (or detoxify) biological reactive chemical intermedi these NADPH oxidases aid the production of superoxide by a ates, repair the damage caused by reactive chemical interme 1-electron reduction of oxygen with NADPH as the electron diates, or maintain the cellular reduction potential most often donor. resulting in apoptosis. US 2014/0079836A1 Mar. 20, 2014
(0197) PARP1 (Poly ADP Ribose Polymerase 1): Chroma of the telomere will increase the replicative capacity and time tin associated enzyme that may signal altered metabolic con until apoptosis in living cells resulting in a prolonged duration ditions to the chromatin. NAD dependent, post translational, of cell “health' and viability. Methods for the isolation and ADP ribosylation plays a role in DNA repair (strand breaks, characterization of polyphenols are well known in the art; in etc.) and recovery of cells from DNA damage. PARP1 acti addition, various purified polyphenols are commercially Vation is required for translation of apoptosis inducing factor available. from the mitochondria to the nucleus (required in PARP1 (0208 POT1 (Protection of Telomeres 1): Codes for a dependant cell death). PARP1 possibly plays a role in many widely conserved protein (across eukaryotes) which binds a other cellular types and functions (spindle cell formation, G rich strand of telomeric DNA and protects chromosome neurons, and gene targeting to list a few). ends. (0198 PARP2 (Poly ADP Ribose Polymerase 2): An ADP (0209 PPARG (Peroxisome Proliferator Activated Protein ribosyltransferase that is activated as an early cellular Gamma): Member of the nuclear hormone receptor subfam response to DNA strand breaks. This class of enzymes modi ily of transcription factors. PPARs form heterodimers with fies nuclear proteins by ADP-ribosylation which is required members of the retinoid receptor family and these structures for DNA repair, regulation of apoptosis and maintaining regulate transcription/activation of a variety of genes. Spe genome stability. cifically, PPARG is thought to be involved in adipocyte dif (0199 PGC-1 Alpha (Peroxisome Proliferator-Activated ferentiation, proinsulin biosynthesis, insulin release and acti Receptor-Gamma, Coactivator 1, Alpha; PPARGC1A): A Vation of inflammatory response genes. transcription coactivator of nuclear receptors which greatly 0210 Proanthocyanidins (Oligomeric Proanthocyanidin; increases the transcriptional activity of PPARgamma, thyroid OPC): A class of flavonoids (plant secondary metabolic prod hormone receptor, activates the expression of key enzymes in ucts including catechins) most commonly found in many the respiratory chain, increases the cellular content of mito plants, with the extracting into wine being the most common chondrial DNA and stimulates mitochondrial biogenesis. occurrence. They area also found in coffee cherry, and (0200 POLB (DNA Polymerase Beta): Another DNA extracts made therefrom, and have been shown to be able to polymerase which is primarily responsible for the base exci absorb many oxygen free radicals. Methods for the isolation sion repair required for DNA maintenance, replication, and characterization of proanthocyanidins are well known in recombination and drug resistance in eukaryotic cells. the art; in addition, specific proanthocyanidin compounds are 0201 POLD3 (DNA Directed Polymerase Delta3): A por commercially available. tion of the DNA polymerase delta complex (along with 0211 Probes and primers: Nucleic acid probes and prim PCNA, POLD1, 2, and 4) involved in replication and repair. ers can be readily prepared based on the nucleic acid mol (0202 POLE (DNA Polymerase Epsilon): Nuclear poly ecules provided as indicators of taste reception or likely taste merase (1 of 4) in eukaryotic cells responsible for DNA repair reception. It is also appropriate to generate probes and prim and replication of chromosomal DNA. ers based on fragments or portions of these nucleic acid 0203 POLG (DNA Polymerase Gamma): An enzyme molecules, particularly in order to distinguish between and present in both the nucleus and mitochondria and plating a among different alleles and haplotypes within a single gene. role in mitochondrial replication. A "proof reading enzyme Also appropriate are probes and primers specific for the that increases the fidelity of mitochondrial replication and reverse complement of these sequences, as well as probes and transcription. primers to 5' or 3' regions. 0204 POLI (DNA Polymerase Iota): Crystal structured 0212 A probe comprises an isolated nucleic acid attached human DNA polymerase that binds to an incoming nucleotide to a detectable label or other reporter molecule. Typical labels and template primer. POLI assists in bypassing DNA damage include radioactive isotopes, enzyme Substrates, co-factors, by incorporating deoxynucleotides directly across from DNA ligands, chemiluminescent or fluorescent agents, haptens, lesions. and enzymes. Methods for labeling and guidance in the 0205 POLL (DNA Polymerase Lambda): On of the many choice of labels appropriate for various purposes are dis DNA polymerases in humans that contributes to both repli cussed, e.g., in Sambrook et al. (In Molecular Cloning. A cation of the entire genome and the DNA repair process Laboratory Manual, CSHL, New York, 1989) and Ausubel et (telomere mediated). al. (In Current Protocols in Molecular Biology, John Wiley & 0206 Polymorphism(s): The difference in DNA Sons, New York, 1998). sequences among a population for the same gene. Generally 0213 Primers are short nucleic acid molecules, for there are two or more alternative forms of a gene (which has instance DNA oligonucleotides 10 nucleotides or more in changes in the nucleotide sequence) that may be harmless or length. Longer DNA oligonucleotides may be about 15, 20, associated with a diseases state. This correlation to possible 25, 30 or 50 nucleotides or more in length. Primers can be disease states has made tracking and identifying polymor annealed to a complementary target DNA strand by nucleic phisms a possible method to determine causative mutations acid hybridization to form a hybrid between the primer and for said disease states. the target DNA strand, and then the primer extended along the 0207 Polyphenols (some of which may be referred to as target DNA strand by a DNA polymerase enzyme. Primer Tea Derived Antioxidants): Ester bond containing polyphe pairs can be used for amplification of a nucleic acid sequence, nols like EGCG (epigallocatechin-3-gallate), EGC (epigallo e.g., by the polymerase chain reaction (PCR) or other in vitro catechin), ECG (epicatechin-3-gallate), EC (epicatechin), nucleic-acid amplification methods known in the art. GCG (gallocatechin gallate), GC (gallocatechin), C (cat 0214 Methods for preparing and using nucleic acid echin) and/or CG (catechin gallate) can be used to extend the probes and primers are described, for example, in Sambrook lifespan of living cells through direct influence over the gene et al. (In Molecular Cloning: A Laboratory Manual. CSHL, expression of the telomere length maintenance unit and New York, 1989), Ausubeletal. (ed.) (In Current Protocols in related proteins. This extension or preservation of the length Molecular Biology, John Wiley & Sons, New York, 1998), US 2014/0079836A1 Mar. 20, 2014
and Innis et al. (PCR Protocols, A Guide to Methods and nucleic acid represents at least 50% of the total nucleic acid Applications, Academic Press, Inc., San Diego, Calif., 1990). content of the preparation. In certain embodiments, a Substan Amplification primer pairs (for instance, for use with poly tially pure nucleic acid will represent at least 60%, at least merase chain reaction amplification) can be derived from a 70%, at least 80%, at least 85%, at least 90%, or at least 95% known sequence Such as any of the bitter taste receptor or more of the total nucleic acid content of the preparation. sequences and specific alleles thereof described herein, for 0222 Quinic Acid (1S,3R,4S.5R)-1,3,4,5-tetrahydroxy example, by using computer programs intended for that pur cyclohexanecarboxylic acid: Discovered in the 1800s, this pose such as PRIMER (Version 0.5, (C) 1991, Whitehead crystalline acid compound is formed synthetically by Institute for Biomedical Research, Cambridge, Mass.). hydrolysis of chlorogenic acid, but is found naturally in the 0215 One of ordinary skill in the art will appreciate that coffee cherry. Thought to provide the “acidity of coffee, this the specificity of a particular probe or primer increases with compound, aside from the usual antioxidant capabilities of its length. Thus, for example, a primer comprising 30 con the other coffee cherry acids, is a versatile starting compound secutive nucleotides of a bitter taste receptor protein encoding for the synthesis of new synthetic compounds as well. Meth nucleotide will anneal to a target sequence, such as homolog ods for the isolation and characterization of quinic acid are of a designated taste receptor protein, with a higher specificity well known in the art; in addition, this compound is commer than a corresponding primer of only 15 nucleotides. Thus, in cially available. order to obtain greater specificity, probes and primers can be 0223 RAD50 (S. cerevisiae; homolog of RAD50): In selected that comprise at least 20, 23, 25, 30, 35, 40, 45, 50 or yeast this gene aids in the repair of double stranded DNA more consecutive nucleotides of a taste receptor gene. breaks by end repair and chromosomal integration (ALT 0216. Also provided are isolated nucleic acid molecules pathway of telomere maintenance). It is thought to have much that comprise specified lengths of bitter taste receptor-encod the same function in humans as it has been found to associate ing nucleotide sequences. Such molecules may comprise at with the TRF2 complex previously described. least 10, 15, 20, 23, 25, 30, 35, 40, 45 or 50 or more (e.g., at 0224 RAD51 (S. cerevisiae; homolog of RAD51): In least 100, 150, 200,250, 300 and so forth) consecutive nucle prokaryotic cells this gene encodes for proteins responsible otides of these sequences or more. These molecules may be for promoting strand exchange between homologous sections obtained from any region of the disclosed sequences (e.g., a of double stranded DNA (this is also part of the ALT pathway specified nucleic acid may be apportioned into halves or of telomere maintenance). In eukaryotic cells, the function is quarters based on sequence length, and isolated nucleic acid also thought to be similar and involved in replication and molecules may be derived from the first or second halves of Strand exchange. the molecules, or any of the four quarters, etc.). A cDNA or 0225 RAP1 (GTPase Activating Protein 1): Encodes for a other encoding sequence also can be divided into Smaller member of a family of p21 proteins whose activities are regions, e.g. about eighths, sixteenths, twentieths, fiftieths, related to binding and hydrolysis of GTP and function in cell and so forth, with similar effect. differentiation and growth. 0217. Another mode of division, provided by way of 0226 Reactive Oxygen Species (ROS): Very small, example, is to divide a bitter taste receptor sequence based on organic or inorganic, highly reactive ions or molecules having the regions of the sequence that are relatively more or less unpaired electrons in a valence shell including but not limited homologous to other bitter taste receptor sequences. to free radicals, peroxides and oxygen ions. 0218 Nucleic acid molecules may be selected that com 0227 Recloning: The process in which a genetically iden prise at least 10, 15, 20, 25, 30,35, 40, 50, 100, 150,200,250, tical organism is made from the genetic material of a previ 300 or more consecutive nucleotides of any of these or other ously cloned (creating genetically identical organisms from portions of a bitter taste receptor nucleic acid molecule or a the genetic material of a single "parent organism through the specific allele thereof, such as those disclosed herein. Thus, use of genetic recombination and in vitro fertilization) organ representative nucleic acid molecules might comprise at least ism 10 consecutive nucleotides of a sequence listed in DATA 0228 Resveratrol (3,5,4'-trihydroxy stilbene) belongs to a TABLE 7 or Array 2. family of compounds known as phytoalexins. These com 0219 Procyanidins: Tannic (polyphenols compounds that pounds are synthesized by various plants including grapes, bind or shrink proteins) compounds found in plants and espe knotweed, blueberries, some pine trees and other plants as cially tea and grape seed. Procyanidins are commonly asso part of their natural defense mechanisms in response to stress, ciated with the bitter, astringent taste of wine. The com injury, invasion by fungi or UV damage. In grapes they are pounds also have a very high antioxidant capacity. Methods concentrated in the grape skin where they protect from UV for the isolation and characterization of procyanidins are well damage and function as antibacterial and anti viral agents. known in the art; in addition, certain procyanidins are com Resveratrol activates the sirtuins which are enzymes which mercially available. produce at least part of the effects of caloric restriction in 0220 PTOP (ACD, Mouse Homolog of ACD): Gene living organisms and caloric restriction has been shown in a responsible for targeting POT1 to telomeres permitting very wide range of species tested to extend the lifespan of telomere extension. PTOP binds both POT1 and TIN2 to the those organisms. TRF1 complex. 0229. The activation of a sirtuin deacetylase protein fam 0221 Purified: The term purified does not require absolute ily member may be used to produce lifespan extension by purity; rather, it is intended as a relative term. Thus, for mimicking caloric restriction in contrast to extending lifespan example, a purified nucleic acid preparation is one in which by protecting or repairing telomeric structure in cells. Acti the specified protein is more enriched than the nucleic acid is Vating compounds may be polyphenol(s) from plants such as in its generative environment, for instance within a cell or in chalcone, stilbene, flavone or other sirtuin modulating com a biochemical reaction chamber. A preparation of Substan pounds derived from plants or created by other synthetic tially pure nucleic acid may be purified such that the desired processes described herein. Methods for the isolation and US 2014/0079836A1 Mar. 20, 2014
characterization of resveratrol are well known in the art; in 0237 SHC1 (SHC Transforming Protein 1): By coupling addition, this compound is commercially available. growth factor receptors to members of the RAS pathway, 0230 Ribosome: A structure of protein and RNA involved mitogenic signal transduction is regulated. The protein in translation, or the expression of genetic code from nucleic encoded by SHC1 acts as an adaptor in many signaling path acid into protein. ways specifically translating reactive oxygen damage into 0231 Sequence identity: The similarity between two cell death. nucleic acid sequences, or two amino acid sequences, is 0238 Small interfering RNAs (siRNAs): Synthetic or expressed in terms of the similarity between the sequences, naturally-produced small double stranded RNAs (dsRNAs) otherwise referred to as sequence identity. Sequence identity that can induce gene-specific inhibition of expression in is frequently measured in terms of percentage identity (or invertebrate and vertebrate species are provided. These RNAs similarity or homology); the higher the percentage, the more are suitable for interference or inhibition of expression of a similar the two sequences are. Homologs or orthologs of target gene and comprise double stranded RNAs of about 15 nucleic acid oramino acid sequences will possess a relatively to about 40 nucleotides (for instance, 20-25 nucleotides), high degree of sequence identity when aligned using standard often containing a 3' and/or 5' overhang on each strand having methods. This homology will be more significant when the a length of 0- to about 5-nucleotides, wherein the sequence of orthologous proteins or nucleic acids are derived from species the double stranded RNAs is substantially identical to a por which are more closely related (e.g., human and chimpanzee tion of a coding region of the target gene for which interfer sequences), compared to species more distantly related (e.g., ence or inhibition of expression is desired. The double human and C. elegans sequences). Typically, orthologs are at stranded RNAs can be formed from complementary ssRNAs least 50% identical at the nucleotide level and at least 50% or from a single stranded RNA that forms a hairpin or from identical at the amino acid level when comparing human expression from a DNA vector. These molecules function in orthologous sequences. RNA silencing a method in which sequence specific gene 0232 Methods of alignment of sequences for comparison expression is reduced/eliminated by the incorporation of the are well known. Various programs and alignment algorithms siRNA in to the RNA induced silencing complex that facili are described in: Smith & Waterman, Adv. Appl. Math. 2:482, tates the degradation of the targeted mRNA. 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; 0239 Specific hybridization: Specific hybridization refers Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, to the binding, duplexing, or hybridizing of a molecule only 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & or Substantially only to a particular nucleotide sequence when Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. that sequence is present in a complex mixture (e.g. total 16:10881-90, 1988; Huang et al. Computer Appls. Biosci. 8, cellular DNA or RNA). Specific hybridization may also occur 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, under conditions of varying stringency. 1994. Altschulet al., J. Mol. Biol. 215:403-10, 1990, presents 0240 Hybridization conditions resulting in particular a detailed consideration of sequence alignment methods and degrees of stringency will vary depending upon the nature of homology calculations. the hybridization method of choice and the composition and length of the hybridizing DNA used. Generally, the tempera 0233. The NCBI Basic Local Alignment Search Tool ture of hybridization and the ionic strength (especially the (BLAST) (Altschulet al., J. Mol. Biol. 215:403-10, 1990) is Na+ concentration) of the hybridization buffer will determine available from several sources, including the National Center the stringency of hybridization. Calculations regarding for Biotechnology Information (NCBI, Bethesda, Md.) and hybridization conditions required for attaining particular on the Internet, for use in connection with the sequence analy degrees of stringency are discussed by Sambrook et al. (In: sis programs blastp, blastin, blastX, thlastn and thlastX. Each of Molecular Cloning: A Laboratory Manual, Cold Spring Har these sources also provides a description of how to determine bor, N.Y., 1989 ch. 9 and 11). By way of illustration only, a sequence identity using this program. hybridization experiment may be performed by hybridization 0234 Homologous sequences are typically characterized of a DNA molecule to a target DNA molecule which has been by possession of at least 60%, 70%, 75%, 80%, 90%. 95% or electrophoresed in an agarose gel and transferred to a nitro at least 98% sequence identity counted over the full length cellulose membrane by Southern blotting (Southern, J. Mol. alignment with a sequence using the NCBI Blast 2.0, gapped Biol. 98:503, 1975), a technique well known in the art and blastp set to default parameters. Queries searched with the described in Sambrook et al. (Molecular Cloning: A Labora blastin program are filtered with DUST (Hancock and Arm tory Manual, Cold Spring Harbor, N.Y., 1989). strong, Comput. Appl. Biosci. 10:67-70, 1994). It will be 0241 Traditional hybridization with a target nucleic acid appreciated that these sequence identity ranges are provided molecule labeled with PI-dCTP is generally carried out in for guidance only; it is entirely possible that strongly signifi a solution of high ionic strength such as 6xSSC at a tempera cant homologs could be obtained that fall outside of the ture that is 20-25° C. below the melting temperature, T. ranges provided. described below. For Southern hybridization experiments 0235 Nucleic acid sequences that do not show a high where the target DNA molecule on the Southern blot contains degree of identity may nevertheless encode similar amino 10 ng of DNA or more, hybridization is typically carried out acid sequences, due to the degeneracy of the genetic code. It for 6-8 hours using 1-2 ng/ml radiolabeled probe (of specific is understood that changes in nucleic acid sequence can be activity equal to 10 CPM/ug or greater). Following hybrid made using this degeneracy to produce multiple nucleic acid ization, the nitrocellulose filter is washed to remove back sequences that all encode Substantially the same protein. ground hybridization. The washing conditions should be as 0236 An alternative indication that two nucleic acid mol stringent as possible to remove background hybridization but ecules are closely related is that the two molecules hybridize to retain a specific hybridization signal. to each other under Stringent conditions, as described under 0242. The term T, represents the temperature (under “specific hybridization.” defined ionic strength, pH and nucleic acid concentration) at US 2014/0079836A1 Mar. 20, 2014 which 50% of the probes complementary to the target quence) of the target sequence. The term “mismatch probe' sequence hybridize to the target sequence at equilibrium. refers to probes whose sequence is deliberately selected not to Because the target sequences are generally present in excess, be perfectly complementary to a particular target sequence. at T 50% of the probes are occupied at equilibrium. The T. 0248 Transcription levels can be quantitated absolutely or of such a hybrid molecule may be estimated from the follow relatively. Absolute quantitation can be accomplished by ing equation (Bolton and McCarthy, Proc. Natl. Acad. Sci. inclusion of known concentrations of one or more target USA 48: 1390, 1962): nucleic acids (for example control nucleic acids or with a T=81.5°C.-16.6(logoNa)+0.41 (% G+C)-0.63(% known amount the target nucleic acids themselves) and ref formamide)-(600/1) erencing the hybridization intensity of unknowns with the where 1=the length of the hybrid in base pairs. known target nucleic acids (for example by generation of a 0243 This equation is valid for concentrations of Na' in standard curve). the range of 0.01 M to 0.4 M, and it is less accurate for 0249 Solid support (or substrate): Any material which is calculations of Tm in solutions of higher Na". The equation insoluble, or can be made insoluble by a Subsequent reaction. is also primarily valid for DNAs whose G+C content is in the Numerous and varied solid Supports are known to those in the range of 30% to 75%, and it applies to hybrids greater than art and include, without limitation, nitrocellulose, the walls of 100 nucleotides in length (the behavior of oligonucleotide wells of a reaction tray, test tubes, polystyrene beads, mag probes is described in detail in Ch. 11 of Sambrook et al. netic beads, membranes, microparticles (such as latex par (Molecular Cloning. A Laboratory Manual, Cold Spring ticles), and sheep (or other animal) red blood cells. Any Harbor, N.Y., 1989). suitable porous material with sufficient porosity to allow 0244 Thus, by way of example, for a 150 base pair DNA access by detector reagents and a suitable Surface affinity to probe derived from a cDNA (with a hypothetical % GC of immobilize capture reagents (e.g., monoclonal antibodies) is 45%), a calculation of hybridization conditions required to contemplated by this term. For example, the porous structure give particular stringencies may be made as follows: For this of nitrocellulose has excellent absorption and adsorption example, it is assumed that the filter will be washed in 0.3x qualities for a wide variety of reagents, for instance, capture SSC solution following hybridization, thereby: Na–0.045 reagents. Nylon possesses similar characteristics and is also M; % GC-45%. Formamide concentration=0; 1=150 base Suitable. Microporous structures are useful, as are materials pairs; Tm=81.5-16.6(logo Na")+(0.41x45)-(600/150); with gel structure in the hydrated state. and so T-74.4° C. 0250) Further examples of useful solid supports include: 0245. The T of double-stranded DNA decreases by 1-1. natural polymeric carbohydrates and their synthetically 5°C. with every 1% decrease in homology (Bonner et al., J. modified, cross-linked or substituted derivatives, such as Mol. Biol. 81:123, 1973). Therefore, for this given example, agar, agarose, cross-linked alginic acid, Substituted and cross washing the filter in 0.3xSSC at 59.4-64.4°C. will produce a linked guar gums, cellulose esters, especially with nitric acid stringency of hybridization equivalent to 90%; that is, DNA and carboxylic acids, mixed cellulose esters, and cellulose molecules with more than 10% sequence variation relative to ethers; natural polymers containing nitrogen, Such as proteins the target cDNA will not hybridize. Alternatively, washing and derivatives, including cross-linked or modified gelatins: the hybridized filter in 0.3xSSC at a temperature of 65.4-68. natural hydrocarbon polymers, such as latex and rubber, Syn 4°C. will yield a hybridization stringency of 94%; that is, thetic polymers which may be prepared with suitably porous DNA molecules with more than 6% sequence variation rela structures, such as vinyl polymers, including polyethylene, tive to the target cloNA molecule will not hybridize. The polypropylene, polystyrene, polyvinylchloride, polyvinylac above example is given entirely by way of theoretical illus etate and its partially hydrolyzed derivatives, polyacryla tration. It will be appreciated that other hybridization tech mides, polymethacrylates, copolymers and terpolymers of niques may be utilized and that variations in experimental the above polycondensates, such as polyesters, polyamides, conditions will necessitate alternative calculations for Strin and other polymers, such as polyurethanes or polyepoxides; gency. porous inorganic materials such as Sulfates or carbonates of 0246 Stringent conditions may be defined as those under alkaline earth metals and magnesium, including barium Sul which DNA molecules with more than 25%, 15%, 10%, 6% fate, calcium sulfate, calcium carbonate, silicates of alkali or 2% sequence variation (also termed “mismatch') will not and alkaline earth metals, aluminum and magnesium; and hybridize. Stringent conditions are sequence dependent and aluminum or silicon oxides or hydrates, such as clays, alu are different in different circumstances. Longer sequences mina, talc, kaolin, Zeolite, silica gel, or glass (these materials hybridize specifically at higher temperatures. Generally, may be used as filters with the above polymeric materials); stringent conditions are selected to be about 5° C. lower than and mixtures or copolymers of the above classes, such as graft the thermal melting point T, for the specific sequence at a copolymers obtained by initializing polymerization of Syn defined ionic strength and pH. An example of stringent con thetic polymers on a pre-existing natural polymer. ditions is a salt concentration of at least about 0.01 to 1.0 M 0251. It is contemplated that porous solid supports, such as Naion concentration (or other salts) at pH 7.0 to 8.3 and a nitrocellulose, described hereinabove are preferably in the temperature of at least about 30°C. for short probes (e.g. 10 form of sheets or strips. The thickness of such sheets or strips to 50 nucleotides). Stringent conditions can also be achieved may vary within widelimits, for example, from about 0.01 to with the addition of destabilizing agents such as formamide. 0.5 mm, from about 0.02 to 0.45 mm, from about 0.05 to 0.3 For example, conditions of 5xSSPE (750 mM. NaCl, 50 mM mm, from about 0.075 to 0.25 mm, from about 0.1 to 0.2 mm, Na phosphate, 5 mM EDTA, pH 7.4) and a temperature of or from about 0.11 to 0.15 mm. The pore size of such sheets 25-30°C. are suitable for allele-specific probe hybridizations. or strips may similarly vary within wide limits, for example 0247. A perfectly matched probe has a sequence perfectly from about 0.025 to 15 microns, or more specifically from complementary to a particular target sequence. The test probe about 0.1 to 3 microns; however, pore size is not intended to is typically perfectly complementary to a portion (Subse be a limiting factor in selection of the solid support. The flow US 2014/0079836A1 Mar. 20, 2014
rate of a Solid Support, where applicable, can also vary within matin structures at the end of chromosomes known as telom widelimits, for example from about 12.5 to 90 sec/cm (i.e., 50 eres. Telomerase adds specific DNA sequence repeats to 300 sec/4 cm), about 22.5 to 62.5 sec/cm (i.e., 90 to 250 (TTAGGG in all vertebrates) to the 3' end of DNA strands in sec/4 cm), about 25 to 62.5 sec/cm (i.e., 100 to 250 sec/4 cm), telomeres, which are found at the ends of eukaryotic chromo about 37.5 to 62.5 sec/cm (i.e., 150 to 250 sec/4 cm), or about Somes. Telomerase functions as a reverse transcriptase, and is 50 to 62.5 sec/cm (i.e., 200 to 250 sec/4 cm). In specific associated with a RNA molecule that acts as a template for embodiments of devices described herein, the flow rate is elongating telomeres that have been shortened after replica about 62.5 sec/cm (i.e., 250 sec/4 cm). In other specific tion. embodiments of devices described herein, the flow rate is 0259 Telomere Unit: The telomere (repetitive sequence at about 37.5 sec/cm (i.e., 150 sec/4 cm). the end of most eukaryotic chromosomes composed of chro 0252. The surface of a solid support may be activated by matin) and all associated proteins, enzymes and genetic chemical processes that cause covalent linkage of an agent sequences including, but not limited to: TERT. TRF1, (e.g., a capture reagent) to the Support. However, any other TERF2, TERF21P, DNA Polymerase, POLG, POLB, Suitable method may be used for immobilizing an agent (e.g., POLD3, POLE, POLI, POLL, PARP1, PARP2, PPARG, a capture reagent) to a solid Support including, without limi SHC1, HSPA1A, HSPA1B, and HSPA1 L. tation, ionic interactions, hydrophobic interactions, covalent 0260 TERC (Telomerase RNA Component): A human interactions and the like. The particular forces that result in gene that serves as the template for the telomeric repeat immobilization of an agent on a solid phase are not important known as the telomerase RNA component. for the methods and devices described herein. 0261 TERF2 (Telomeric Repeat Binding Factor 2): Plays 0253) A solid phase can be chosen for its intrinsic ability to a vital role in the protective activity of telomeres. TERF2 may attract and immobilize an agent, such as a capture reagent. convert the telomeres into large duplex loops (called t loops) Alternatively, the Solid phase can possess a factor that has the that may provide a general mechanism for the protection and ability to attract and immobilize an agent, Such as a capture replication of telomeres. reagent. The factor can include a charged substance that is 0262 TERF2IP (TERF2 Interacting Protein): A ubiqui oppositely charged with respect to, for example, the capture tously expressed protein recruited to telomeres by TERF2 to reagent itself or to a charged substance conjugated to the regulate telomere length. This protein is involved in activa capture reagent. In another embodiment, a specific binding tion of RNA polymerase II and central to cellular function and member may be immobilized upon the Solid phase to immo efficiency during rapid growth events. bilize its binding partner (e.g., a capture reagent). In this 0263 TFAM (Transcription Factor A, mitochondrial): example, therefore, the specific binding member enables the Activates mitochondrial transcription by binding to nucle indirect binding of the capture reagent to a solid phase mate otides present in both light and heavy promoters. Also plays a rial. promoter role in the mitochondrial replication process 0254 Except as otherwise physically constrained, a solid through formation of an RNA primer. Support may be used in any Suitable shapes, such as films, 0264. TIN2 (TRF1 Interacting Nuclear Factor 2): Telom sheets, strips, or plates, or it may be coated onto or bonded or ere length in humans is partly controlled by a feedback laminated to appropriate inert carriers, such as paper, glass, mechanism in which telomere elongation by telomerase is plastic films, or fabrics. limited by the accumulation of the TRF1 complex at chromo 0255 Stressed Cells: Cells not able to function fully in some ends. TRF1 itself can be inhibited by PARP activity of their expected capacity either through chemical, biological, its interacting partner tankyrase-1 which disables binding or mechanical interference by an outside agent including but capabilities and removes TRF1 complex from telomeres. not limited to: free radicals, ROS, Toxins, UV radiation and TIN2 is ma mediator of tankyrase 1 and the TRF1 complex. genetic inhibitors like siRNAs. Transient inhibition of TIN2 with small interfering RNA led 0256 Suffruticosol A and B: Stilbenes (a hydrocarbon to diminished telomeric TRF1 signals. These and other data with a trans ethane double bond substituted with a phenyl identified TIN2 as a PARP modulator in the TRF1 complex group on both carbon atoms of the double bond), resveratrol and explained how TIN2 contributes to the regulation of derivatives from peony seeds having antioxidant properties telomere length. and mimicking the effects of resveratrol. 0265 Transcription: The process by which DNA directs 0257 Tea: An aqueous extract of plant material, usually the synthesis of RNA by serving as the nucleotide sequence temperature modulated (hot or cold); often the extracted template for the formation of the RNA nucleotide sequence. material is leaves (commonly, but not limited to dried and/or 0266 TPP1 (Tripeptidyl Peptidase 1): Lysosomal protein fermented leaves of Camelia sinensis green or black tea; that catalyzes the removal of an amino acid from a polypep including white tea), though teas can be made from other tide chain, specifically it sequentially removes tripeptides plant material including bark, flowers, seeds, seed hulls, and from the N termini of proteins. so forth. EGCG a primary element of the tea extract (as well 0267 TRF1 (Telomeric Repeat Binding Factor 1): Regu as all ester-bond containing polyphenols) has been able to lates telomere length via binding to the TTAGGG sites and show a pronounced inhibitory effect on certain types of can tankyrase, TIN2 and PINX1. The TRF1 complex interacts cer cells thought to be through a proteosome inhibition with POT1 (protection of telomeres-1; a single stranded telo mechanism, while the non ester bond containing polyphenols meric binding protein) which controls telomerase mediated have shown diminished or no such effect. In the case of telomere elongation. lifespan extension through telomere length maintenance 0268 Ubiquinone (also known as Coenzyme Q10): A key mechanisms no such distinction is made with the belief that component of the electron transport/cellular respiration/en all forms of polyphenols have an effect. ergy production mechanism, ubiquinone is found in the mito 0258 Telomerase: The enzyme (DNA polymerase) pri chondria of most eukaryotic cells and in great abundance in marily responsible for repairing damage to the special chro cells that have high energy requirements (heart, liver, etc.). US 2014/0079836A1 Mar. 20, 2014 20
Through the process of aerobic cellular respiration ATP is include plural referents unless context clearly indicates oth created for use by the cell (95% of all energy in the human erwise. Similarly, the word 'or' is intended to include “and” body is created in this fashion). Ubiquinone has an affinity for unless the context clearly indicates otherwise. Hence “com electron transfer and is intimately involved in mitochondrial prising A or B' means including A, or B, or A and B. It is cellular respiration specifically between Complex II and III further to be understood that all base sizes oramino acid sizes, where it acts as a transfer agent. Since ubiquinone is a Redox and all molecular weight or molecular mass values, given for (oxidative reduction) agent, it demonstrates free radical nucleic acids or polypeptides are approximate, and are pro quenching capabilities. The fully oxidized form of the com vided for description. Although methods and materials simi pound is known as ubiquinone, when absorbed into the body lar or equivalent to those described herein can be used in the 90% of it converts to the “active' antioxidant form of practice or testing of the present invention, Suitable methods ubiquinol. Methods for the isolation and characterization of and materials are described below. All publications, patent ubiquinone are well known in the art; in addition, this com applications, patents, and other references mentioned herein pound is commercially available. are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and Coenzyme Q10 examples are illustrative only and not intended to be limiting. OH III. Overview of Several Embodiments H3CO CH3 0272 Provided herein in a first embodiment is a method for modulating the lifespan of a cell, tissue, organ or organ ism, comprising contacting the cell, tissue, organ or organism HCO N H with one (or more) of the compounds or compositions dis OH CH3 cussed herein, such as idebenone, or an analog or derivative thereof: a cocoa extract; a coffee cherry extract; quinic acid, Ubiquinol (CoQH2) oran analog orderivative thereof ferulic acid, oran analog or Oo derivative thereof a proanthocyanidin, anthocyanidin, pro cyanidin, or cyanidin; chlorogenic acid, or an analog or HCO CH derivative thereof; a tea extract; or resveratrol or a composi tion derived from or chemically related to resveratrol. By way of example, the coffee cherry extract in Some instances com H3CO N H prises one or more of chlorogenic acid, quinic acid, ferulic acid, caffeic acid or proanthocyanidins. In another example, OH CH tea extract comprises one or more polyphenols selected from EGCG (epigallocatechin-3-gallate), EGC (epigallocatechin), Semiquinone radical (CoQH) ECG (epicatechin-3-gallate), EC (epicatechin). GCG (gallo O catechin gallate), GC (gallocatechin), C (catechin) and CG H3CO CH3 (catechin gallate). In yet another example, the composition derived from or chemically related to resveratrol is selected from the group consisting of viniferin, gnetin H, and Suffru HCO N H ticosol B. In another example, the cocoa extract comprises a polyphenol and/or procyanidin selected from (+) catechin, O CH3 (-) epicatechin, procyanidin oligomers 2 through 18, procya nidin B-5, procyanidin B-2, procyanidin A-2 and/or procya Ubiquinone (CoQ) nidin C-1. 0273. In various of the provided embodiments, modulat 0269 UVA 1: A subset of wavelengths in one of the three ing the lifespan comprises modulating the level and/or activ “bands” of solar lights Ultraviolet Radiation (UVA, UVB and ity of at least one gene selected from the group consisting of UVC) in the relatively higher power, longer wavelength range those listed in Data Table 7 and those listed as part of Array 2. of 340 nm-400 nm. UVA2: Solar radiation wavelength range For instance, modulating comprises (in Some cases) increas of 320 nm-340 nm. UVB: Solar radiation between the wave ing the level of activity of the at least one listed gene. In other lengths of 280 nm-315 nm, capable of causing direct damage cases, there is provided a method wherein modulating com to the DNA of cells. UVC: The short, highest energy wave prises decreasing the level of activity of the at least one listed length radiation (100 nm-280 nm) that is generally filtered by gene. the atmosphere. 0274 Also provided are methods for modulating the lifespan of a cell, tissue, organ or organism, wherein modu 0270. Viniferin: A stilbene (a hydrocarbon with a trans lating comprises modulating the level and/or activity of ten ethane double bond substituted with a phenyl group on both or more of the genes listed as part of Array 2; the genes listed carbonatoms of the double bond), resveratrol derivative from as part of Array 1; VEGFA, HMOX1, CCL4L1, DDC, peony seeds having antioxidant properties and mimicking the NOS2A, SIRT1, TERT, PTGS2, or IFI44; four or more of effects of resveratrol. TERT, TERC, NRF2, POT1, TRF1, TRF2, TIN2, TPP1, 0271 Unless otherwise explained, all technical and scien RAP1, TNKS, TNKS 2, TERF2, TERF2IP, POLG, POLB, tific terms used herein have the same meaning as commonly POLD3, POLE, POLI, POLL, PARP2, PPARG, SHC1, understood by one of ordinary skill in the art to which this PTOP, IFI44, NFKB1, HSPA1A, HSPA1B, HSPA1L, invention belongs. The singular terms “a” “an,” and “the MTND5, HPGD, IDH2, MDH1, MDH2, ME1, ME2, ME3, US 2014/0079836A1 Mar. 20, 2014
MTHD1, MTHFD1 L, MTHFR, NADK, NADSYN1, NDUFB7, NDUFB8, NDUFB9, NDUFC2, NDUFS2, NDUFA2, NDUFA3, NDUFA4, NDUFA4L2, NDUFA5, NDUFS4, NDUFS5, NDUFS7, NDUFS8, NDUFV2, NDUFA6, NDUFA7, NDUFA9, NDUFA10, NDUFA12, NDUFV3, NOX1, NOX3, NOX4, NOX5, NOXA1, NOXO1, NDUFB2, NDUFB3, NDUFB5, NDUFB6, NDUFB7, NQO1, FOXO1, FOXO3, FOXO4, LMNA, NHP2L1, NDUFB8, NDUFB9, NDUFC2, NDUFS2, NDUFS4, RAD50, RAD51, KL and KU70; BCL2, SOD1, TP53, and NDUFS5, NDUFS7, NDUFS8, NDUFV2, NDUFV3, SOD2: BCL2, SOD1, TP53, SOD2, BCL2L1, TIMM22, NOX1, NOX3, NOX4, NOX5, NOXA1, NOXO1, NQO1, TOMM40, IMMP1L, CDKN2A, GADPH, ACTB, HRP1, FOXO1, FOXO3, FOXO4, LMNA, NHP2L1, RAD50, and HGDC; PARP1, PARP2, TERT, TEP1, TPS3, JUN, RAD51, KL and KU70; BCL2, SOD1, TP53, and SOD2: PARP3, PARP4, TERF2, TINF2, and CDKN2A; PARP1, BCL2, SOD1, TP53, SOD2, BCL2L1, TIMM22, TOMM40, PARP2, TERT, TEP1, and TP53; TERF2, POT1, TERT, and IMMP1L, CDKN2A, GADPH, ACTB, HRP1, and HGDC: TPP1; PAPR1, PARP2, PARP3, and PARP4; PARP2, PARP1, PARP2, TERT, TEP1, TPS3, JUN, PARP3, PARP4, CYP19A1, TEP1, BCL2, HSPA1A, ACE, TP53, and TERF2, TINF2, and CDKN2A; PARP1, PARP2, TERT, NFKB1; IGF1, IGF2, PPARG, IL10, APOE, TERT, TNF, TEP1, and TP53; TERF2, POT1, TERT, and TPP1; PAPR1, HLA-DRA, DDC, CCL4L1, NOS2A, and GH1; PARP1, IL6, PARP2, PARP3, and PARP4; PARP2, CYP19A1, TEP1, SIRTT1, KRAS, and HSPA1L: IGF1, IL6, PPARG, IL10, BCL2, HSPA1A, ACE, TP53, and NFKB1; IGF1, IGF2, TERT, TNF, TEP1, HSPA1A, SIRT1, TP53, GH1, NOS2A, PPARG, IL10, APOE, TERT, TNF, HLA-DRA, DDC, and PPC; or another list of genes described herein; or a CCL4L1, NOS2A, and GH1: PARP1, IL6, SIRTT1, KRAS, combination of two or more of these specific lists. By way of and HSPA1L: IGF1, IL6, PPARG, IL10, TERT, TNF, TEP1, example, modulating in Such methods may involve increasing HSPA1A, SIRT1, TP53, GH1, NOS2A, and PPC; or another the level of activity of the at least one listed gene, or decreas list of genes described herein; or a combination of two or ing the level of activity of the at least one listed gene, or more of these specific lists. increasing some while decreasing others. 0275. In one embodiment, modulating the lifespan com 0279. Yet another embodiment is a method of increasing prises modulating the activity or level of at least one of the or decreasing cellular respiration and/or capacity and/or bio telomere length maintenance genes, for instance increasing genesis of mitochondria in a cell, by contacting the cell with the level or activity of at least one telomere length mainte at least one lifespan modulating agent discussed herein, Such nance gene or decreasing the level or activity of at least one as idebenone, or an analog or derivative thereof; a cocoa telomere length maintenance gene. extract; a coffee cherry extract; quinic acid, or an analog or 0276. In another embodiment, modulating the lifespan derivative thereof: ferulic acid, or an analog or derivative comprises modulating the activity or level off telomerase, for thereof: a proanthocyanidin, anthocyanidin, procyanidin, or instance increasing the level or activity of telomerase or cyanidin; chlorogenic acid, oran analog orderivative thereof. decreasing the level or activity of telomerase. Also provided a tea extract; or resveratrol or a composition derived from or are methods that involve differentially modulating the activ chemically related to resveratrol. By way of example, the ity of one or more telomere length maintenance genes so that coffee cherry extract in Some instances comprises one or the lifespan of healthy cells is increased and/or the lifespan of more of chlorogenic acid, quinic acid, ferulic acid, caffeic unhealthy, diseased, damaged or cancerous cells is decreased. acid or proanthocyanidins. In another example, tea extract 0277. In any of the provided methods, the method can take comprises one or more polyphenols selected from EGCG place in a cell that is in vitro. Optionally, the cell is a mam (epigallocatechin-3-gallate), EGC (epigallocatechin), ECG malian cell (e.g., cells are selected from keratinocytes, fibro (epicatechin-3-gallate), EC (epicatechin). GCG (gallocat blasts, melanocytes, endothelial cells, langerhans cells, mer echin gallate), GC (gallocatechin), C (catechin) and CG (cat kel cells, adipocytes, nerve cells, hair, Sweat, oil, stem cells echin gallate). In yet another example, the composition and/or muscle cells), a plant cell, a microbial cell, a stem cell, derived from or chemically related to resveratrol is selected an autologous cell or an allograft cell, an embryo or in vitro from the group consisting of viniferin, gnetin H, and Suffru ticosol B. In another example, the cocoa extract comprises a fertilization cell. In different embodiments, the cell is a polyphenol and/or procyanidin selected from (+) catechin, eukaryotic cell or a a prokaryotic cell. (-) epicatechin, procyanidin oligomers 2 through 18, procya 0278 Provided in yet another embodiment is a method for modulating response or resistance to stress of a cell, tissue, nidin B-5, procyanidin B-2, procyanidin A-2 and/or procya organ or organism, comprising modulating the level and/or nidin C-1. activity of at least one gene selected from the group consisting 0280. In another example of such methods, the method of those listed in Data Table 7 and those listed as part of Array comprises increasing the lifespan of a cell through modulat 2. Also provided are methods for modulating response or ing biogenesis of, or respiratory efficiency of mitochondria, resistance to stress, wherein modulating comprises modulat lengthening telomeres, and/or modulating at least one gene ing the level and/or activity of ten or more of the genes listed affecting the same. as part of Array 2; the genes listed as part of Array 1; VEGFA, 0281 Also provided are such methods, comprising HMOX1, CCL4L1, DDC, NOS2A, SIRT1, TERT, PTGS2, or increasing or decreasing proliferation or biogenesis of mito IFI44; four or more of TERT, TERC, NRF2, POT1, TRF1, chondria through modulation of at least one of PGC 1C. TRF2, TIN2, TPP1, RAPT, TNKS, TNKS 2, TERF2, SIRT1, SIRT3, SIRT4, SIRT5, NRF1 and/or Tfam. TERF2IP, POLG, POLB, POLD3, POLE, POLI, POLL, 0282 Any of the provided methods optionally also PARP2, PPARG, SHC1, PTOP, IFI44, NFKB1, HSPA1A, includes inducing mitochondrial regeneration, or new mito HSPA1B, HSPA1L, MTND5, HPGD, IDH2, MDH1, MDH2, chondrial biosynthesis in at least one cell. ME1, ME2, ME3, MTHD1, MTHFD1 L, MTHFR, NADK, 0283 Yet another embodiment is a method for modulat NADSYN1, NDUFA2, NDUFA3, NDUFA4, NDUFA4L2, ing, preventing, delaying, or reversing acute cell death or NDUFA5, NDUFA6, NDUFA7, NDUFA9, NDUFA10, apoptosis, or prolonging the Survival of a cell, tissue, organ or NDUFA12, NDUFB2, NDUFB3, NDUFB5, NDUFB6, organism comprising modulating the level and/or activity of US 2014/0079836A1 Mar. 20, 2014 22 at least one gene selected from the group consisting of those chondrial damage, comprising: contacting an cell with an listed in Data Table 7 and those listed as part of Array 2. For agent; and detecting the level of a nucleic acid molecule instance, modulating acute cell death or apoptosis comprises corresponding to AIFM2, AIP. BAK1, BBC3, BID, BNIP3, increasing or upregulating acute cell death or apoptosis. CLK1, HSPA1A, HSPA1B, HSPA1L, IMMP1L, IMMP2L, 0284 Another provided method is for modulating, MIPEP PARP1, PARP2, PMAIP1, RPL13A, SOD1, SOD2, enhancing, maintaining or producing a more youthful or SFN, SH3GLB1, UXT or another gene indicated herein as function of the skin and/or associated tissues, comprising beneficial for mitochondrial health or maintenance when modulating the level and/or activity of at least one gene decreased, or the level or activity of a protein encoded selected from the group consisting of those listed in Data thereby, in the presence and absence of the agent, wherein a Table 7 and those listed as part of Array 2. decrease in the level or activity in the presence of the agent as 0285. The methods provided herein may involve modulat compared to in the absence of the agent indicates that the ing the level or activity of the at least one gene comprising agent has potential to reverse or inhibit mitochondrial dam contacting a cell with an antisense or siRNA molecule. age. 0286 Also provided are collections of lifespan-influenc 0290 Still another method described herein is a method ing nucleic acid molecules, which collection comprises a for identifying an agent with potential to increase or acceler plurality of nucleic acid molecules selected from those listed ate mitochondrial damage, comprising: contacting an cell in Data Table 7 or Array 2, or fragments of those listed in Data with an agent; and detecting the level of a nucleic acid mol Table 7 or Array 2. Optionally, such collections are affixed to ecule corresponding to ACTB, BCL2, BCL2L1, CDKN2A, Solid Surface in an array Such as for instance a microarray. COX10, COX18, CPT1B, CPT2, DNAJC19, EGF, EGR2, 0287 Also provided are methods of screening compounds FIS1, GAPDH, GRPEL1, HSP90AA1, LRPPRC, MFN1, useful for modulating lifespan, the methods involving con MFN2, NOS3, OPA1, PARP3, PARP4, PPARGC1A, SIRT2, tacting a test compound with a host cell expresses a lifespan SIRT4, SLC25A1 SLC25A1 SLC24A2, SLC25A3, influencing protein encoded by an isolated nucleic acid mol SLC25A4, SCL25A5, SLC25A10, SLC25A12, SLC25A13, ecule listed in Data Table 7 or listed as part of Array 2 and SLC25A14, SLC25A15, SLC25A16, SLC25A17, detecting a change in the expression of the nucleotide SLC25A19, SLC25A2, SLC25A20, SLC25A21, sequence or a change in activity of encoded protein, wherein SLC25A22, SLC25A 23, SLC25A24, SLC25A25, Such a change indicates the test compound is useful for modu SLC25A27, SLC25A3, SLC25A30, SLC25A31, lating lifespan. Optionally, such methods are high throughput SLC25A37, SLC25A4, SLC25A5, TIMM10, TIMM17A, methods (ror instance, in an array format), involving contact TIMM17B, TIMM22, TIMM23, TIMM44, TIMM50, ing in parallel a test compound with a collection of host cells TIMM8A, TIMM8B, TIMM9, TOMM20, TOMM22, each of which expresses a different lifespan-influencing pro TOMM34, TOMM40, TOMM40L, TOMM70A, UCP1, tein encoded by an isolated nucleic acid molecule in listed in UCP2, UCP3 or another gene indicated herein as beneficial Data Table 7 or listed as part of Array 2; and detecting a for mitochondrial health or maintenance when increased, or change in the expression of at least one of the nucleotide the level or activity of a protein encoded thereby, in the sequences or a change in activity of at least one of the encod presence and absence of the agent, wherein a decrease in the ing proteins, wherein such a change indicates the test com level or activity in the presence of the agent as compared to in pound(s) are useful for modulating lifespan. the absence of the agent indicates that the agent has potential 0288 Another method described herein is a method for to increase or accelerate mitochondrial damage. identifying an agent with potential to reverse or inhibit mito 0291 Another method described herein is a method for chondrial damage, comprising: contacting an cell with an identifying an agent with potential to increase or accelerate agent; and detecting the level of a nucleic acid molecule mitochondrial damage, comprising: contacting an cell with corresponding to ACTB, BCL2, BCL2L1, CDKN2A, an agent; and detecting the level of a nucleic acid molecule COX10, COX18, CPT1B, CPT2, DNAJC19, EGF, EGR2, corresponding to AIFM2, AIP. BAK1, BBC3, BID, BNIP3, FIST, GAPDH, GRPEL1, HSP90AA1, LRPPRC, MFN1, CLK1, HSPA1A, HSPA1B, HSPA1L, IMMP1L, IMMP2L, MFN2, NOS3, OPA1, PARP3, PARP4, PPARGC1A, SIRT2, MIPEP PARP1, PARP2, PMAIP1, RPL13A, SOD1, SOD2, SIRT4, SLC25A1 SLC25A1 SLC24A2, SLC25A3, SFN, SH3GLB 1, UXT or another gene indicated herein as SLC25A4, SCL25A5, SLC25A10, SLC25A12, SLC25A13, beneficial for mitochondrial health or maintenance when SLC25A14, SLC25A15, SLC25A16, SLC25A17, decreased, or the level or activity of a protein encoded SLC25A19, SLC25A2, SLC25A20, SLC25A21, thereby, in the presence and absence of the agent, wherein an SLC25A22, SLC25A 23, SLC25A24, SLC25A25, increase in the level or activity in the presence of the agent as SLC25A27, SLC25A3, SLC25A30, SLC25A31, compared to in the absence of the agent indicates that the SLC25A37, SLC25A4, SLC25A5, TIMM10, TIMM17A, agent has potential to increase or accelerate mitochondrial TIMM17B, TIMM22, TIMM23, TIMM44, TIMM50, damage. TIMM8A, TIMM8B, TIMM9, TOMM20, TOMM22, 0292. In another embodiment, there is provided a method TOMM34, TOMM40, TOMM40L, TOMM70A, UCP1, for identifying an agent with potential to reverse or inhibit UCP2, UCP3 or another gene indicated herein as beneficial DNA damage or telomere shortening, comprising: contacting for mitochondrial health or maintenance when increased, or ancell with an agent, and detecting the level of a nucleic acid the level or activity of a protein encoded thereby, in the molecule corresponding to AK3, APEX1, APEX2, ATF2, presence and absence of the agent, wherein an increase in the ATM, ATR, ATRX, BARD1, BLM, BRIP1, CCNH, CDK7, level or activity in the presence of the agent as compared to in CDKN2A, CHEK1, CHEK2, CSF2, CTPS, DDB1, DDB2, the absence of the agent indicates that the agent has potential DHFR, DMC1, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, to reverse or inhibit mitochondrial damage. ERCC6, ERCC8, EXO1, FANCA, FANCC, FANCF, 0289. Yet another method described herein is a method for FANCG, FEN1, GADD45A, GADD45G, GTF2H1, identifying an agent with potential to reverse or inhibit mito GTF2H2, GTF2H3, GTF2H4, JUN, LIG1, LIG3, LIG4, US 2014/0079836A1 Mar. 20, 2014 23
MAP2K6, MAPKAPK2, MLH1, MLH3, MRE 11A, MSH2, CIDEA, CIDEB, DDIT3, DKC1, GTSE1, MDM2, PCBP4, MSH3, MSH4, MSH5, MSH6, NBN, NEIL1, NEIL2, PDCD8, PINX1, PPP1R15A, RAD17, RELATELO2, TEP1 NEIL3, NFKB1, NFKBIA, HK1, NUDT1, NUDT2, ODC1, or another gene indicated herein as beneficial for DNA or PAPSS1, PAPSS2, PARP1, PARP3, PCNA, PMS1, PMS2, telomere maintenance when decreased, or the level or activity PNKP, POLB, POLD3, POLE, POLI, POLL PRKDC, of a protein encoded thereby, in the presence and absence of RAD1, RAD18, RAD21, RAD23A, RAD50, RAD51C, the agent, wherein an increase in the level or activity in the RAD51L1, RAD51L3, RAD52, RAD54B, RAD54L, presence of the agent as compared to in the absence of the RBBP8, SESN1, SLC23A2, TDG, TYMS, UBE2V2, UNG2, agent indicates that the agent has potential to accelerate or WRN, XAB2, XPA, XPC, XRCC1, XRCC2, XRCC3, cause or enhance DNA damage or telomere shortening. XRCC4, XRCC5, XRCC6, ZNRD1 or another gene indi 0296. Further embodiments exploit the discovery made cated herein as beneficial for DNA or telomere maintenance herein that the dosage of compounds applied has a profound when increased, or the level or activity of a protein encoded effect on the up and/or down regulation of various genes. thereby, in the presence and absence of the agent, wherein an Thus, there is provided a first method for inducing expression increase in the level or activity in the presence of the agent as of TERT, POT1, TPP1 and TERF2 in a cell, by applying to the compared to in the absence of the agent indicates that the cell or an organism comprising the cell a composition com agent has potential to reverse or inhibit DNA damage or prising between about 0.000001% and about 10% (by weight) telomere shortening. coffee cherry extract. In certain embodiments of this method 0293 Another embodiment is a method for identifying an the composition comprises no more than about 0.01% (by agent with potential to reverse or inhibit DNA damage or weight) coffee cherry extract. Alternatively, the composition telomere shortening, comprising: contacting an cell with an further comprises green tea extract, a component of green tea agent; and detecting the level of a nucleic acid molecule extract, or idebenone. Such as for instance one or more of corresponding to B2M, BRCA1, BRCA2, BTG2, CIDEA, about 0.001% (by weight) green tea extract or about CIDEB, DDIT3, DKC1, GTSE1, MDM2, PCBP4, PDCD8, 0.00004% (by weight) idebenone. PINX1, PPP1R 15A, RAD17, RELA, TELO2, TEP1 or 0297 Also provided is a method inducing expression of another gene indicated hereinas beneficial for DNA or telom PARP1, BCL2 and p53 in a cell, by applying to the cell or an ere maintenance when decreased, or the level or activity of a organism comprising the cell a composition comprising protein encoded thereby, in the presence and absence of the between about 0.000001% and about 10% (by weight) coffee agent, wherein a decrease in the level or activity in the pres cherry extract. In certain examples of this method, the com ence of the agent as compared to in the absence of the agent position comprises no more than about 0.000005% (by indicates that the agent has potential to reverse or inhibit DNA weight) chlorogenic acid. damage or telomere shortening. 0298. Yet another embodiment is a method of inducing 0294 Also provided is a method for identifying an agent expression of NOS2A, NOS1, and NOS3 in a cell, by apply with potential to accelerate or cause or enhance DNA damage ing to the cell or an organism comprising the cell a composi or telomere shortening, comprising: contacting an cell with tion comprising between about 0.000001% and about 10% an agent; and detecting the level of a nucleic acid molecule (by weight) coffee cherry extract, or wherein the composition corresponding to AK3, APEX1, APEX2, ATF2, ATM, ATR, comprises no more than about 0.01% coffee cherry extract. ATRX, BARD1, BLM, BRIP1, CCNH, CDK7, CDKN2A, 0299. In another embodiment, there is provide method of CHEK1, CHEK2, CSF2, CTPS, DDB1, DDB2, DHFR, inducing expression of CCL4L1 in a cell, by applying to the DMC1, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, cell or an organism comprising the cell a composition com ERCC6, ERCC8, EXO1, FANCA, FANCC, FANCF, prising between about 0.000001% and about 10% (by weight) FANCG, FEN1, GADD45A, GADD45G, GTF2H1, coffee cherry extract. Examples of this method involve using GTF2H2, GTF2H3, GTF2H4, JUN, LIG1, LIG3, LIG4, a composition that comprises no more than about 0.01% (by MAP2K6, MAPKAPK2, MLH1, MLH3, MRE 11A, MSH2, weight) coffee cherry extract. MSH3, MSH4, MSH5, MSH6, NBN, NEIL1, NEIL2, 0300 Still other embodiments are described herein, and NEIL3, NFKB1, NFKBIA, HK1, NUDT1, NUDT2, ODC1, this list is not intended to be exhaustive. PAPSS1, PAPSS2, PARP1, PARP3, PCNA, PMS1, PMS2, PNKP, POLB, POLD3, POLE, POLI, POLL PRKDC, IV. Lifespan Extension RAD1, RAD18, RAD21, RAD23A, RAD50, RAD51C, 0301 Many factors have been shown to diminish lifespan RAD51L1, RAD51L3, RAD52, RAD54B, RAD54L, in living creatures, but few have clearly demonstrated exten RBBP8, SESN1, SLC23A2, TDG, TYMS, UBE2V2, UNG2, sion of lifespan. WRN, XAB2, XPA, XPC, XRCC1, XRCC2, XRCC3, 0302 One example of lifespan extension however, is a XRCC4, XRCC5, XRCC6, ZNRD1 or another gene indi dietary program termed caloric restriction which is well cated herein as beneficial for DNA or telomere maintenance documented to extend the lifespan in a variety of living organ when increased, or the level or activity of a protein encoded isms. Agents which can mimic Some of the effects of caloric thereby, in the presence and absence of the agent, wherein a restriction or increase a cells resistance to stress have been decrease in the level or activity in the presence of the agent as described with the NAD+salvage pathway. Members of a compared to in the absence of the agent indicates that the family of genes termed Silent Information Regulators (SIR) agent has potential to accelerate or cause or enhance DNA are involved in various processes of gene silencing and DNA damage or telomere shortening. repair. It has been shown that yeasts which are lacking the 0295) Another provided method is a method for identify SIR2 gene do not live longer when calorically restricted and ing an agent with potential to accelerate or cause or enhance thus it is believed that the SIR2 gene mediates some of the DNA damage or telomere shortening, comprising: contacting beneficial lifespan extending effects of caloric restriction. ancell with an agent, and detecting the level of a nucleic acid Other genes in this family include SIRT1 which may have a molecule corresponding to B2M, BRCA1, BRCA2, BTG2, similar effect. US 2014/0079836A1 Mar. 20, 2014 24
0303 Sirtuin modulating compounds then have use in enables balancing of healthy and unhealthy influences to extending the lifespan of certain living cells as well as having yield healthier longevity. The identified genes can be repro the potential to treat and/or prevent various diseases related to grammed (either up or down, depending on the gene and the aging. One Such example is resveratrol which is a naturally circumstance); where they are not amenable to reprogram occurring Substance in red wine which has been shown to ming directly, the cell expressing them can be removed, inca increase lifespan in mice and which appears to affect in some pacitated, killed (e.g., through apoptosis) or disabled; and genes of the sirtuin family (although it also alters the gene where that is not readily feasible, other genes that counteract expression and/or protein production by many other genes). or balance the unhealthy influence(s) through offsetting 0304 Relatively minor changes at a genetic level have expression of healthy factors, either in the same or another been shown to significantly alter the aging process as have cell. Likewise, the counteracting influence may be biogenesis various environmental factors. The rate of aging, the health of of new cells, repair of DNA damage, and/or prevention of the organism as the aging process progresses as well as the DNA damage. All of these in different ways may be exploited total lifespan are complexly controlled and are the subject of to influence lifespan of cells, tissues, organs and organism— various theories of aging. whether to extend lifespan or reduce it by causing lethal damage or triggering apoptosis is another way to get rid of V. Modification of Lifespan cells. 0305 Provided herein are the results of comprehensive 0310. The extracts, compounds or combination of com analyses of gene expression changes in the presence of anti pounds derived therefrom are prepared by methods com oxidant compounds, with and without pre-stimulation with a monly known and many naturally derived compounds are stressor (e.g., an environmental stress Such as ultraviolet commercially available. Since naturally derived compounds radiation exposure). Also provided are dosage response are not the only way to achieve the concentrations of active analyses, illustrating the changes in gene responses with compounds, the invention comprehends synthetic forms can changes in the amount of antioxidant compounds. Based on be prepared from isolation from other plant species as well as the results provided herein, methods are now enabled for from synthetic routes, which are all covered in these claims. affecting such expression changes in order to influence (in Also the skilled artisan will be able to envision additional crease or decrease) the health or lifespan of cells, tissues, routes of synthesis, based on knowledge in the art, without organs and organisms, by intentionally altering the expres undue experimentation. For instance, given the phenolic sion of one or more of the identified genes. character of the compounds, variable methods of selective (0306 Methods herein apply to extending the health and protection, coupled with organometallic additions, phenolic lifespan of human cells (and tissues, organs, and organisms), couplings and photochemical reactions, e.g., in a convergent, as well as cells (and tissues, organism, and organisms) of linear or biomimetic approach, together with standard well non-human animals, unicellular and multicellular organisms, known reactions for synthetic organic chemists could pro plants, and so forth. Thus, it will be understood when a gene duce synthetic derivatives that perform the desired telomeric is referred to, that reference includes the orthologous length maintenance alterations. sequence(s) from other species etc. 0311 Data from telomerase activity experiments show 0307 Healthy longevity includes causing cells to offset that green tea increases measurable activity of telomerase in age or environmental damage or disease, for instance related most tested circumstances. Cells given green tea before UVB to decline in function (e.g., when mitochondria do not make exposure show a decrease intelomerase activity. That is when as much ATP any longer, improving mitochondria respiration the cells are stressed with UVB and given green tea, the or increasing number of mitochondria or both addresses this), measurable telomerase activity increases and when the cells or reducing or eliminating expression/activity of an are not stressed and contacted with green tea the telomerase unhealthy factor (e.g., MMP1 collagenase can be consid activity also increases (younger cells are more responsive to ered an unhealthy factor, as it degrades collagen which in turn the increase than older cells). Conversely, cells both stressed damages the structural integrity of skin, joints, etc.). In this and unstressed, which receive treatment withidebenone show latter example, changing the programming of gene expres a decreased level of telomerase activity. These results indicate sion improves health, for instance reducing or reversing the that by administering green tea to the cells, before stress, or chronic response to injury (e.g., environmental or other even to normal unstressed cells, there is an increase intelom wise—UV light exposure, Smoking, inflammation, etc.) that erase activity that is higher in the younger cell lines, indicat had caused/induced overproduction of MMP1, which prema ing an increase in the ability to maintain the length of the turely ages cells and organs and organism. telomere and to better protect against/combat oxidative stress 0308 The methods provided herein are useful also to based damage to the DNA, thereby reducing and/or prevent modulate gene activity/expression in order to shorten lifespan ing cellular damage and apoptosis signaling events. of unhealthy cells, for instance in order to kill cancer or other 0312. In custom microarray experiments, the data illus unwanted cells or to eliminate cells that are sending wrong trate that the chosen antioxidant compounds (green tea, cof genetic or molecular signals. When that is accomplished, you fee cherry and idebenone) are biologically active, showing can replace the eliminated or down-modulated cells with cells statistically significant changes in expression levels for some that are healthy (e.g., through biogenesis or using stem cells). longevity related genes in all the compounds tested. The 36 Alternatively, cells can be programmed to offset the negative year old cells seem to be active with large statistical changes signals—for instance, responding to overproduction of in the expression of PARP1, NADSYN1, IFI44, TERT, and MMP1 by modulating the expression of a cell in order to NFKB1. All of these genes were downregulated when produce collagen to replace that which the MMP1 is degrad exposed to UVB stress, but upregulated when exposed to cells ing. given the tested antioxidant compounds (for various time 0309 The discoveries herein regarding gene expression in intervals) and then stressed. The increase in TERT (respon response to antioxidant induction provides a system that sible for telomerase enzyme activity) compared to a decrease US 2014/0079836A1 Mar. 20, 2014
in the UVB alone (stressed) cells indicates very strongly that 1557286 at 1557287 at 1557302 at, 1557341 x at, the antioxidant compounds are enabling more enzyme activ 1557348 at 1557383 a at 1557667 at 1557740 a at, ity to keep telomere length intact (and thus extend the cellular 1558105 a at, 1558236 at, 1558237 x at 1558250 s at, lifespan) via the mediation of UV damage to the telomere. A 1558401 at 1558445 at 1558515 at, 1558604 a at, second interesting finding is in older (presumably more envi 1558605 at 1558750 a at 1558801 at 1558836 at, ronmentally damaged and less efficient at telomere repair and 1558837 a at, 1558890 at 1558906 a at, 1558920 at, total telomere length) cells the idebenone and coffee cherry 1559229 at 1559867 at 1560071 a at 1560208 at, treated cells, and not green tea, were able to reverse the expression levels of UVB stressed aged cells. In this case, the 1560579 s at, 1561064 a at, 1562012 at, 1562013 a at, TERT reduction from UVB stress was even greater, consid 1562056 at 1562098 at 1562777 at, 1563012 x at, ering the age of the cell relative to the younger cell, but even 1563414 at 1565495 at 1565577 s at 1565783 at, more interestingly, the antioxidant response was even stron 1568633 a at, 1569 129 s at, 1569765 at, 1570061 at, ger than in the younger cell implying that, at least for ide 1570 100 at, 182-FIP, 208187 s at 210230 at 213156_at, benone and coffee cherry, the older/more damaged/less effi 213158 at, 213567 at, 213817 at, 213832 at 214202 at, cient the cellular mechanism of telomere length maintenance 214808 at, 214862 x at, 214967 at, 21.5128 at, 215287 at, is, the greater the ability for certain antioxidants to effect 217166 at, 217536 X at, 217540 at, 217554 at, 217604 at, changes towards longevity. 220494 sat, 220726 at, 221200 at, 222184 at, 222284 at, 0313 Green tea shows, through significant downregula 224769 at, 224778 s at, 224811 at, 225256 at, 225356 at, tion of TNF 18 fold, a role in combating the damage caused 225725 at 225893 at 225917 at 226203 at 226250 at, by this pro-inflammatory cytokine, which could also lead to 226282 at, 226316 at, 226362 at, 226365 at, 226392 at, extending the lifespan of the cell by preventing apoptosis 226457 at, 226458 at, 226520 at, 226532 at, 226542 at, 226546 at, 226550 at, 226773 at, 226885 at, 226964 at, caused by inflammatory signaling cascades. 227041 at, 227044 at, 227051 at, 227061 at, 227082 at, 0314. The RT-PCR primer assays examined genes specifi 227121 at, 227126 at, 227184 at, 227.193 at, 227221 at, cally related to the telomere complex itself, and show that 227252 at, 227283 at, 227306 at, 227422 at, 227503 at, coffee cherry was able to downregulate the expression of 227531 at 227533 at 227565 at 227623 at 227655 at, TINF2 which, when expressed is a negative regulator of 227663 at, 227682 at, 227929 at, 227955 s at, 228032 S telomere length maintenance, and indicates a directional at, 228045 at 228049 X at, 228084 at, 2281.56 at, change toward the lengthening of telomeres and cellularlon 228159 at, 228216 at, 228242 at, 228304 at 228315 at, gevity. 228333 at, 228346 at, 228390 at, 228478 at, 228528 at, 0315. In Human Genome arrays the experiments show the 228694 at, 228740 at, 228742 at, 228750 at, 228773 at, effect of antioxidant activity (idebenone and coffee cherry) in 228781 at, 2288.11 at, 228812 at, 228850 s at, 228955 at, stressed and unstressed cells on the entire genetic expression 228963 at, 229024 at, 229072 at, 229121 at, 22.9189 sat, profile. The data showed both compounds to be biologically 229.190 at 229281 at 229297 at 229315 at, 229319 at, active and affecting many genes with a variety of functions 229333 at, 229359 at, 229384 at, 22.9460 at, 2294.79 at, focused on the aging/longevity groups and indicate the ability 229512 at, 229569 at, 229572 at, 22.9602 at, 2296.15 at, for the application of antioxidants to not only effect the free 229641 at 229699 at 2297.05 at 229710 at 229756 at, radical metabolism, but also directly affect gene expression 2297.57 at 229795 at, 229810 at 229815 at, 229948 at, profiles responsible for telomere length maintenance, cellular 229994 at 230003 at 230090 at 230127 at 230183 at, metabolism, mitochondrial function and inflammation all of 230211 at, 230227 at, 230240 at, 230304 at, 230345 at, which when modulated properly would lead to the potential 230383 X at, 230406 at, 230407 at, 230431 at, 230446 at, extension of and improvement in the quality of cellular 230449 X at, 230483 at, 230503 at, 230683 at, 230741 at, lifespan. 230766 at 230773 at 230860 at 230927 at 230968 at, 0316 The application of the desired antioxidants, before 231055 at, 231069 at, 231193 s at, 231238 at, 231576 at, UV stress or without UV stress, can directly affect the expres 231597 X at, 231890 at, 231963 at, 231993 at, 232088 x sion levels of genes responsible for telomere length mainte at, 232125 at, 232156 at, 232484 at, 232535 at, 232538 nance and modulate the cellular lifespan. Telomere length at, 232656 at 232795 at 232903 at, 233105 at, 233335 maintenance is not the only factor involved in cell longevity, at, 233354 at, 233376 at, 233485 at, 233518 at, 233723 and antioxidants have shown the ability to modulate those at, 233814 at, 234340 at, 234578 at, 234723 x at, expression levels as well, with effects on energy production 234983 at, 235000 at, 235028 at, 235046 at, 235072 s at, and inflammation responses indicating multiple methods for 235078 at 235123 at 235124 at, 235.171 at 235207 at, extension of lifespan through a single antioxidant compound. 235224 S at, 235227 at, 235264 at, 235279 at, 235299 at, 0317 Based on the work presented herein, it is now rec 235302 at 235304 at 235352 at, 235407 at 235427 at, ognized that that at least all of the genes listed in Table 1 and 235428 at, 235434 at, 235438 at, 235459 at, 235532 at, in Array 2 (described below) are involved in lifespan, longev 235556 at 235571 at 235581 at 235585 at 235612 at, ity, mitochondrial biogenesis or health, cellular respiratory 235655 at 235658 at 235696 at 235733 at 235761 at, health, and/or DNA or telomere maintenance. Thus, it is 235764 at 235830 at 235831 at 235889 at 235890 at, contemplated that modification of the level or expression of 235919 at 235931 at 235938 at 236.004 at, 23.6038 at, any one or more of these genes may be useful in modulating 236089 at 236097 at, 236105 at 236174 at, 23.6180 at, Such process. At least the following genes are therefore rec 2361.94 at, 236.196 at, 236335 at, 236344 at, 236350 at, ognized as lifespan-influencing genes and useful in one or 236364 at, 23.6423 at, 236433 at, 236452 at, 236619 at, more of the methods and/or compositions described herein: 236685 at 236787 at, 236856 x at 236875 at, 236898 at, 1553575 at 1554007 at 1554948 at 1555846 a at, 236922 at 236996 at 237071 at 237212 at 237290 at, 1555875 at 1556097 at, 1556216 s at 1556242 a at, 2373.15 at, 237416 at, 237435 at, 237622 at, 237768 x at, 1556332 at 1556545 at 1556936 at 15571 18 a at, 238030 at 238050 at 238109 at 238178 at, 23.8250 at,
US 2014/0079836A1 Mar. 20, 2014 33
YPEL2, YPEL3, YPEL4, YRDC, YTHDC2, YTHDF3, ZNF597, ZNF599, ZNF605, ZNF606, ZNF615, ZNF618, Z28739, ZAK, ZBED1, ZBED3, ZBED5, ZBTB10, ZNF621, ZNF622, ZNF623, ZNF627, ZNF630, ZNF652, ZBTB20, ZBTB24, ZBTB26, ZBTB3, ZBTB34, ZBTB41, ZNF655, ZNF658, ZNF662, ZNF672, ZNF675, ZNF677, ZBTB43, ZBTB44, ZC3H13, ZC3H6, ZC3HAV1L, ZNF680, ZNF681, ZNF684, ZNF688, ZNF691, ZNF697, ZCCHC10, ZCCHC11, ZCCHC3, ZDBF2, ZDHHC14, ZNF70, ZNF702, ZNF708, ZNF709, ZNF710, ZNF713, ZDHHC17, ZDHHC2, ZDHHC21, ZDHHC22, ZDHHC3, ZNF717, ZNF75, ZNF768, ZNF770, ZNF783, ZNF785, ZDHHC5, ZEB1, ZEB2, ZFAND2A, ZFAND2B, ZFAND5, ZNF792, ZNF805, ZNF81, ZNF814, ZNF818, ZNF84, ZFAND6, ZFHX3, ZFHX4, ZFP1, ZFP106, ZFP3, ZNF85, ZNF91, ZNF92, ZNF93, ZRF1, ZSCAN29, ZFP36L2, ZFP90, ZFPL1, ZFPM2, ZFX, ZFY, ZFYVE16, ZSWIM6, ZWILCH, ZWINT, ZXDB, ZYG 11B, and ZYX. ZGPAT, ZHX1, ZHX2, ZHX3, ZIK1, ZKSCAN1, ZMAT3, 0318. It is also recognized that the change in expression is MIZ1, ZMYM2, ZMYM4, ZMYM5, ZMYND10, directional, in that for some genes it is beneficial to increase MYND11, ZNF101, ZNF107, ZNFLJ7, ZNF12, ZNF131, expression in order to enhance (or reduce) longevity, health, NF14, ZNF148, ZNF160, ZNF165, ZNF167, ZNF174, or biological wellbeing while the expression of other genes NF182, ZNF189, ZNF19, ZNF192, ZNF20, ZNF 207, needs to be decreased for the same purpose. For instance, with NF217, ZNF223, ZNF224, ZNF225, ZNF226, ZNF228, regard to telomeres, it is beneficial (for maintenance of the NF230, ZNF232, ZNF236, ZNF24, ZNF248, ZNF252, telomere and therefore increased longevity/health/wellbeing) NF253, ZNF259, ZNF264, ZNF267, ZNF273, ZNF277, to upregulate (increase the expression of) TNKS and POT1, NF280D, ZNF282, ZNF286A, ZNF289, ZNF292, while it is beneficial to downregulated (decrease the expres NF294, ZNF297B, ZNF302, ZNF313, ZNF317, ZNF323, sion of) TNKS2, TRF1, TIN2, and/or TRF2. Likewise, the NF326, ZNF331, ZNF333, ZNF334, ZNF33A, ZNF345, following four tables provide beneficial up-and down-regu lation indications for genes found on two specific arrays NF347, ZNF350, ZNF354C, ZNF367, ZNF37B, ZNF395, provided herein (Array 1 and Array 2), as well as the genes F397, ZNF404, ZNF408, ZNF415, ZNF420, ZNF423, involved in mitochondrial maintenance and DNA repair. In F43, ZNF430, ZNF432, ZNF438, ZNF439, ZNF440, each table, the shaded genes are beneficially downregulated F441, ZNF446, ZNF449, ZNF462, ZNF468, ZNF473, for longevity/health/etc., while the unshaded genes are ben F512, ZNF512B, ZNF516, ZNF517, ZNF521, ZNF529, eficially upregulated. Methods and compositions are pro F532, ZNF551, ZNF555, ZNF557, ZNF560, ZNF564, vided herein that can accomplish up-and down-regulation of F567, ZNF569, ZNF57, ZNF573, ZNF585A, ZNF587, these genes.
Array #1
APOE e4 allele promotes premature atherosclerosis BAX upregulated in psoriation epidermis, regulates neutrophil apoptosis BCL2 anti-apoptotic gene, promotes cell viability
deficiency – premature aging, shortened lifespan, impaired hair growth, bones loss CASP9 Suppression of tumor growth
Slows down general cellular aging, affects metabolic rate responsible for producing cytoprotective prostaglandins which is critical in maintaining integrity of gastric mucosa CREBBP glucose homeostasis Dopa decarboxylase controls synthesis of neurotransmitters, dopamine and serotonin Growth Hormone necessary for longevity regulates immune responses cellular repair and maintenance One of the stress response genes, cellular maintenance & repair US 2014/0079836A1 Mar. 20, 2014 34
-continued
Array #1
One of the stress response genes, cellular maintenance & repair regulation of carbohydrate metabolism and pancreatic control of glucose homeostasis. regulation of carbohydrate metabolism and pancreatic control of glucose homeostatis. important tumor suppressor gene normally preventing cancer development via apoptosis MAPK14 immune response gene, regulates longevity of neutrophils NFKB1 marker of genetic disorders affecting immune response and cell differentiation. NOS2A regulates endothelial function, hypertension DNA repair, apoptosis, maintenance of optimal niacin status in skin. DNA repair, apoptosis, maintenance of optimal niacin status in skin. regulator of adipose tissue metabolism, insulin sensitivity and inflammatory response. p66 she is highly expressed in fibroblasts from centenarians, increases resistance to oxidative and hypoxic stress represses p53-mediated transactivation, regulates apoptotic response to DNA damage ROS Scavenging, apoptosis ROS Scavenging, apoptosis tumor Suppresser, deletions of this gene associated with a variety of human cancers. TERT gene for telomerase reverse transcription, controls cellular response to stress. TP53 important tumor suppressor gene normally preventing cancer development via apoptosis
Array 2.0
ACTB Cell differentiation APOE e4 allele promotes premature atherosclerosis BAX upregulated in psoriation epidermis, regulates neutrophil apoptosis BCL2 anti-apoptotic gene, promotes cell viability BCL2L1 positive/negative regulation of apoptosis deficiency – premature aging, shortened lifespan, impaired hair growth, bones loss CASP9 Suppression of tumor growth CCL4L1 CDKN2A TP53 and RAB pathway regulator Slows down general cellular aging, affects metabolic rate COL1A1 ECM deposition COL3A1 Fetal and internal organ ECM
responsible for producing cytoprotective prostaglandins which is critical in maintaining integrity of gastric mucosa US 2014/0079836A1 Mar. 20, 2014 35
-continued
Array 2.0
CREBBP glucose homeostasis issue injury or inflammation Dopa decarboxylase controls synthesis of neurotransmitters, dopamine and serotonin induced in response to serum deprivation and oxidative stress, EGF Mitogen EGR2 Associated with mitogens Cell cycle progression GAPDH Carbohydrate metabolism GH1 Growth Hormone necessary for longevity GPX1 protection against Some oxidative stressors and in protection of neurons against peroxide HBEGF activates EGFR regulates immune responses HMOX1 shows antioxidative effects
cellular repair and maintenance One of the stress response genes, cellular maintenance & repair One of the stress response genes, cellular maintenance & repair regulation of carbohydrate metabolism and pancreatic control of glucose homeostasis. regulation of carbohydrate metabolism and pancreatic control of glucose homeostasis. Bone cell proliferation Pro inflammatory for joint disease Inflammatory response Activates proapoptotic protein AP-1 complex necessary for cell cycle reentry of ultraviolet (UV)-irradiated cell KIT function in hematopoiesis, melanogenesis, and gametogenesis
KL regulation of calcium metabolism
important tumor suppressor gene normally preventing cancer development via apoptosis MAPK14 immune response gene, regulates longevity of neutrophils
Collagenase NEIL1 initiate the first step in base excision repair NFKB1 marker of genetic disorders affecting immune response and cell differentiation. NOS2A regulates endothelial function, hypertension NOS3 Nitric Oxide Synthase, endothelial triggers mitosynthesis PARP1 DNA repair, apoptosis, maintenance of optimal niacin status in skin. PARP2 DNA repair, apoptosis, maintenance of optimal niacin status in skin. PARP3 DNA repair, apoptosis PARP4 DNA repair, apoptosis US 2014/0079836A1 Mar. 20, 2014 36
-continued Array 2.0 migration and dissemination of cancer Protection of telomeres regulator of adipose tissue metabolism, insulin sensitivity and inflammatory response. PPARGC1A Energy metabolism chemotactic inflammatory protein, psoriasis p66 she is highly expressed in fibroblasts from centenarians, increases resistance to oxidative and hypoxic stress represses p53-mediated transactivation, regulates apoptotic response to DNA damage contribute to free-radical defense mitochondrial ADP-ribosyltransferase ROS Scavenging, apoptosis ROS Scavenging, apoptosis tumor Suppresser, deletions of this gene associated with a variety of human cancers. Protection and replication of chromosome ends TERT gene for telomerase reverse transcription, controls cellular response to stress. TGFB1 Cell growth and proliferation TIMM22 Mitochondrial inner membrane chaperones TIMP3 Collagenase Inhibitor TIN2 negative regulator of telomerase length TOMM40 Mitochondrial inner membrane chaperones important tumor suppressor gene normally preventing cancer development via apoptosis tumor cell proliferation, invasion, and metastasis Tumor angiogenesis
Mitochondria biogenesis, maintenance, etc. -continued
Activates proapoptotic protein Cell differentiation Activates proapoptotic protein Mito Chaperone Mito fusion Apoptotic stimulator Mito fusion Pro Apoptosis
Mito membrane Apoptosis induction CDKN2A Upregulated in leukemia Induced by DNA damaging agents Pro Apoptotic SLC25A1 Mitochondrial membrane transport CPT2 Fatty acid oxidation SLC25A10 Mitochondrial membrane transport DNAJC19 Mitochondrial protein import motor SLC25A12 Mitochondrial membrane transport FIS1 Promotes mitochondrial fission SLC25A13 Mitochondrial membrane transport GAPDH Carbohydrate metabolism GRPEL1 Mitochondrial chaperone US 2014/0079836A1 Mar. 20, 2014 37
-continued -continued
SLC25A31 Mitochondrial membrane transport SLC25A14 Mitochondrial membrane transport SLC25A37 Mitochondrial membrane transport SLC25A15 Mitochondrial membrane transport SLC25A4 Mitochondrial membrane transport SLC25A16 Mitochondrial membrane transport SLC25A5 Mitochondrial membrane transport LC25A17 itochondrial mem transport TIMM1() Mitochondrial inner membrane chaperones SLC25A19 Mitochondrial mem transport TIMM17A Mitochondrial inner membrane chaperones SLC25A2 Mitochondrial membrane transport TIMM17B Mitochondrial inner membrane chaperones SLC25A2O Mitochondrial mem transport TIMM22 Mitochondrial inner membrane chaperones C25A21 Mitochondrial mem transport TIMM23 Mitochondrial inner membrane chaperones TIMM44 Mitochondrial inner membrane chaperones SLC25A22 M OCOC8 le. anSport TIMM50 ial inner mem (Oile:S SLC25A23 Mitochondrial membrane transport TIMM8A Mitochondrial inner membrane chaperones SLC25A24 Mitochondrial membrane transport IMM8B Mitochondrial inner membrane chaperones SLC25A25 Mitochondrial mem transport TIMM9 Mitochondrial inner membrane chaperones SLC25A27 Mitochondrial mem transport inner mem (Oile:S SLC25A3 Mitochondrial membrane transport inner mem (Oile:S SLC25A30 Mitochondrial membrane transport ille le. (Oile:S
inner mem (Oile:S TOMM4OL ial inner mem (Oile:S TOMMTOA ial inner mem (Oile:S Decreases ROS in mitochondria Decreases ROS in mitochondria Decreases ROS in mitochondria Tumorigenesis
DNA Repair, maintenance, etc.
AK3 APEX1 APEX2 ATF2 ATM ATR ATRX
BARD1
Negative Regulator of cell growth Negative Regulator of cell growth BRIP1
CCNH CDK7 Regulates Cell Cycle Progression CDKN2A US 2014/0079836A1 Mar. 20, 2014 38
-continued
CHEK1 Monitors meiotic recombination CHEK2 DNA damage repair Activate apoptosis Activate apoptosis CSF2 colony stimulating factor CTPS biosynthesis of phospholipids and nucleic acids DDB1 DNA repair DDB2 DNA repair
deletion protected cells from ER stress by decreasing ER protein load and changing redox condition DHFR Nitric Oxide production
apoptosis in higher cell cycles DMC1 promote DNA strand exchange ERCC1 damage recognition and incision activities. ERCC2 damage recognition and incision activities. ERCC3 damage recognition and incision activities. ERCC4 damage recognition and incision activities. ERCC5 damage recognition and incision activities.
ERCC6 damage recognition and incision activities. ERCC8 damage recognition and incision activities. EXO1 DNA replication, repair, and recombination. FANCA repair of DNA damage FANCC repair of DNA damage FANCF repair of DNA damage FANCG repair of DNA damage FEN1 DNA replication, repair, and recombination GADD45A DNA repair GADD45G | DNA repair GTF2H1 Transcription GTF2H2 Transcription GTF2H3 Transcription GTF2H4 Transcription
induce G2/M-phase accumulation when overexpressed.
LIG1 DNA ligase US 2014/0079836A1 Mar. 20, 2014 39
-continued
LIG3 DNA ligase LIG4 DNA lagase MAP2K6 activation of p38 MAPKAPK2 activation of p38 major regulator of p53 MLH1 involved in DNA mismatch repair MLH3 DNA mismatch repair protein MRE11A blocks meiotic recombination MSH2 DNA mismatch repair MSH3 DNA mismatch repair MSH4 DNA mismatch repair MSHS DNA mismatch repair MSH6 DNA mismatch repair NBN MRE11/RAD50 double-strand break (DSB) repair complex
initiate the first step in base excision repair initiate the first step in base excision repair initiate the first step in base excision repair
NFKB1 cell differentiation NFKBIA cell differentiation HK1 first step in glucose metabolism, utilizing ATP NUDT1 preventing occurrence of mutations caused by misincorporation NU D T2 preventing occurrence of mutations caused by misincorporation ODC1 transcriptional target of MYC PAPSS1 Sulfonation of endobiotics and xenobiotics PAPSS2 Sulfonation of endobiotics and xenobiotics PARP1 PARP3
necessary to induce apoptosis and cell cycle arrest maintenance of the fidelity of mammalian DNA replication apoptosis-inducing factor inhibit telomerase activity PMS1 mismatch repair of dinucleotide and trinucleotide repeat sequences PMS2 mismatch repair of dinucleotide and trinucleotide repeat sequences PNKP DNA repair following ionizing radiation or oxidative damage US 2014/0079836A1 Mar. 20, 2014 40
-continued POLB performs base excision repair (BER) POLD3 DNA replication and repair POLE replication of chromosomal DNA POLI
replication of the genome and DNA repair processes mediates growth arrest and apoptosis PRKDC modulating transcription required for DNA repair and replication Cell cycle checkpoint RAD18 Post-replication repair functions sister chromatid cohesion during mitosis genome-overall repair pathway essential for double-stranded DNA break repair RAD51C recombinational repair of DNA damage and in meiotic recombination RAD51L1 double-stranded break repair RAD51L3 double-stranded break repair RAD52 responsible for DNA double-strand break repair
RAD54B responsible for DNA double-strand break repair RAD54L responsible for DNA double-strand break repair RBBP8 required for tumor Suppression
SESN1 reestablishing the antioxidant firewall SLC23A2 protecting metabolically active tissues from oxidative stress TDG initiates repair of G.T and G/U mismatches
checkpoint responses to cellular stress tumor Suppresser, deletions of this gene associated with variety of cancers. TYMS DNA repair UBE2W2 DNA repair
UNG2 DNA repair WRN DNA repair
XPA repair of UV radiation-induced photoproducts and DNA adducts induced by chemical carcinogens XPC May play a part in DNA damage recognition XRCC1 The complex may be involved in the repair of nonhomologous DNA ends such The complex may be involved in the repair of nonhomologous DNA ends such The complex may be involved in the repair of nonhomologous DNA ends such The complex may be involved in the repair of nonhomologous DNA ends such The complex may be involved in the repair of nonhomologous DNA ends such The complex may be involved in the repair of nonhomologous DNA ends such DNA-directed RNA polymerase activity
0319. Additional contemplated sets of lifespan or longev MTND5, HPGD, IDH2, MDH1, MDH2, ME1, ME2, ME3, ity responsive genes include (without limitation): TERT, MTHD1, MTHFD1 L, MTHFR, NADK, NADSYN1, TERC, NRF2, POT1, TRF1, TRF2, TIN2, TPP1, RAPT, NDUFA2, NDUFA3, NDUFA4, NDUFA4L2, NDUFA5, TNKS, and TNKS 2: TERF2, TERF2IP, POLG, POLB, NDUFA6, NDUFA7, NDUFA9, NDUFA10, NDUFA12, POLD3, POLE, POLI, POLL, PARP2, PPARG, SHC1, PTOP, NDUFB2, NDUFB3, NDUFB5, NDUFB6, NDUFB7, IFI44 and NFKB1; HSPA1A, HSPA1B, and HSPA1L: NDUFB8, NDUFB9, NDUFC2, NDUFS2, NDUFS4, US 2014/0079836A1 Mar. 20, 2014 41
NDUFS5, NDUFS7, NDUFS8, NDUFV2, NDUFV3, tives from naturally occurring, biosynthetic or bioengineered NOX1, NOX3, NOX4, NOX5, NOXA1, NOXO1, NQO1, Sources. Exemplary routes of achieving contact with Such FOXO1, FOXO3, FOXO4, LMNA, NHP2L1, RAD50, modulating agent or agents may involve any known method RAD51, KL and KU70; TERT, TERC, NRF2, PARP1, POT1, of delivery or contact for at least one cell, tissue, organ or TRF1, TRF2, TIN2, TPP1, RAP1, Tankyrase 1, Tankyrase 2, organism in vivo or ex vivo or in vitro. TERF2, TERF2IP, POLG, POLB, POLD3, POLE, POLI, 0322. It is believed that plants from any plant Division, POLL, PARP2, PPARG, SHC1, PTOP, IFI44, NFKB1, including Bryophyta, Psilophyta, Lycophyta, Equisetophyta, MTND5, HPGD, IDH2, MDH1, MDH2, ME1, ME2, ME3, Filicophyta, Coniferophyta, Ginkgophyta, Cycadophyta, MTHD1, MTHFD1 L, MTHFR, NADK, NADSYN1, Gnetophyta, and Angiospermophyta. NDUFA2, NDUFA3, NDUFA4, NDUFA4L2, NDUFA5, 0323 Without intending to be limited to compounds or NDUFA6, NDUFA7, NDUFA9, NDUFA10, NDUFA12, compositions derived from particular plants, the following NDUFB2, NDUFB3, NDUFB5, NDUFB6, NDUFB7, specific plants are contemplated for preparing lifespan influ NDUFB8, NDUFB9, NDUFC2, NDUFS2, NDUFS4, encing compositions: coffee (e.g., coffee cherry extract), NDUFS5, NDUFS7, NDUFS8, NDUFV2, NDUFV3, green tea (e.g., green tea extract), blueberries (Alaskan, for NOX1, NOX3, NOX4, NOX5, NOXA1, NOXO1, NQO1, instance), cranberries, huckleberries, acai berries, goji ber FOXO1, FOXO3, FOXO4, LMNA, NHP2L1, RAD50, ries, blackberries, raspberries, grapes (scupernog), Strawber RAD51, KL and KU70; TERF2, TERF2IP, POLG, POLB, ries, persimmon, pomegranate, lingonberry, bearberry, mul POLD3, POLE, POLI, POLL, PARP2, PPARG, SHC1, berry, bilberry, choke cherry, sea buckthorn berries, goji HSPA1A, HSPA1B, and HSPA1L: PARP1, POT1, TRF1, berry, tart cherry, kiwi, plum, apricot, apple, banana, berry, TRF2, TIN2, TPP1, RAP1, Tankyrase 1, Tankyrase 2, blackberry, blueberry, cherry, cranberry, currant, greengage, TERF2, TERF2IP, POLG, POLB, POLD3, POLE, POLI, grape, grapefruit, gooseberry, lemon, mandarin, melon, POLL, PARP2, PPARG, SHC1, PTOP, IFI44, NFKB1, orange, pear, peach, pineapple, plum, raspberry, Strawberry, MTND5, HPGD, IDH2, MDH1, MDH2, ME1, ME2, ME3, sweet cherry, watermelon, and wild strawberry. In addition, MTHD1, MTHFD1 L, MTHFR, NADK, NADSYN1, extracts from trees and bushes are also contemplated, includ NDUFA2, NDUFA3, NDUFA4, NDUFA4L2, NDUFA5, ing for instance sequoia, coastal redwood, bristlecome pine, NDUFA6, NDUFA7, NDUFA9, NDUFA10, NDUFA12, birch, cedar of Lebanon, frankincense, and so forth. NDUFB2, NDUFB3, NDUFB5, NDUFB6, NDUFB7, 0324 By way of additional examples, compositions may NDUFB8, NDUFB9, NDUFC2, NDUFS2, NDUFS4, be from leafy or salad vegetables e.g., Amaranth (Amaran NDUFS5, NDUFS7, NDUFS8, NDUFV2, NDUFV3, thus cruentus), Arugula (Eruca sativa), Beet greens (Beta NOX1, NOX3, NOX4, NOX5, NOXA1, NOXO1, NQO1, vulgaris Subsp. vulgaris), Bitterleaf (Vermonia calvoana), FOXO1, FOXO3, FOXO4, LMNA, NHP2L1, RAD50, Bok choy (Brassica rapa Chinensis group), Broccoli Rabe RAD51, KL, KU70, HSPA1A, HSPA1B, and HSPA1L: (Brassica rapa Subsp. rapa), Brussels sprout (Brassica olera TERT, TERC, NRF2, PARP1, POT1, TRF1, TRF2, TIN2, cea Gemmifera group), Cabbage (Brassica oleracea Capitata TPP1, RAP1, TNKS, TNKS 2, TERF2, TERF2IP, POLG, group), Catsear (Hypochaeris radicata), Celery (Apium gra POLB, POLD3, POLE, POLI, POLL, PARP2, PPARG, veolens), Celtuce (Lactuca sativa var.asparagina), Ceylon SHC1, PTOP, IFI44, NFKB1, MTND5, HPGD, IDH2, spinach (Basella alba), Chard (Beta vulgaris var. cicla), MDH1, MDH2, ME1, ME2, ME3, MTHD1, MTHFD1 L, Chaya (Cnidoscolus aconitifolius Subsp. aconitifolius), MTHFR, NADK, NADSYN1, NDUFA2, NDUFA3, Chickweed (Stellaria), Chicory (Cichorium intybus), Chinese NDUFA4, NDUFA4L2, NDUFA5, NDUFA6, NDUFA7, cabbage (Brassica rapa Pekinensis group), Chinese Mallow NDUFA9, NDUFA10, NDUFA12, NDUFB2, NDUFB3, (Malva verticillata), Chrysanthemum leaves (Chrysanthe NDUFB5, NDUFB6, NDUFB7, NDUFB8, NDUFB9, mum coronarium), Collard greens (Brassica oleracea), Corn NDUFC2, NDUFS2, NDUFS4, NDUFS5, NDUFS7, salad (Valerianella locusta), Cress (Lepidium sativum), Dan NDUFS8, NDUFV2, NDUFV3, NOX1, NOX3, NOX4, delion (Taraxacum officinale), Endive (Cichorium endivia), NOX5, NOXA1, NOXO1, NQO1, FOXO1, FOXO3, Epazote (Chenopodium ambrosioides), Fathen (Chenopo FOXO4, LMNA, NHP2L1, RAD50, RAD51, KL, KU70, dium album), Fiddlehead (Pteridium aquilinum, Athyrium HSPA1A, HSPA1B, and HSPA; PGC1C, SIRT1, SIRT3, esculentum), Fluted pumpkin (Telfairia Occidentalis), Gar SIRT4, SIRT5, NRF1 and Tfam; and TNKS, TNKS2, TRF1, den Rocket (Eruca sativa), Golden Samphire (Inula crith TIN2, TPP1, POT1, RAP1, TRF2, and TERT. Also contem moides), Good King Henry (Chenopodium bonus-henricus), plated are subsets of any of these lists. Greater Plantain (Plantago major), Kai-lan (Brassica rapa Exemplary Methods and Compositions Alboglabra group), Kale (Brassica oleracea Acephala group), Komatsuna (Brassica rapa PerVidis or Komatsuna 0320 Provided herein are various methods and composi group), Kuka (Adamsonia spp.), Lagos bologi (Talinum fru tions for modulating gene expression or protein production or ticosum), Land cress (Barbarea verna), Lettuce (Lactuca cell signaling which controls the maintenance of the telomere sativa), Lizard's tail (Houttuynia cordata), Melokhia (Cor and/or which controls the biogenesis or respiratory activity of chorus Olitorius, Corchorus capsularis), Mizuna greens mitochondria and/or which control the lifespan, rate of aging, (Brassica rapa Nipposinica group), Mustard (Sinapis alba), senescence, onset of disease states, or response to stress New Zealand Spinach (Tetragonia tetragonioides), Orache including apoptosis and cell death for a living cell, tissue, (Atriplex hortensis), Paracress (Acmella oleracea), Pea organ or organism. sprouts/leaves (Pisum sativum), Polk (Phytolacca ameri 0321. The methods comprise contacting at least one cell cana), Radicchio (Cichorium intybus), Samphire (Crithmum with a sufficient amount of a modulating compound, or com maritimum), Sea beet (Beta vulgaris Subsp. maritima), bination of compounds either simultaneously exposed or Seakale (Crambe maritima), Siena Leone bologi (Crasso sequentially exposed. These compositions include the cephalum spp.), Soko (Celosia argentea), Sorrel (Rumex described modulating agents as well as their analogs, deriva acetosa), Spinach (Spinacia oleracea), Summer purslane US 2014/0079836A1 Mar. 20, 2014 42
(Portulaca oleracea), Swiss chard (Beta vulgaris Subsp. cicla latifolia), Camas (Camassia), Canna (Canna spp.), Carrot var. flavescens), Tatsoi (Brassica rapa RoSularis group), Tur (Daucus carota), Cassaya (Manihot esculenta), Chinese arti nip greens (Brassica rapa Rapifera group), Watercress (Nas choke (Stachys affinis), Daikon (Raphanus sativus Longipin turtium officinale), Water spinach (Ipomoea aquatica), Win natus group), Earthnut pea (Lathyrus tuberosus), Elephant ter purslane (Claytonia perfoliata), Yarrow (Achillea Foot yam (Amorphophallus paeoniifolius), Ensete (Ensete millefolium); fruiting and flowering vegetables, such as ventricosum), Ginger (Zingiber officinale), Gobo (Arctium those from trees e.g., Avocado (Persea americana), Bread lappa), Hamburg parsley (Petroselinum crispum var. tubero fruit (Artocarpus altilis); or from annual or perennial plants sum), Jerusalem artichoke (Helianthus tuberosus), Jicama e.g., Acorn squash (Cucurbita pepo), Armenian cucumber (Pachyrhizus erosus), Parsnip (Pastinaca sativa), Pignut (Co (Cucumis melo Flexuosus group), Aubergine (Solanum mel nopodium majus), Plectranthus (Plectranthus spp.), Potato Ongena), Bell pepper (Capsicum annuum), Bitter melon (Mo (Solanum tuberosum), Prairie turnip (Psoralea esculenta), mordica charantia), Caigua (Cyclanthera pedata), Cape Radish (Raphanus sativus), Rutabaga (Brassica napus Napo Gooseberry (Physalis peruviana), Capsicum (Capsicum brassica group), Salsify (Tragopogon porrifolius), ScorZon annuum), Cayenne pepper (Capsicum frutescens), Chayote era (Scorzonera hispanica), Skirret (Sium sisarum), Sweet (Sechium edule), Chili pepper (Capsicum annuum Longum Potato or Kumara (Ipomoea batatas), Taro (Colocasia escu group), Courgette (Cucurbita pepo), Cucumber (Cucumis lenta), Ti (Cordyline fruticosa), Tigernut (Cyperus esculen sativus), Eggplant (Solanum melongena), Lufa (Lufa acu tus), Turnip (Brassica rapa Rapifera group), Uluco (Ulucus tangula, Lufa aegyptiaca), Malabar gourd (Cucurbitaficifo tuberosus), Wasabi (Wasabia japonica), Water chestnut lia), Parwal (Trichosanthes dioica), Pattypan squash (Cucur (Eleocharis dulcis), Yacon (Smallanthus sonchifolius), Yam bitapepo), Perennial cucumber (Coccinia grandis), Pumpkin (Dioscorea spp.); and even sea vegetables e.g., Aonori (Cucurbita maxima, Cucurbita pepo), Snake gourd (Tricho (Monostroma spp., Enteromorpha spp.), Carola (Callophyllis Santhes cucumerina), Squash aka marrow (Cucurbita pepo), variegata), Dabberlocks aka badderlocks (Alaria esculenta), Sweet corn aka corn, aka maize (Zea mays), Sweet pepper Dulse (Palmaria palmata), Gim (Porphyra spp.), Hijiki (Hiz (Capsicum annuum Grossum group), Tinda (Praecitrullus ikia fusiformis), Kombu (Laminaria japonica), layer (Por fistulosus), Tomatillo (Physalis philadelphica), Tomato (Ly phyra spp.), MoZuku (Cladosiphon Okamuranus), Nori (Por copersicon esculentum var), Winter melon (Benincasa his phyra spp.), Ogonori (Graciliaria spp.), Seagrape (Caulerpa pida), West Indian gherkin (Cucumis anguria), Zucchini (Cu spp.), Seakale (Crambe maritima), Sea lettuce (Ulva lac curbita pepo); the flower buds of perennial or annual plants tuca), Wakame (Undaria pinnatifida). Some of which are not e.g., Artichoke (Cynara cardunculus, C. Scolymus), Broccoli even plants in the taxonomic sense. (Brassica oleracea), Cauliflower (Brassica oleracea), Squash blossoms (Cucurbita spp.); podded vegetables e.g., 0325 Of particular interest for the method described American groundnut (Apios americana), AZuki bean (Vigna herein are compounds and compositions derived from berry angularis), Black-eyed pea (Vigna unguiculata Subsp. fruits, which are recognized as producing a wide array of unguiculata), Chickpea (Cicer arietinum), Common bean (beneficial) phytochemicals. The botanical definition of a (Phaseolus vulgaris), Drumstick (Moringa oleifera), Doli berry is a simple fruit produced from a single ovary, such as a chos bean (Lablab purpureus), Fava bean (Vicia faba), Green grape. The berry is the most common type of fleshy fruit in bean (Phaseolus vulgaris), Guar (Cyanopsis tetragonoloba), which the entire ovary wall ripens into an edible pericarp. The Horse gram (Macrotyloma uniflorum), Indian pea (Lathyrus flowers of these plants have a superior ovary formed by the sativus), Lentil (Lens culinaris), Lima Bean (Phaseolus luna fusion of two or more carpels. The seeds are embedded in the tus), Moth bean (Vigna acontifolia), Mung bean (Vigna flesh of the ovary. However, the term “berry' as used herein is radiata), Okra (Abelmoschus esculentus), Pea (Pisum sati broader than the botanical definition and encompasses, for vum), Peanut (Arachis hypogaea), Pigeon pea (Cajanus instance, false berries (e.g., blueberries), aggregate fruits cajan), Ricebean (Vigna umbellata), Runner bean (Phaseolus (e.g., blackberries and raspberries), drupes (e.g., hackberries coccineus), Soybean (Glycine max), Tarwi (tarhui, chocho; and Agai palm), and accessory fruits (e.g., Strawberries). Lupinus mutabilis), Tepary bean (Phaseolus acutifolius), 0326 Examples of true berries include: grape (Vitis vin Urad bean (Vigna mungo), Velvet bean (Mucuna pruriens), ifera), tomato (Lycopersicon esculentum and other species of Winged bean (Psophocarpus tetragonolobus), Yardlong bean the family Solanaceae, many of which are commercial impor (Vigna unguiculata Subsp. sesquipedalis); bulb and stem tance, Such as Capsicum, and aubergine?eggplant (Solanum Vegetables e.g., Asparagus (Asparagus officinalis), Cardoon melongena), wolfberry or Goji berries (Lycium barbarum, (Cynara cardunculus), Celeriac (Apium graveolens var. Lycium spp.; Solanaceae), garberry (Berberis; Berberi rapaceum), Celery (Apium graveolens), Elephant Garlic (Al daceae), red, black, and white currant (Ribes spp.; Grossu lium ampeloprasum var. ampeloprasum), Florence fennel lariaceae), elderberry (Sambucus niger; Caprifoliaceae), (Foeniculum vulgare var. dulce), Garlic (Allium sativum), gooseberry (Ribes spp.; Grossulariaceae), honeysuckle Kohlrabi (Brassica oleracea Gongylodes group), Kurrat (Al (Lonicera spp., Caprifoliaceae) (the berries of Some species lium ampeloprasum var. kurrat), Leek (Allium porrum), (e.g., honeyberries) are edible, and even though others are Lotus root (Nelumbo nucifera), Nopal (Opuntia ficus-indica), poisonous they may provide useful phytochemicals if prop Onion (Allium cepa), Prussian asparagus (Ornithogalum erly purified), mayapple (Podophyllum spp.; Berberidaceae), pyrenaicum), Shallot (Allium cepa Aggregatum group), nannyberry or sheepberry (Viburnum spp., Caprifoliaceae), Welsh onion (Allium fistulosum), Wild leek (Allium tricoc Oregon-grape (Mahonia aquifolium; Berberidaceae), and cum); root and tuberous vegetables e.g., Ahipa (Pachyrhizus sea-buckthorn (Hippophae rhamnoides; Elaeagnaceae). Also ahipa), Arracacha (Arracacia xanthorrhiza), Bamboo shoot contemplated herein within the term “berries' are the modi (Bambusa vulgaris and Phyllostachys edulis), Beetroot (Beta fied, juicy berries, such as the fruit of citrus. Such fruits, vulgaris Subsp. vulgaris), Black cumin (Bunium persicum), including orange, kumquat, grapefruit, lime, and lemon, are Burdock (Arctium lappa), Broadleaf arrowhead (Sagittaria modified berries referred to botanically as hesperidium. US 2014/0079836A1 Mar. 20, 2014
0327. Also specifically contemplated herein is the choke foods called “superfruits’ and is identified by DataMonitor as berry (Aronia melanocarpa; commonly called black choke one of the top 10 food categories for growth in 2008 (Food berry), which has attracted scientific interest due to its deep Navigator-USA.com, “Fresh, Super and organic top trends for purple, almost black pigmentation that arises from dense 2008”, Nov. 28, 2007). contents of phenolic phytochemicals, and especially antho 0330. Additional sources for modulating compounds, and cyanins. Total anthocyanin content in chokeberries is 1480 methods for preparing compositions containing such, can be mg per 100 g of fresh berries, and proanthocyanidin concen found in the literature. See, for instance, European published tration is 664 mg per 100 g (Wu et al., JAgric Food Chem. 52: application EP 1985,280; Schmid et al., “Plant Stem Cell 7846-7856, 2004: Wu et al., J Agric Food Chem. 54: 4069 Extract for Longevity of Skin and Hair SOFW-Journal, 134: 4075, 2006). Both values are among the highest measured in 30-35, 2008; U.S. Pat. Nos. 7,544,497, 7,582,674; and Inter plants to date. Chokecherry produces these pigments mainly national Patent Publication No. WO/2007/084861. in the skin of the berries to protect the pulp and seeds from 0331 Further exemplary modulating compounds include constant exposure to ultraviolet radiation (Simon, Hort for instance stress-induced phenylpropanoids (see, e.g., FIG. Science 32(1):12-13, 1997). By absorbing UV rays in the 2 and Dixon et al., The Plant Cell 7:1085-1097, 1995). blue-purple spectrum, pigments filter intense Sunlight. Scien 0332 Exemplary modulating compounds or agents tific measurement of ORAC antioxidant strength demon strates chokeberry with one of the highest values yet include those selected from the group of compounds con recorded—16,062 micromoles of Trolox equivalents per 100 tained in coffee cherry acids or extracts including the antioxi g (Nutrient Data Laboratory, Agriculture Research Service, dant compounds chlorogenic acid, quinic acid, caffeic acid, US Department of Agriculture, 2007 publication entitled ferulic acid and proanthocyanidins. “Oxygen Radical Absorbance Capacity (ORAC) of Selected 0333 Exemplary modulating compounds or agents Foods, available on-line; see this ORAC reference also pro include ubiquinone, idebenone and the analogs and deriva vides antioxidant scores for 277 common foods). Analysis of tives thereof including various esters and conjugated com anthocyanins in chokeberries has identified the following pounds. individual chemicals (among hundreds known to exist in the 0334 Exemplary modulating compounds or agents plant kingdom): cyanidin-3-galactoside, epicatechin, caffeic include extracts and the analogs and derivatives obtained acid, quercetin, delphinidin, petunidin, pelargonidin, peoni from cocoa. The extracts, compounds or combinations of din, and malvidin. All these are members of the flavonoid compounds derived from the cocoa beans from various iso category of antioxidant phenolics, and they are found in lation or purification processes are derived from any species myriad other plants in differing concentrations. of Theobroma, Herrania or inter- or intra-species hybrid 0328 Many “berries' as referenced herein are not true crosses thereof. It is also understood that similarly such berries by the scientific definition, but are in fact drupes, extracts or compounds are included if derived from geneti epigynous fruits, or compound fruits. Drupes are fruits pro cally engineered versions of these species or hybrids. Further duced from a single-seeded ovary or achene; example drupes more synthetic formulations, analogs or derivatives of these are hackberry (Celtis spp., Cannabaceae) and Agai (Euterpe), compounds are similarly included as well as compounds a palm fruit native to the Amazon region. Epigynous fruits are derived from natural or synthetic fermentation processes. berry-like fruits formed from inferior ovaries, in which the These extracts or compounds preferably comprise polyphe receptacle is included. Notable examples are the fruits of the nol(s) such as cocoa procyanidin(s). Such as at least one cocoa Ericaceae, including blueberry, huckleberry, and cranberry. procyanidin selected from (+) catechin, (-) epicatechin, pro Other epigynous fruits include: bearberry (Arctostaphylos cyanidin oligomers 2 through 18, procyanidin B-5, procyani spp.), crowberry (Empetrum spp.), lingonberry (Vaccinum din B-2, procyanidin A-2 and procyanidin C-1. vitis-idaea), Strawberry tree (Arbutus unedo), and sea grape 0335 Exemplary modulating compounds or agents (Coccoloba uvifera; Polygonaceae). The fruit of cucumbers, include extracts and the analogs and derivatives obtained melons and their relatives are modified berries called "pep from Camelia sinensis, Camelia sinensis sinensis, Camellia oes. Compound fruits are groups or aggregates of multiple Sinensis assamica or Camelia oleifera either naturally or individual fruits with seeds from different ovaries of a single synthetically derived. flower, and include: blackberry, dewberry, boysenberry, ola 0336 Exemplary modulating compounds or agents lieberry, and tayberry (genus Rubus), cloudberry (Rubus include resveratrol and the analogs and derivatives thereof, chamaemorus), loganberry (Rubus loganobaccus), rasp including viniferin, gnetin H, and Suffruticosol B. berry, Rubus idaeus and other species of Rubus, salmonberry 0337 For methods of preparing (green) tea extracts, see, (Rubus spectabilis), thimbleberry (Rubus parviflorus), for instance, Perva-Uzunalic et al., Food Chemistry 96(4): wineberry (Rubus phoenicolasius), bayberry, and boysen 597-605, 2006: Koiway & Masuzawa, Jpn. J. Appl. Phys berry. Multiple fruit are the fruits of separate flowers, packed 46:4936-4938, 2007; U.S. Pat. Nos. 4,668,525 and 3,080, closely together, Such as the mulberry. Others are accessory 237. Tea extracts containing polyphenols, as well as indi fruit, where the edible portion is not generated by the ovary, vidual tea-derived polyphenols, are commercially available such as the strawberry. from many sources. By way of example only, one source is 0329 Berry colors are due to natural plant pigments. Pharma Cosmetix Research, LLC (Richmond, Va.), the Sup Many are polyphenols such as the flavonoids, anthocyanins, plier of Premier Green Tea Extract Loti 10783 that was used and tannins localized mainly in berry skins and seeds. Berry in various examples described herein. pigments are usually antioxidants and thus have oxygen radi 0338 Idebenone (CAS no. 58186-27-9) is commercially cal absorbance capacity (“ORAC) that is high among plant available from myriad Suppliers, including for instance foods (Wu et al., J. Agric. Food Chem. 52(12):4026-4037, Pharma Cosmetix Research, LLC (Richmond, Va.), the Sup 2004). Together with good nutrient content, ORAC distin plier of idebenone Lot #27816 that was used in various guishes several berries within a new category of functional examples described herein. US 2014/0079836A1 Mar. 20, 2014 44
0339 Coffee cherry extract can be prepared using art rec agents or therapies to improve or enhance or increase the ognized methods; see, for instance U.S. Patent Publication destruction of the cancer and thus improve the cure rate of No. 2007/028.1048 (published Dec. 6, 2007). In addition, the Such a therapy. coffee cherry extract referred to as COFFEEBERRYR) can be 0349 Yet another topical embodiment may target other purchased from VDF FutureCeuticals, Inc. (Momence, Ill.); structures in the skin Such as hair or nails. One such applica for several of the experiments described herein, COFFEE tion is to prevent, delay or reverse hair loss or other disorders BERRYR) Beauty LothiO2480000x5729 from VDF was used. of aging Such as graying of the hair. The modulating agent 0340. In one embodiment a modulating compound or may be used to contact the hair directly or it may modulate the combination of modulating compounds may be used to aging or damage to the skin in which the hair follicle is extend the lifespan of one or more types of cells in the skin or located thus indirectly reducing hair loss. Subcutaneous tissue under the skin including fat, fascia, 0350 One embodiment utilizes modulation to protect or muscle and blood vessels. Such a treatment may be topical or repair the telomere structure so as to extend the lifespan of at systemic and may be delivered, with or without penetration least one cell. Alternatively modulation may be directed to enhancing agents or therapies, in many forms well known to shorten the lifespan of at least one cell. While extending the one skilled in the art of skin medications. lifespan of living cells is one very important function of the 0341 Topical delivery of modulating agents may uniquely invention, in certain cases such as diseased, damaged or can extend the lifespan of contacted cells in the skin or subcuta cerous cells it may be desirable to accelerate the death of such neous tissues without necessarily modulating the lifespan of cells or to turn immortalized cells back into mortal cells so the entire organism. Systemic delivery of modulating agents that they may be killed or be more responsive to other thera may also reach the skin to produce a lifespan modulating pies. effect. 0351. There are various skin cells which may be targeted 0342 Topical formulations may include but are not lim alone or in various combinations to contact a modulating ited to creams, emollients, gels, lotions, Solutions, micro compound. These includebut are not limited to keratinocytes, emulsions, Suspensions, ointments, spray mists, delayed or fibroblasts, melanocytes, Langerhans cells, merkel cells, time release formulations, patches, injectable, implantable, nerve cells, endothelial cells, adipocyte or fat cells, muscle depot, mask or other formulations. cells and the various specialized cells of Sweat and oil glands, 0343 Various methods may be utilized to enhance pen hair structure cells, nail and other skin appendage cells. It may etration including liposomal or polymer or other matrix deliv also be desirable to modulate the lifespan of stem and pro ery systems, agents which enhance delivery or disrupt skin genitor cells. Subcellular organelles including mitochondria, barrier function, ultrasound or acoustic assisted delivery, ribosomes, and Golgi apparatus may also be indirect targets laser or mechanical disruption of the skin or other energy for the modulating agent as well as nuclear and mitochondrial based devices which enhance delivery or disrupt skin barrier DNA function thus indirectly enhancing delivery into the skin or 0352 Another embodiment involves contacting at least through the skin into the Subcutaneous tissues. one cell of the organism with a sufficient amount of modu 0344) Other embodiments may include delivery combined lating compound to protect, defend, reverse, rescue, revive, with skin care products such as cosmetic foundations, resuscitate, or repair acute stress from the environment, from makeup, lipstick, shampoo, cleansers, Sunscreen, and body oxidative stress, from acute or chronic injury or disease lotions. Systemic delivery may include but not limited to oral, including acutely injured and dying cells. These cells may be parenteral, intravenous, intradermal, intramuscular, rectal, skin cells or they may be cells from any or all parts of the buccal, Sublingual, Vaginal, ophthalmic, otic, intranasal, tissue, organ or organism. nebulizer, injectable, depot, catheter, endoscopic or incorpo 0353. In yet another embodiment the modulating com rated onto or into implantable devices or agents. pound may be used to contact one or more cells when they are 0345. In one embodiment the modulating agent is used to not present in the living organism but rather they are ex vivo reduce one or more factors which create the appearance of Such as an organ being prepared for transplant, or in vitro. For aging or prematurely aging skin Such as fine lines, wrinkles, example a donor organ being transported is subjected to vari uneven pigment, skin radiance, skin elasticity, skin thickness, ous cellular stresses and has a finite time span of viability and pore size, skin sagging, loss of subcutaneous fat or Volume in the modulating agent may be utilized to extend this time span the skin collagen and abnormal vascularity. and/or to increase the number of healthy functioning cells 0346. In yet another embodiment the modulating agent present during the same time span. Another application with may be used in combination with agents or methods which transplanted cells, tissues or organs is to repair the telomere protect the skin from UV ultraviolet or IR infrared damage structure so that the lifespan is extended before or after trans from any light source to enhance, facilitate or produce DNA plantation. For example a donor kidney from an older donor repair or telomere structure protection or repair or to prevent, might be treated with a modulating agent prior to transplan diminish or avoid apoptosis. The concomitant use either tation in order to extend the lifespan of the kidney for trans simultaneously in the same formulation or serially within 24 plant into a younger transplant recipient or to make the trans hours of compounds which function as antioxidants may be planted kidney less Vulnerable to apoptosis or damage from utilized. either the procedure itselfor to the immunosuppressive thera 0347 A preferred embodiment may include the use of an pies given after the transplantation. agent which modulates the maintenance of telomere in com 0354. In vitro fertilization and embryo and stem cell bination with an agent which mimics caloric restriction, Such research are yet other embodiments wherein the modulating as resveratrol or a sirtuin pathway modulating agent. agent may be used to extend the lifespan of cells. 0348 Other topical embodiments may utilize modulation 0355. In another embodiment the modulating agent may of lifespan to decrease or shorten the lifespan of cancerous be used to extend the lifespan of the progeny or a cloned cells either alone or in combination with various anticancer derivative of an organism. It is known that Somatic and US 2014/0079836A1 Mar. 20, 2014 embryonic cloning may produce cloned organisms with with an agent or compound which mimics or produces shorter lifespan than the original organism that was cloned. directly or indirectly caloric restriction biochemical and/or The modulating agent may be used to extend the lifespan of a cellular processes in living organisms. cloned organism directly. Another option is to use the modu 0363 Another similar embodiment is the use of a modu lating agent to repair the telomere structure in a recloning lating agent or compound in association with (either com event either of the original organism or of the clone itself. bined, co-administered, or sequentially administered) other 0356. In one embodiment the modulating agent may con lifespan modulating compounds which modulate the biogen tact plant cells which are being cloned or expanded by a esis and/or respiratory capacity of mitochondria. meristematic process in which the cell line has become senes 0364 Premature or accelerated aging as a result of direct cent and the agent may help restore viability to extend the or indirect interaction of cells with environmental factors lifespan of the plant cell culture allowing continued commer which injure at least one cell or which produce cellular stress cial production of copies of the plant. and/or cellular inflammatory processes and/or oxidative 0357. A useful embodiment may utilize the modulating stress and/or DNA or telomere structure damage and/or cel agent to treat autoimmune disease where autoimmune or lular apoptosis may be delayed, retarded, diminished, pre inflammatory processes shorten the lifespan of cells thus vented, or even repaired or reversed by use of effective com producing disease, disability, premature aging or even death. binations and concentrations of at least one of a telomere 0358. One preferred embodiment incorporates other structure modulating compound, a caloric restriction mim lifespan modulating agents with the modulating agent or icking compound and a compound which stimulates more agents described in this invention for the purpose of extending efficient mitochondrial respiratory activity and/oran increase the lifespan or shortening the lifespan of at least one cell. For in the number of mitochondria. example a modulating agent to shorten lifespan might be 0365. In a further embodiment a telomere structure modu included with an anti cancer therapeutic agent or treatment lating compound may be utilized in combination with at least with the purpose of making the cancerous target cell more one mitochondrial biogenesis modulating compound so that Vulnerable to the therapy. Another example would be to dif not only is the telomere structure of either the mitochondrial ferentially modulate lifespan so that the lifespan of the can DNA and/or the nuclear DNA protected but also the number cerous cells was shortened but the lifespan of the non cancer of mitochondrial organelles is also modulated. To further the ous normal cells was extended or at least protected. goal of lifespan extension the maintenance of the telomere 0359 An embodiment to extend the lifespan of cardiac structure would be stimulated, activated or enhanced as well muscle cells could be used to extend the lifespan of the entire as stimulating an increase in the actual number of mitochon organism Such as a human or an animal such as a horse or dria. companion animal Such as a cat or dog. The use of a modu 0366. In a further embodiment, in the case of diseased or lating agent to prevent, diminish or reverse apoptosis in car cancerous cells the opposite goal would be desirable in that diac muscle cells during an acute injury Such as a myocardial accelerating the death of these abnormal cells would be the infarction or heart attack or ischemic episode not only pre goal and thus impairing the telomere structure maintenance serves these cells but also may prevent disability or death of and decreasing the number or the respiratory efficiency of the the entire organism. diseased or cancerous cells would be desirable. 0360. Other embodiments include diverse and novel meth 0367. In a further embodiment it may be desirable to ods of producing contact of the modulating agent with a cell extend the lifespan of healthy cells and shorten the lifespan of including aerosolizing into a steam sauna or humidifier for diseased or cancerous cells in order to maximize the healthy inhalation of the agent, impregnating clothing for contact lifespan of the tissue, organ or entire organism. with the agent, impregnating implantable devices such as 0368. In one embodiment a modulating agent such as ide vascular stents or joint replacements or ocular lens implants. benone, or its derivatives or analogs which transfer electrons Intraocular injections of a modulating agent might be used to rather than terminate electron transfer, may be used to reduce extend the lifespan of retinal cells. Injectable filling agents are oxidative stress on mitochondria by transferring electrons commonly used for the skin and Subcutaneous tissue and a down the electron transport system within the mitochondria modulating agent may be incorporated into the implant or bypassing complex I and instead transferring the electron to agent in a time or delayed release formulation. Transdermal complex III. Complex I creates much of the ROS and oxida patches for hormonal therapy might incorporate a modulating tive stress within the mitochondria that is internally generated agent for systemic delivery as part of an anti aging hormone (in contrast to ROS created by exposure to outside environ replacement therapy. Novel oral delivery may include incor mental stress or other injuries) and mitochondria have limited poration into toothpaste, mouthwash, oral lozenges, chew ability to neutralize oxidative stress thus mitochondria respi able items, or dental floss. ratory efficiency declines over time and is responsible for part 0361. An embodiment for oral delivery may include of the premature aging or senescence or dysfunction or dis diverse forms known in the art including but not limited to ease states in cells so affected. This bypass of electrons then nutritional Supplements or vitamins, additives for food or may contribute to lifespan extension of the cell as well as beverages, in combination with various drugs. The incorpo contribute to a healthier lifespan. ration of a modulating agent via genetically engineered plant 0369. In a further embodiment a modulating agent may oranimal or other food products is another route to administer improve or protect the function or even produce repair of a modulating agent. damage to the ribosomes. Ribosomes are responsible for the 0362. One key embodiment is the use of a modulating translation of instructions from the DNA during the synthesis agent or compound in association with (either combined, of proteins. Thus, maintaining the accuracy of ribosome co-administered, or sequentially administered) other lifespan translational activity may prolong lifespan. It has recently modulating compounds. A telomere structure maintenance been reported that UVB radiation induces persistent activa modulating compound may be combined in Such a manner tion of ribosome and oxidative phosphorylation pathways US 2014/0079836A1 Mar. 20, 2014 46
(Tsai et al., Radiat. Res. 171 (6):716-724, 2009. Those authors 0375. In another embodiment this data from the index noted that ultraviolet B (UVB) radiation has strong biological could be used to assess both the need for treatment interven effects and modulates the expression of many genes. Though tion with a modulating agent, but also to guide the therapeutic the major biological pathways affected by UVB radiation treatment doses and routes of administration and protocols. It remain controversial, Tsai et al. used a loop-design microar could also be used for risk assessment or for predictive appli ray approach and applied rigorous statistical analyses to iden cations. A lifespan extension factor or age protection or pro tify differentially regulated genes at 4, 8, 16 or 24 hours after tective factor or an anti aging protection or protective factor UVB irradiation. The most prominent biological categories in could also be created to guide therapy or to assign a value to lists of differentially regulated gene sets were extracted by the efficacy of a modulating agent. functional enrichment analysis. With this approach, the 0376. In an illustrative embodiment a human or animal is authors determined that genes participating in two prime tested diagnostically for the level of a lifespan modulating cellular processes, the ribosome pathway and the oxidative protein or factor and then rated or graded relative to other phosphorylation pathway, were persistently activated after human or like animal populations and how they compare UVB irradiation. Mitochondrial activity assays confirmed relative to this group provides a relative risk factor for greater increased activity for up to 24 h after UVB irradiation. These or shorter lifespan than the comparison group. A lifespan results suggest that the persistent activation of ribosome and modulating compound or group of compounds might then be oxidative phosphorylation pathways may have a key role in selected to treat the human or animal based on the lifespan UVB-radiation-induced cellular responses. extension factor. This could be used in an attempt to repair or 0370. Also contemplated are methods that improve correct existing damage or it could be used as a lifespan immune function, for instance by modifying the expression of protective factorina preventive way. Diagnostic testing could one or more genes involved in a nitric oxide pathway. Syn then be utilized to assess the efficacy of the treatment and thesis of nitric oxide (NO) is one of the important effector guide ongoing therapeutic efforts using the modulating factor functions of innate immune cells. Although several reports (s). have indicated mistletoelectins induce immune cells to pro 0377. In another embodiment a buccal Swab, punch or duce cytokines, studies regarding the activities of the lectins shave biopsy from the skin or any internal organ or system, for in the production of NO have been very limited. It has the purpose of assessment of anti aging gene expression pro recently been reported (Bong-Kang et al., J. Biomed. Sci., files through the use of human genome, or specialized custom 197-204, 2007), for instance, that Korean mistletoe (e.g., cDNA microarrays is collected and compared to a control Viscum album coloratum) lectin (KML-IIU) induces NO syn sample, which can be from an age matched subject, a pooled thesis in a murine macrophage cell line. When the macroph collection of subjects or the same subject taken years earlier age cells were treated with KML-IIU in the presence of a or later. The comparison of this profile would enable a deter suboptimal concentration of IFN-gamma, NO production mination to be made on the relative effects of aging on lon was induced in a concentration-dependent manner (Id.). gevity/mitochondrial related genetic factors. 0371. In a further embodiment modulating the rate of pro 0378. In a further embodiment a buccal swab, punch or tein synthesis through ribosomal activity modulation may be shave biopsy from the skin or any internal organ or system, for utilized to increase the lifespan of a cell. the purpose of assessment of anti-aging gene expression pro 0372. In a further embodiment a nucleic acid may be intro files through the use of human genome, or specialized cDNA duced into a cell to modulate the level of a modulating agent microarray, is collected from a treated and untreated location that is at least about 70%, 80%, 90%, 99% identical to the on the same Subject. Comparison of this genome expression sequence of a modulating agent target such as telomerase or profile can be used to assess the ability of the treatment sirtuin or electron transport protein. modality to alter/extend the longevity or mitochondrial func 0373) In another embodiment various methods of diagnos tion. This embodiment will allow for the testing of formula ing the level of a telomere structure maintenance protein are tion levels, combinations of, and sequential application of utilized such methods which are well known to those skilled modulating compounds as viable interventions. One example in the art of these diagnostics. Using Such diagnosis one may of this would be the inclusion of a modulating agent in a determine if an organism is likely to have accelerated aging or Sunscreen that is applied to a subject and then tested and shortened lifespan. After Such a diagnosis is made, then a through the aforementioned genomic data a relative level of therapeutically effective amount of a modulating agent may efficacy can be determined. be used to treat that organism. The efficacy of this treatment may then be measured again at periodic appropriate intervals Compositions, Including Pharmaceutical Compositions to assess the progress of the treatment. Such diagnostic meth ods may also be used in screening compounds and formula 0379 Compositions for use in accordance with the present tions of compounds and efficacy of delivery methods and methods may be formulated in conventional manner using optimal concentrations of modulating agents. one or more physiologically acceptable carriers. Methods and 0374. In another embodiment such diagnostic information formats for cosmetic and cosmeceutical compositions are may be combined with various other data obtained from the well known. For non-limiting examples, see for instance US organism in order to create a profile or index that gives a publication no. 2009/0208433, Japan publications no. relative value scale foraging or lifespan for benchmarking an JP08092057, JP200031915.4; and United Kingdom publica individual organism relative to a larger population of the same tion no. GB2445265A. organism or to a historical database of the same organisms or 0380 Compounds and their physiologically acceptable any other subset of data which might be of interest. This index salts and Solvates may be formulated for administration by, might be viewed as an aging index or anaging ageing index or for example, injection, inhalation or insufflation (either a longevity or lifespan index. through the mouth or the nose) or oral, buccal, parenteral or US 2014/0079836A1 Mar. 20, 2014 47 rectal administration. The compound is administered locally, the skin than could be expected from application of the for at the site where the target cells, e.g., diseased or aged cells, mulation alone. These processes may also enhance the effect are present. of the compound(s) through increased absorption or chemi 0381 Compounds can be formulated for a variety of dis cal/physical change making the compound(s) more reactive pensation methods, including systemic (injectable, pill form, or effective. Suppository, inhalant) and topical (creams, lotions, gel, wrap, 0389 Pharmaceutical compositions (including cosmetic coated bandage or adhesive strip) or localized administration. preparations) may comprise from about 0.00001 to 100%, For systemic administration, injection is preferred, including such as from 0.001 to 10% or from 0.1% to 5% by weight or intramuscular, intravenous, intraperitoneal, and Subcutane Volume of one or more compounds described herein, such as ous. The injectable can be formulated in liquid Solutions, for instance coffee cherry, idebenone, carnosine, green tea preferably in physiologically compatible buffers such as extract, or another plant extract or component thereof. Ringer's Solution. In addition, the compounds may beformu 0390. In one embodiment, a compound described herein, lated in solid form and redissolved or suspended immediately is incorporated into a topical formulation containing a topical prior to use. Lyophilized forms are also included. carrier that is generally Suited to topical drug administration 0382 For oral administration, compositions may take the and comprising any Such material known in the art. The form of for example, tablets, lozenges, or capsules prepared topical carrier may be selected so as to provide the composi by conventional means with pharmaceutically acceptable tion in the desired form, e.g., as an ointment, lotion, cream, excipients. The tablets may be coated by methods well known microemulsion, gel, oil, Solution, or the like, and may be in the art. Liquid preparations for oral administration may comprised of a material of either naturally occurring or Syn take the form of for example, Solutions, syrups or Suspen thetic origin. sions, or they may be presented as a dry product for consti 0391) Formulations may be colorless, odorless ointments, tution with water or other suitable vehicle before use. Such lotions, creams, micro-emulsions and gels. liquid preparations may be prepared by conventional means 0392 Compounds may be incorporated into ointments, with pharmaceutically acceptable additives such as Suspend which generally are semisolid preparations which are typi ing agents (e.g., Sorbitol syrup, cellulose derivatives or hydro cally based on petrolatum or other petroleum derivatives. The genated edible fats); emulsifying agents (e.g., lecithin or aca specific ointment base to be used, as will be appreciated by cia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl those skilled in the art, is one that will provide for optimum alcohol or fractionated vegetable oils); preservatives (e.g., drug delivery, and, preferably, will provide for other desired methyl or propyl-p-hydroxybenzoates or sorbic acid). The characteristics as well, e.g., emolliency or the like. Emulsion preparations may also contain buffer salts, flavoring, coloring ointment bases are either water-in-oil (W/O) emulsions or and Sweetening agents as appropriate. Preparations for oral oil-in-water (O/W) emulsions, and include, for example, administration may be suitably formulated to give controlled cetyl alcohol, glyceryl monostearate, lanolin and Stearic acid. release of the active compound. Exemplary water-soluble ointment bases are prepared from 0383 For administration by inhalation, the compounds polyethylene glycols (PEGs) of varying molecular weight. may be conveniently delivered in the form of an aerosol spray 0393 Compounds may be incorporated into lotions, presentation from pressurized packs or a nebulizer. In the case which generally are preparations to be applied to the skin of a pressurized aerosol the dosage unit may be determined by Surface without friction, and are typically liquid or semi liq providing a valve to deliver a metered amount. The compound uid preparations in which Solid particles, including the active can be prepped for use in an inhaler or insufflator and may be agent, are present in a water or alcohol base. Lotions are formulated containing a powder mix of the compound. usually suspensions of solids, and may comprise a liquid oily 0384 The compounds may be formulated for parenteral emulsion of the oil-in-water type. administration by injection, e.g., by bolus injection or con 0394 Compounds may be incorporated into creams, tinuous infusion. The compositions may contain formulatory which generally are viscous liquid or semisolid emulsions, agents such as Suspending, stabilizing and/or dispersing either oil-in-water or water-in-oil. Cream bases are water agents. Alternatively, the active ingredient may be in powder washable, and contain an oil phase, an emulsifier and an form for constitution with a suitable vehicle, e.g., sterile aqueous phase. pyrogen-free water, before use. 0395 Compounds may be incorporated into micro-emul 0385 Slow release implantable formulations may include sions, which generally are thermodynamically stable, isotro coated devices Such as vascular stents or grafts, dermal or pically clear dispersions of two immiscible liquids, such as oil Subcutaneous implants, cervical rings, dental implants or and water, stabilized by an interfacial film of surfactant mol other implant or infusion pump delivery methods. ecules. 0386 The compounds may also be formulated in rectal 0396 Compounds may be incorporated into gel formula compositions such as Suppositories or retention enemas. tions, which generally are semisolid systems consisting of 0387. In addition to the formulations described previously, either Suspensions made up of small inorganic particles (two the compounds may also be formulated as a depot prepara phase systems) or large organic molecules distributed Sub tion. Such long acting formulations may be administered by stantially uniformly throughout a carrier liquid (single phase implantation (for example Subcutaneously or intramuscu gels). Single phase gels can be made, for example, by com larly) or by intramuscular injection. bining the active agent, a carrier liquid and a suitable gelling 0388. The compound(s) may also be formulated so that agent together and mixing until a characteristic semisolid Subcutaneous delivery through application or addition of product is produced. Although gels commonly employ aque ultrasound, iontophoresis, occlusion, Sonication and/or other ous carrier liquid, alcohols and oils can be used as the carrier mechanisms that enlarge the pore size, disrupt the epidermal liquid as well. barrier, alter the chemical structure or otherwise drive the 0397 Various additives, known to those skilled in the art, compound(s) further through, or enhance the absorption of may be included in formulations, e.g., topical formulations. US 2014/0079836A1 Mar. 20, 2014 48
Examples of additives include, but are not limited to, solubi U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206: U.S. Pat. lizers, skin permeation enhancers, opacifiers, preservatives No. 5,271,961; U.S. Pat. No. 5,254,342; and U.S. Pat. No. (e.g., anti-oxidants), gelling agents, buffering agents, Surfac 5,534,496). tants (particularly nonionic and amphoteric Surfactants), 0402 Compounds described herein may be stored in oxy emulsifiers, emollients, thickening agents, stabilizers, gen free environment according to methods in the art. For humectants, colorants, fragrance, and the like. Inclusion of example, resveratrol or analog thereof can be prepared in an solubilizers and/or skin permeation enhancers is particularly airtight capsule for oral administration. preferred, along with emulsifiers, emollients and preserva 0403 Cells, e.g., treated ex vivo with a compound tives. described herein, can be administered according to methods 0398. Other active agents may also be included in formu for administering a graft to a subject. lations, e.g., other anti-inflammatory agents, analgesics, anti 04.04. It is also contemplated that the compositions microbial agents, antifungal agents, antibiotics, vitamins, described herein can be used in combination with other com antioxidants, and Sun block agents commonly found in Sun pounds or drugs, for instance other recognized antioxidant screen formulations. compounds, Sunscreens, anticancer and anti-infective agents, anti-inflammatory Substances, and so forth. 0399 Topical skin treatment compositions can be pack aged in a suitable container to Suit its viscosity and intended use by the consumer. For example, a lotion or cream can be Arrays packaged in a bottle or a roll-ball applicator, or a propellant 04.05 Also provided herein are collections of genes that driven aerosol device or a container fitted with a pump Suit have been found to be influenced by antioxidant(s), and/or able for finger operation. Novel pumps or dispensers which that are now recognized as being involved in lifespan exten mix products from separate chambers at the time of dispens Sion, cell longevity or health, mitochondrial biogenesis or ing may be used. When the composition is a cream, it can function, telomere maintenance or DNA fidelity or repair, and simply be stored in a non-deformable bottle or squeeze con so forth. The identification of sets of genes that are responsive tainer, Such as a tube or a liddedjar. The composition may also to antioxidant treatment and that act in a concerted manner be included in capsules such as those described in U.S. Pat. (e.g., in a recognized pathway, in a similar manner as to No. 5,063,507. magnitude and/or direction of change in gene expression, (0400. In an alternative embodiment, a pharmaceutical for etc.) enables the production of tailored arrays. Such arrays mulation is provided for oral or parenteral administration, in can be used in myriad ways, including but not limited to which case the formulation may comprises an activating com characterizing the activities of known antioxidants, studying pound-containing microemulsion as described above, but and identifying potential new antioxidant compositions, may contain alternative pharmaceutically acceptable carriers, tracking the biological effect (e.g., on an experimental system vehicles, additives, etc. particularly Suited to oral or or a Subject) of an antioxidant treatment regimen, and analy parenteral drug administration. Alternatively, an activating sis of, e.g., skin biopsy, blood, and other various body com compound-containing microemulsion may be administered ponents. orally or parenterally substantially as described above, with 0406. The specific arrays described herein were con out modification. structed at the inventor's specifications by SABiosciences 04.01 Conditions can be treated or prevented by, e.g., sys (Fredrick, Mass.) (information relevant to their procedures is temic, topical, intraocular injection of a compound described available on-line at Sabiosciences.com/customarray biomar herein, or by insertion of a sustained release device that ker.phpihiw). The genes in the first custom microarray (“Ar releases a compound described herein. Polymers can be used ray 1) were selected based on an exhaustive literature search for controlled release. Various degradable and nondegradable for previously recognized longevity genes and lifespan alter polymeric matrices for use in controlled drug delivery are ing genes. The second microarray (Array 2) includes the known in the art (Langer. Accounts Chem. Res. 26:537, genes from the first array, plus genes related to mitochondrial 1993). For example, the block copolymer, polaxamer 407 biogenesis, respiration efficiency, telomere maintenance, and exists as a viscous yet mobile liquid at low temperatures but genes that had a large significant response the Agilent and/or forms a semisolid gel at body temperature. It has shown to be Affymetrix Human Genome array analyses described herein. an effective vehicle for formulation and sustained delivery of This customization of the array permits focused genetic recombinant interleukin-2 and urease (Johnston et al., analysis that is significantly faster than analyzing the entire Pharm. Res. 9:425, 1992: Pec, J. Parent. Sci. Tech. 44(2):58, human genome. The array style selected was a 96 well plate 1990). Alternatively, hydroxyapatite has been used as a suited for a BioRadicycler. The initial array was a 48 gene set microcarrier for controlled release of proteins (Intema et al., (including all required controls and QC checks recommended Int. J. Pharm. 112:215, 1994). In yet another aspect, lipo by the manufacturer) and allowed two samples to be run on Somes are used for controlled release as well as drug targeting each plate. The second array had 91 genes of interest (the of lipid-capsulated compounds (Betageri et al., Liposome remaining spaces were controls and QC checks). The genes Drug Delivery Systems, Technomic Publishing Co., Inc., were selected using the SABioscience custom array online Lancaster, Pa., 1993). Numerous additional systems for con design tool, which gave a RefSeq number once the gene trolled delivery of therapeutic proteins are known (e.g., U.S. symbol of interest was entered. Pat. No. 5,055.303; U.S. Pat. No. 5,188,837; U.S. Pat. No. 0407. By way of example, the following lists provide two 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; different arrays of genes identified hereinas being associated U.S. Pat. No. 4,957,735; and U.S. Pat. No. 5,019,369; U.S. with or linked to some aspect of lifespan extension. Design Pat. No. 5,055.303; U.S. Pat. No. 5,514,670; U.S. Pat. No. and use of these exemplified arrays are described in the 5,413,797; U.S. Pat. No. 5,268, 164; U.S. Pat. No. 5,004,697: Examples.
US 2014/0079836A1 Mar 20, 2014 50
-continued Array 2 Gene Symbol Alias Refseq # Official FullName IL11 AGIFIL-11 NM 000641 interleukin 11 IL1A NM 000575 interleukin 1, alpha IL33 NM 033439 interleukin 33 NM OOO600 interleukin 6 (interferon, beta 2) CXCL8, GCP NM 000584 interleukin 8 1 GCP1, LECTLUCTLYNAPSMDNCFMO NAPNAFANAP-1 NAP1 IMMP1L, FL2SOS9 IMP1 IMP1-LIKE NM 144981 MP1 inner mitochondrial membrane peptidase ike (S. cerevisiae) JUN AP-1, AP1, c-Jun NM OO2228 un. Oncogene KIT C-Kit CD117 PBTSCFR NM 000222 V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog KL NM 004.795 Klotho KRAS C-K-RASK-RAS2AK-RAS2BK NM OO4985 V-Ki-ras2 Kirsten rat Sarcoma viral oncogene RAS4AK-RAS4B, KI homolog RASKRAS1 KRAS2NS3RASK2 MAPK14 CSBP1, CSBP2. CSPB1 EXIP.MX2fPRKM1 NM 001315 Mitogen-activated protein kinase 14 4PRKM15/RK/SAPK2Ap38/p38ALPHA MMP1 CLG CLGN NM 002421 Matrix metallopeptidase 1 (interstitial collagenase) NADSYN NM 018161 NAD synthetase 1 1 NEIL1 FLJ22402 FPG1 NEI1 FPG1 NM O24608 Nei endonuclease VIII-like 1 (E. coli) NFKB1 DKFZp686C01211/EBP NM OO3998 Nuclear factor of kappa light polypeptide gene /KBF1/MGC54151/NF-kappa-B/NFKB enhancer in B-cells 1 p105/NFKB-p50?p105/p50 NOS1 HPS1, NOSINOS NM 000620 Nitric oxide synthase 1 (neuronal) NOS2 HEP-NOSINOS NOSNOS2A NM 000625 Nitric oxide synthase 2, inducible NOS3 ECNOS, eNOS NM OOO603 Nitric oxide synthase 3 (endothelial cell) PARP1 ADPRTADPRT1 PARPPARP NM OO1618 Poly (ADP-ribose) polymerase 1 /PPOL pADPRT-1 PARP2 ADPRT2 ADPRTL2 ADPRTL3 PARP NM 005484 Poly (ADP-ribose) polymerase 2 2.p.ADPRT-2 PARP3 ADPRT3, ADPRTL2 ADPRTL3 IRT1 PAD NM 005485 Poly (ADP-ribose) polymerase family, member 3 PRT3 PARP4 ADPRTL1 PARPLPHSPVAULT3 VPARP, NM OO6437 Poly (ADP-ribose) polymerase family, member 4 VWA5C/p193 PARP9 BAL/BAL1/DKFZp666BO810/DKFZp686M NM 031458 Poly (ADP-ribose) polymerase family, member 9 S238, FLJ26637, FLJ35310, FLJ41418 FLJ4 3593, MGC: 7868 PDGFRL PDGRLFPRLTS NM O06207 Platelet-derived growth factor receptor-like POT1 NM O15450 POT1 protection of telomeres 1 homolog (S. pombe) PPARG CIMT1 NR1C3 PPARG1 PPARG2. NM O15869 Peroxisome proliferator-activated receptor PPARgamma gamma PPARGC1 LEM6/PGC-1 (alpha)/PGC NM 013261 Peroxisome proliferator-activated receptor A. wiRGC1APGC1APPARGC1 gamma, coactivator 1 alpha PPC PPC SA 00103 Positive PCR Control PPC SA 00103 Positive PCR Control PTGS2 NM OOO963 Prostaglandin-endoperoxide synthase 2 (prostaglandin GH synthase and cyclooxygenase) RAP1A KREV-1 KREV1 RAP1ASMGP21 NM 002884 RAP1A, member of RAS oncogene family RTC RTC SA OO104 Reverse Transcription Control RTC RTC SA 00104 Reverse Transcription Control S100A7 PSOR1, S100A7c NM OO2963 S100 calcium binding protein A7 ERPINB HST12O1 PAIPAI-2fPAI2FPLANEH2 NM OO2575 Serpin peptidase inhibitor, clade B (ovalbumin), member 2 FLJ26SO4fSHCASHCA NM OO3O29 SHC (Src homology 2 domain containing) transforming protein 1 SIRT1 SIR2L1 NM 012238 Sirtuin (silent mating type information regulation 2 homolog) 1 (S. cerevisiae) SIRT2 SIR2, SIR2LSIR2L2 NM 012237 Sirtuin (silent mating type information regulation 2 homolog) 2 (S. cerevisiae) SIRT3 SIR2L3 NM 012239 Sirtuin (silent mating type information regulation 2 homolog) 3 (S. cerevisiae) SIRT4 MGC13OO46, MGC130047, MGCS7437 SIR2 NM 012240 Sirtuin (silent mating type information regulation L4 2 homolog) 4 (S. cerevisiae) S OD1 ALSALS1 IPOASOD homodimer NM 000454 Superoxide dismutase 1, soluble S OD2 IPO-BNMNSOD, MI-SOD NM OOO636 Superoxide dismutase 2, mitochondrial EP1 TLP1/TP1/TROVE1/VAULT2, p240 NM 007110 Telomerase-associated protein 1 I ERF2 TRBF2TRF2 NM OO5652 Telomeric repeat binding factor 2 US 2014/0079836A1 Mar. 20, 2014 51
-continued Array 2 Gene Symbol Alias Refseq # Official FullName TERT EST2, TCS1 TP2, TRT. hEST2 NM 198255 Telomerase reverse transcriptase TGFB1 CEDDPD1 TGFBTGFbeta NM OOO660 Transforming growth factor, beta 1 TIMM22 TEX4, TIM22 NM 013337 Translocase of inner mitochondrial membrane 22 homolog (yeast) TIMP3 HSMRK222 K222 K222TA2;SFD NM OOO362 TIMP metallopeptidase inhibitor 3 TINF2 TIN2fTIN2L NM 012461 TERF1 (TRF1)-interacting nuclear factor 2 TNF DIFTNF-alpha/TNFATNFSF2 NM 000594 Tumor necrosis factor (TNF Superfamily, member 2) TOMM40 C19Crf1 D19S1177EPER- NM OO6114 Translocase of outer mitochondrial membrane 40 EC1 PEREC1 TOM40 homolog (yeast) TP53 FLJ92943/LFS1/TRP53/p53 NM 000546 Tumor protein p53 TPP1 CLN2, GIG1 LPICMGC21297 NM OOO391 Tripeptidyl peptidase I UBE2S E2-EPFE2EPF/EPFS NM 0145O1 Ubiquitin-conjugating enzyme E2S VEGFA MGC7O609, MVCD1 VEGF, VEGF-AVPF NM OO3376 Vascular endothelial growth factor A
Kits tions. The design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art. 0408. Also provided herein are kits, e.g., kits for therapeu 0415. Alternatively, kits may be provided with the neces tic purposes or kits for modulating the lifespan of cells or sary reagents to carry out quantitative or semi-quantitative modulating apoptosis. A kit may comprise one or more acti Northern analysis of mRNA. Such kits include, for instance, Vating or inhibitory compounds described herein, e.g., in at least one target sequence-specific oligonucleotide for use premeasured doses. A kit may optionally comprise devices as a probe. This oligonucleotide may be labeled in any con for contacting cells with the compounds and instructions for ventional way, including with a selected radioactive isotope, use. Devices include syringes, and other devices for introduc enzyme substrate, co-factor, ligand, chemiluminescent or ing a compound into a subject or applying it to the skin of a fluorescent agent, hapten, or enzyme. subject. 0416. Also contemplated are kits containing an array, 0409 Kits are provided which contain the necessary where the feature(s) of the array correspond to genes identi reagents for determining the level of expression of one or fied herein as associated with lifespan, longevity, mitochon more genes (or the proteins encoded thereby) associated with drial health/maintenance/biogenesis, and/or telomere or longevity, mitochondrial biogenesis or health, and/or telom DNA health or maintenance. ere or DNA repair or maintenance. Provided herein are lists 0417 B. Kits for Detection of Protein or Peptide Expres and sets of genes the detection (and/or quantitation) of sion expression of which can be accomplished using kits. Instruc 0418 Kits for the detection of protein expression, include tions provided in the kits can include calibration curves, dia for instance at least one target protein specific binding agent grams, illustrations, or charts or the like to compare with the (e.g., a polyclonal or monoclonal antibody or antibody frag determined (e.g., experimentally measured) values or other ment) for each protein target to be detected, and may include results. at least one control. The protein specific binding agent and 0410 A. Kits for Detection of mRNA Expression control may be contained in separate containers. The kits may 0411 Kits can be used to detect mRNA expression levels. also include means for detecting target:agent complexes, for Such kits may include an appropriate amount of one or more instance the agent may be detectably labeled. If the detectable of the oligonucleotide primers for use in reverse transcription agent is not labeled, it may be detected by second antibodies amplification reactions, similarly to those provided above, or protein. A for example which may also be provided in some with art-obvious modifications for use with RNA. kits in one or more separate containers. Such techniques are well known. 0412. In some embodiments, kits for detection of mRNA 0419. Additional components in some kits include expression levels may also include the reagents necessary to instructions for carrying out the assay. Instructions will allow carry out RT-PCR in vitro amplification reactions, including, the tester to determine whether protein expression levels are for instance, RNA sample preparation reagents (including altered, for instance in comparison to a control sample. Reac e.g., an RNAse inhibitor), appropriate buffers (e.g., poly tion vessels and auxiliary reagents such as chromogens, buff merase buffer), salts (e.g., magnesium chloride), and deox ers, enzymes, etc. may also be included in the kits. yribonucleotides (dNTPs). Written instructions may also be 0420. By way of example only, an effective and conve included. nient immunoassay kit such as an enzyme-linked immun 0413 Kits in addition may include either labeled or unla osorbent assay can be constructed to test anti-target protein beled oligonucleotide probes for use in detection of the in antibody in human serum. Expression vectors can be con vitro amplified target sequences. The appropriate sequences structed using a human target cDNA to produce the recom for such a probe will be any sequence that falls between the binant human target protein in either bacteria or baculovirus. annealing sites of the two provided oligonucleotide primers, By affinity purification, unlimited amounts of pure recombi Such that the sequence the probe is complementary to is nant protein can be produced. amplified during the PCR reaction. 0421 Assay kits in some embodiments provide the recom 0414. It also may be advantageous to provided in the kit binant protein(s) as an antigen and enzyme-conjugated e.g., one or more control sequences for use in the RT-PCR reac goat anti-human IgG as a second antibody as well as enzy US 2014/0079836A1 Mar. 20, 2014 52 matic substrates. Such kits can be used to test if a subjects medium, but without the fetal bovine serum. During the 24 serum contains antibodies against a target lifespan extension hour experimental phase, cells were maintained in the same associated protein (or a collection of them). medium, but with only 1% fetal bovine serum. All cultures 0422 The present description is further illustrated by the were incubated at 37°C. with 5% CO, in a humidified cham following examples, which should not be construed as limit ber. ing in any way. The contents of all cited references (including 0428 Seeding of cells: On Day 1, each cell culture was literature references, issued patents, and published patent seeded into 6 well cluster dishes at ~1.5x10 cells in 4 mls applications as cited throughout this application) are hereby medium per well. Three wells were seeded per test condition expressly incorporated by reference. Any publicly available or control. sequences referenced herein are incorporated by reference 0429 Experimental phase: On Day 3, wells were washed from the public database as they were available on Dec. 1, 1x with 2 mls medium, fed back 2 mls medium without fetal 2008 (the date offiling of the first priority application). bovine serum and preincubated for 30-60 minutes before challenge with test or control conditions. After the preincu EXAMPLES bation, medium was aspirated from the wells and 2.5 mls of test or control condition in medium with 1% fetal bovine 0423. The effects of oxidative stress, environmental dam serum was pipetted into the appropriate wells. age and premature aging are almost as diverse as their caus ative agents (FIGS. 4, 5, and 6). The mechanistic pathways 0430. The green tea polyphenols used were Premier Green most commonly affecting premature aging and anti-Longev Tea Extract Lot #10783, obtained from Pharma Cosmetix Research, LLC (Richmond, Va.). The Green Tea extract was ity effects involve the AP1 matrix regulation pathway, mito measured into a stock solution of the above described Mini chondrial DNA damage/deletions, telomere shortening, mal Essential Media+1%. Fetal Bovine Serum in a w/v ratio inflammation, and cancer cell creation. These pathways are and then serially diluted into the testing concentrations with affected through environmental damage in the form(s) of UV MEM+1% Fetal Bovine Serum. radiation (all types and full spectrum), thermal injury, chronic 0431. Three conditions were tested: (1) cells were exposed or acute disease states/conditions, Smoking, chemicals, to the test condition 4 hours before UVB exposure, but not dietary habits, and oxidative stress/free radical formation. All during or after UVB exposure (2) cells were exposed to the of these effects can modulate the cells gene responses, mito test condition after UVB exposure, but not before or during chondrial numbers and/or efficiency, and elimination of ROS (3) cells were exposed to the test condition 4 hours before, from the cellular environment in a negative fashion, much like then during and after UVB exposure. UVB exposed and unex antioxidants and the other described compounds can modu posed cells without test condition were used as controls. The late the same responses in the direction of increased lifespan. experimental phase ended at 24 hours post UVB exposure. 0424 The following experimental examples illustrate the Cells from each test and control parameter were then use of UV radiation to affect the negative or lifespan short trypsinized, collected by centrifugation, washed 3 times in ening affects previously described. UV radiation was selected PBS, and a cell count made using a hemacytometer. The final as an injury producing agent for several reasons: 1) it is a pellet was stored at -80°C. until processed further. classic model used in the literature to injure cells in culture, 2) its dosage is easily controlled and directed, and 3) it is one of 0432 UVB exposure: Cells exposed to UVB received 200 the most ubiquitous sources of environmental injury that cells mJ/cm UVB using ThermoOriel solar simulator model and tissues will face on a daily basis in real world settings. SP66923-3056. Cells were exposed from the bottom of the The use of UV and H2O is by no means meant to limit the culture dish. The UVB dose delivered to the cells was application or interpretation of these results, and are meant to adjusted for interference from the plastic in the culture dish. serve as examples of the type of pro-longevity modulation 0433 Preparation of cell extract: Extract from the cell that can be achieved through proper application of the pellets stored at -80C post experimental phase was prepared described compounds. Any method of the previously recog for PCR according to the instructions in the Allied Biotech, nized environmental agents (oxidative stress, thermal injury, Inc., Quantitative Telomerase Detection Kit. Briefly, pellets Smoking, hypoxia, and so forth) could have been and may be were thawed and immediately resuspended in 200ul 1x Lysis used in the future expansion of these experimental examples Buffer per 10 to 10 cells. The suspension was incubated that follow. on ice for 30 minutes, then microcentrifuged at 12,000xg for 30 minutes at 4°C. The supernatant was aliquoted and stored Example 1 at 80° C. 0434. Detection of Telomerase: The extract from the cells 0425 This example illustrates protection of telomere allows for determination of telomerase activity by coupling length maintenance and/or lifespan extension through appli the extracts ability to form telomeric repeats onto an oligo cation of modulating compounds (exemplified by green tea nucleotide substrate and the resultant extended product are polyphenols). amplified using Polymerase Chain Reaction. These products 0426 Cell cultures: Two human skin fibroblast cell cul are then visualized with SYBR green a fluorescent detection tures were obtained through the Coriell Cell Repository from agent that emits green fluorescence when bound to the double the National Institute on Aging Cell Repository. The cultures stranded DNA product. Each 25ul PCR assay included 12.5 were established from biopsies of a Caucasian female, at 36 ul of 2xOTD Premix, 1.0 ul of Cell Extract, and 11.5 ul of years and again at 50 years of age (AG7308 and AG14271 Molecular GradeTM HO (distilled, deionized, sterile-filtered respectively). water, which is ultrapure and DNase, RNase and protease 0427 Culture media: Cells were grown in Minimal Essen free). tial Medium supplemented with 10% fetal bovine serum, 2 0435 The samples were run in a BioRad iCycler for 20 mM L-glutamine, 2 mM Glutamax I, and 1x MEM non minutes at 25° C. to complete the telomerase reaction. The essential amino acids solution. Cells were washed in the same PCR initial activation step followed immediately and was of US 2014/0079836A1 Mar. 20, 2014
10 minute duration at 60°C. The iCycler then ran the dena DATA TABLE 1-continued turing, annealing, and extension (30 seconds at 95, 60 and 72 C. respectively) series for 40 cycles. The SYBR green detec (Level of telomerase -- ability to form telomeric repeats onto tion occurred during the extension phase. an oligonucleotide Substrate: treated with Green Tea Extracts p fold 0436 Telomerase Detection Results: The results for N=3 value change were downloaded from the iCycler into a modified array 36 yo UVB + green tea ContVs. 50 yo green tea O4537 4.48 analysis program and the results examined for statistical sig Before nificance. The raw analysis is provided in DATA TABLE 1. 50 yo UVB + green tea ContVs. 36 yo green tea O.1969 1.30 Before DATA TABLE 1. 36 yo UVB + green tea ContVs. 50 yo green tea. After O.O995 - 1.44 50 yo UVB + green tea ContVs. 36 yo green tea. After OO678 -1.48 (Level of telomerase -- ability to form telomeric repeats onto 36 yo green tea. Before Vs. 50 yo green tea. After O.4730 4.17 an oligonucleotide substrate; treated with Green Tea Extracts 50 yo green tea. Before Vs. 36 yo green tea. After O3131 -8.OO p fold value change Example 2 Compared to UnTx Control (36 yo) 0437. This example illustrates protection of telomere 36 yo UVB Alone O.1515 - 1.43 length maintenance and/or lifespan extension through appli 36 yo UVB + gren tea Continuous Exposure O.1770 -1.39 36 yo UVB + green tea. After O.1SSO 29 cation of modulating compounds (idebenone and coffee 36 yo UVB + green tea. Before O.O634 -1.49 cherry). 36 yo Untreated 1.OOOO -1.OO 0438 Cell cultures: Two human skin fibroblast cell cul 50 yo UVB Alone O4305 -1.78 50 yo UVB + green tea Continuous Exposure O.S156 - 1.15 tures were obtained through the Coriell Cell Repository from 50 yo UVB + green tea. After O.8.192 .04 the National Institute on Aging Cell Repository. The cultures 50 yo UVB + green tea. Before O.3690 -6.22 were established from biopsies of a Caucasian female, at 36 50 yo Untreated O.2718 -130 years and again at 50 years of age, AG7308 and AG14271 Compared to UnTx Control (50 yo) respectively. 36 yo UVB Alone O.6966 -110 0439 Culture media: Cells were grown in Minimal Essen 36 yo UVB + green tea Continuous Exposure O.7937 - 1.06 tial Medium supplemented with 10% fetal bovine serum, 2 36 yo UVB + green tea. After O.O411 68 mM L-glutamine, 2 mM Glutamax I, and 1xMEM non-es 36 yo UVB + green tea. Before OSO86 -1.14 sential amino acids solution. Cells were washed in the same 36 yo Untreated O.2718 30 Compared too UnTX Control (50 yo) medium, but without the fetal bovine serum. During the 24 hour experimental phase, cells were maintained in the same 50 yo UVB Alone O.6642 -1.37 50 yo UVB + green tea Continuous Exposure O.S909 .14 medium, but with only 1% fetal bovine serum. All cultures 50 yo UVB + green tea. After O.1686 35 were incubated at 37°C. with 5% CO, in a humidified cham 50 yo UVB + green tea. Before O.4367 -4.77 ber 50 yo Untreated 1.OOOO -1.OO 0440 Seeding of cells: On Day 1, each cell culture was Compared to UVB Tx Control (36 yo) seeded into 6 well cluster dishes at ~1.5x10 cells in 4 mls 36 yo UVB Alone 1.OOOO -1.OO medium per well. Three wells were seeded per test condition 36 yo UVB + green tea Continuous Exposure O.8894 O3 or control. 36 yo UVB + green tea. After O.O220 84 0441 Experimental phase: On Day 3, wells were washed 36 yo UVB + green tea. Before O.8372 -1.04 36 yo Untreated O.1515 43 1x with 2 mls medium, fed back 2 mls medium without fetal 50 yo UVB Alone O.7604 -1.24 bovine serum and preincubated for 30-60 minutes before 50 yo UVB + green tea Continuous Exposure O.3566 25 challenge with test or control conditions. After the preincu 50 yo UVB + green tea. After O.O851 49 bation, medium was aspirated from the wells and 2.5 mls of 50 yo UVB + green tea. Before 0.4628 -4.34 test or control condition in medium with 1% fetal bovine 50 yo Untreated O.6966 1O Compared to UVB Tx Control (50 yo) serum was pipetted into the appropriate wells. 0442. The coffee cherry Beauty extract (COFFEE 36 yo UVB Alone O.7604 .24 36 yo UVB + green tea Continuous Exposure O.7262 28 BERRYR); VDF FutureCeuticals, Inc., Momence, Ill.) was 36 yo UVB + green tea. After 0.2705 2.29 placed into a stock solution of 1% coffee cherry (w/v) in the 36 yo UVB + green tea. Before 0.7985 19 previously described Minimal Essential Medium+1% Fetal 36 yo Untreated O.43OS .78 Bovine Serum and DMSO. This stock solution was diluted in 50 yo UVB Alone 1.OOOO -1.OO 50 yo UVB + green tea Continuous Exposure O.S437 55 a 1:10 ratio with the MEM+1% FBS previously described 50 yo UVB + green tea. After O.3986 .85 until the testing concentrations were reached. At the testing 50 yo UVB + green tea. Before O.5495 -3.49 concentrations, the DMSO content was less than 0.01%– 50 yo Untreated O.6642 37 well within safe limits for tissue culture. Idebenone dilutions 36 yo Vs. 50 yo Untreated O.2718 30 36 yo green tea. Before Vs. green tea. After O.OO37 - 1.92 were prepared in a similar fashion with the stock 1% solution 50 yo green tea. Before Vs. green tea. After O.3597 -6.45 being dissolved in sterile alcohol. The 1% stock solution was 36 yo UVB + green tea ContVs. 36 yo green tea. After O.O243 -1.79 also diluted with MEM+1% FBS until the testing concentra 50 yo UVB + green tea ContVs. 50 yo green tea. After O.3443 -1.19 tions were reached. 36 yo UVB + green tea ContVs. 36 yo green tea O.7042 O7 Before 0443) Three conditions were tested: (1) cells were exposed 50 yo UVB + green tea ContVs. 50 yo green tea O.4031 5.41 to the test condition 4 hours before UVB exposure, but not Before during or after UVB exposure (2) cells were exposed to the test condition after UVB exposure, but not before or during US 2014/0079836A1 Mar. 20, 2014 54
(3) cells were exposed to the test condition 4 hours before, DATA TABLE 2-continued then during and after UVB exposure. UVB exposed and unex posed cells without test condition were used as controls. The (Level of telomerase -- ability to form telomeric experimental phase ended at 24 hours post UVB exposure. repeats onto an oligonucleotide Substrate; treated Cells from each test and control parameter were then with idebenone & Coffee cherry extracts) trypsinized, collected by centrifugation, washed 3 times in 50 yo UVAVs. 50 yo UVB NA -111.43 PBS, and a cell count made using a hemacytometer. The final 36 yo Untx Vs. 36 yo UVA O6440 -1.20 pellet was stored at -80°C. until processed further. 36 yo UVAVs. 50 yo UVA O.OS32 1.68 0444 UVB exposure: Cells exposed to UVB received 200 Conclusions (with Good p Values/Statistical Significance): mJ/cm UVB using Thermo Oriel solar simulator model 0449 36 year old cells (that is, cells from a 36 year old SP66923-3056. Cells were exposed from the bottom of the person) given green tea have a higher level of telomerase culture dish. The UVB dose delivered to the cells was activity than 50 year old cells given the same dose ofgreen tea adjusted for interference from the plastic in the culture dish. (+1.33 fold or 33% increase in telomerase activity) 0445 Preparation of cell extract: Extract from the cell 0450 36 year old cells given green tea before being pellets stored at -80C post experimental phase was prepared stressed with 1 MED of UVB have a +2.8 fold (roughly for PCR according to the instructions in the Allied Biotech, 18.0%) increase in telomerase activity when compared to the Inc., Quantitative Telomerase Detection Kit. Briefly, pellets same cells given Idebenone. were thawed and immediately resuspended in 200ul 1x Lysis 0451 50 year old cells given green tea before being Buffer per 10 to 10 cells. The suspension was incubated stressed with 1 MED of UVB have a +3.36 fold (roughly on ice for 30 minutes, then microcentrifuged at 12,000xg for 23.6%) increase in telomerase activity when compared to the 30 minutes at 4°C. The supernatant was aliquoted and stored same cells given Idebenone. at 80° C. 0452 36 year old cells given Idebenone have roughly 53% 0446 Detection of Telomerase: The extract from the cells less activity than untreated cells. allows for determination of telomerase activity by coupling 0453 36 year old cells given Idebenone and stressed with the extracts ability to form telomeric repeats onto an oligo 1 MED UVB have -2.4 fold less activity than untreated cells, nucleotide substrate and the resultant extended product are and -1.7 fold less activity than UVB stressed cells alone. amplified using Polymerase Chain Reaction. These products 0454 50 year old cells given Idebenone and stressed with are then visualized with SYBR green a fluorescent detection 1 MED of UVB have roughly -2.61 fold decrease in telom agent that emits green fluorescence when bound to the double erase activity when compared to untreated controls, and stranded DNA product. Each 25ul PCR assay included 12.5 -2.65 fold less when compared to UVB treated age matched ul of 2xOTD Premix, 1.0 ul of Cell Extract, and 11.5 ul of controls. Molecular Grade H2O. 0455 There is a slight increase intelomerase activity in 36 y.o. cells treated with green tea alone and UVB stressed 0447 The samples were run in a BioRad iCycler for 20 green tea when compared to UVB treated cells. (+1.55 and minutes at 25° C. to complete the telomerase reaction. The +1.65 respectively) PCR initial activation step followed immediately and was of 0456. When UVB unstressed 36 y.o. cells are given green 10 minute duration at 60°C. The iCycler then ran the dena tea there is an increase of telomerase activity (+1.69 fold or turing, annealing, and extension (30 seconds at 95, 60 and 72 69%) compared to age matched unstressed cells receiving C. respectively) series for 40 cycles. The SYBR green detec Idebenone. tion occurred during the extension phase. 0457. A final trend with lower p value and statistical sig 0448 Telomerase Detection Results: The results for N=3 nificance shows that telomerase activity is lower in 36 year were downloaded from the iCycler into a modified array old untreated cells than in 50 year old untreated cells. analysis program and the results examined for statistical sig nificance. The raw analysis is provided in DATA TABLE 2. Example 3 0458. This example describes examination of the gene DATA TABLE 2 expression profile related to aging, lifespan and telomerase length maintenance of cells contacted with coffee cherry (Level of telomerase -- ability to form telomeric extract and idebenone demonstrate lifespan and/or telomere repeats onto an oligonucleotide Substrate; treated length extension characteristics. with idebenone & Coffee cherry extracts) 0459 Cell cultures: Two human skin fibroblast cell cul Compared to UnTx Control tures were obtained through the Coriell Cell Repository from the National Institute on Aging Cell Repository. The cultures 36 yo UVB Alone O.OOO1 97.01 36 yo UVB + idebenone 4 hr O.OOO1 90.51 were established from biopsies of a Caucasian female, at 36 36 yo UVB + idebenone Continuous O.OOO1 97.01 years and again at 50 years of age, AG7308 and AG14271 36 yo UVB + coffee cherry 4 hr O.OOO1 78.79 respectively. 36 yo UVB + coffee cherry Continuous O.OOO2 101.59 0460 Culture media: Cells were grown in Minimal Essen Compared to UVB Tx Control tial Medium supplemented with 10% fetal bovine serum, 2 36 yo UVB Alone 1.OOOO -1.00 mM L-glutamine, 2 mM Glutamax I, and 1xMEM non-es 36 yo UVB + idebenone 4 hr 0.7625 -1.07 sential amino acids solution. Cells were washed in the same 36 yo UVB + idebenone Continuous 1.OOOO -1.00 36 yo UVB + coffee cherry 4 hr O.3712 -1.23 medium, but without the fetal bovine serum. During the 24 36 yo UVB + coffee cherry Continuous O.8971 1.OS hour experimental phase, cells were maintained in the same 36 yo idebenone Vs. 50 yo idebenone 4 hr O.2102 1.46 medium, but with only 1% fetal bovine serum. All cultures 36 yo UVAVs. 36 yo UVB O.S631 -1.19 were incubated at 37°C. with 5% CO in a humidified cham ber. US 2014/0079836A1 Mar. 20, 2014
0461 Seeding of cells: On Day 1, each cell culture was were established from biopsies of a Caucasian female, at 36 seeded into 6 well cluster dishes at ~1.5x10 cells in 4 mls years and again at 50 years of age, AG7308 and AG14271 medium per well. Three wells were seeded per test condition respectively. or control. 0470 Culture media: Cells were grown in Minimal Essen 0462 Experimental phase: On Day 3, wells were washed tial Medium supplemented with 10% fetal bovine serum, 2 1x with 2 mls medium, fed back 2 mls medium without fetal mM L-glutamine, 2 mM Glutamax I, and 1xMEM non-es bovine serum and preincubated for 30-60 minutes before sential amino acids solution. Cells were washed in the same challenge with test or control conditions. After the preincu medium, but without the fetal bovine serum. During the 24 bation, medium was aspirated from the wells and 2.5 mls of hour experimental phase, cells were maintained in the same test or control condition in medium with 1% fetal bovine medium, but with only 1% fetal bovine serum. All cultures serum was pipetted into the appropriate wells. were incubated at 37C with 5% CO in a humidified chamber. 0463 Nine conditions were tested: (1) cells were exposed 0471) Seeding of cells: On Day 1, each cell culture was to one of the test conditions (1 uM Idebenone, 0.001% coffee seeded into 6 well cluster dishes at ~1.5x10 cells in 4 mls cherry, or 0.001% green tea extract; sourced and prepared as medium per well. Three wells were seeded per test condition described above) 4 hours before UV exposure, but not during or control. or after UV exposure (2) cells were exposed to one of the test 0472 Experimental phase: On Day 3, wells were washed conditions 4 hours before, then during and after UV exposure. 1x with 2 mls medium, fed back 2 mls medium without fetal (3) UVA1. UVB exposed and unexposed cells without test bovine serum and preincubated for 30-60 minutes before condition were used as controls. The experimental phase challenge with test or control conditions. After the preincu ended at 24 hours post UV exposure. Cells from each testand bation, medium was aspirated from the wells and 2.5 mls of control parameter were then prepped for RNA isolation as test or control condition in medium with 1% fetal bovine described below. serum was pipetted into the appropriate wells. 0464 UVB/UVA1 exposure: Cells exposed to UV 0473 Nine conditions were tested: (1) cells were exposed received 200 ml/cm UVB/1 MED UVA1 using Thermo to one of the test conditions (1 uM Idebenone, 0.001% coffee Oriel Solar simulator model SP66923-3056. Cells were cherry, or 0.001% Green tea)4 hours before UV exposure, but exposed from the bottom of the culture dish. The UV dose not during or after UV exposure (2) cells were exposed to one delivered to the cells was adjusted for interference from the of the test conditions 4 hours before, then during and after UV plastic in the culture dish. exposure. (3) UVA1. UVB exposed and unexposed cells 0465 Preparation of RNA: The RNA was isolated using without test condition were used as controls. The experimen the Qiagen RNEasy Plus Mini Kit according to the manufac tal phase ended at 24 hours post UV exposure. Cells from turer's protocol which can be found on the World Wilde Web each test and control parameter were then prepped for RNA at qiagen.com/literature/ isolation as described below. Defaultaspx?Term=&Language=EN&LiteratureType=4& 0474 UVB/UVA1 exposure: Cells exposed to UV ProductCategory=10162. The RNA was quantified and received 200 m.J/cm UVB/1 MED UVA1 using Thermo examined for purity using the 260/280 nm read method in a L Oriel Solar simulator model SP66923-3056. Cells were Quant spectrophotometer before use in the array. exposed from the bottom of the culture dish. The UV dose 0466 Creation and Performance of a Custom Microarray delivered to the cells was adjusted for interference from the ('Array 1): Through the Custom Array design service of plastic in the culture dish. Superarray Bioscience Corporation, a 96 well RT-PCR 0475 Preparation of RNA: The RNA was isolated using microarray (Array 1) was developed to illustrate genetic the Qiagen RNEasy Plus Mini Kit according to the manufac responses in cells treated as above for specific genes involved turer's protocol which can be found on the World WideWeb in longevity as well as the relevant quality controls. The array at qiagen.com/literature/Default. was performed in compliance with manufacturer's guidelines aspx?Term=&Language=EN&LiteratureType=4&Product and by recommended manufacturer's protocol as can be Category=10162. found on the World Wilde Web at superarray.com/Manual/ perarrayplate.pdf 0476. The RNA was quantified and examined for purity 0467 Custom Microarray Results: The results from the using the 260/280 nm read method in a LL Quant spectropho microarrays were determined using the delta CT method tometer before use in the array. which uses comparisons of control genes and threshold 0477 Creation and Performance of the Custom Microar cycles of genes of interest to generate relative expression ray: Through the Custom Array design service of Superarray values. The full set of experimental conditions was cross Bioscience Corporation, a 96 well RT-PCR Microarray was referenced and the data is provided herewith in DATA TABLE developed to illustrate genetic responses in cells treated as 3. above for specific genes involved in longevity as well as the relevant quality controls. The array was performed in com Example 4 pliance with manufacturer's guidelines and by recommended manufacturer's protocol as can be found on the World Wide 0468. This example describes examination of the gene Web at superarray.com/Manual/perarrayplate.pdf. expression profile related to aging, lifespan and telomerase 0478 Custom Microarray Results: The results from the length maintenance of cells contacted with green tea polyphe microarrays were determined using the delta CT method nols and idebenone demonstrate lifespan and/or telomere which uses comparisons of control genes and threshold length extension characteristics. cycles of genes of interest to generate relative expression 0469 Cell cultures: Two human skin fibroblast cell cul values. The full set of experimental conditions was cross tures were obtained through the Coriell Cell Repository from referenced and the data is provided herewith in DATA TABLE the National Institute on Aging Cell Repository. The cultures 4. US 2014/0079836A1 Mar. 20, 2014 56
Example 5 manufacturer's protocol as can be found on the World Wide Web at superarray.com/Manual/perarrayplate.pdf. 0479. This example describes examination of the gene 0488 Custom Microarray Results: The results from the expression profile related to aging, lifespan and telomerase microarrays were determined using the delta CT method length maintenance of cells contacted with green tea polyphe which uses comparisons of control genes and threshold nols and coffee cherry extract demonstrate lifespan and/or cycles of genes of interest to generate relative expression telomere length extension characteristics. values. The full set of experimental conditions was cross 0480 Cell cultures: Two human skin fibroblast cell cul referenced and the data is provided herewith in DATA TABLE tures were obtained through the Coriell Cell Repository from 5. the National Institute on Aging Cell Repository. The cultures were established from biopsies of a Caucasian female, at 36 Example 6 years and again at 50 years of age, AG7308 and AG14271 respectively. 0489. This example describes application of UVA/UVB 0481 Culture media: Cells were grown in Minimal Essen injury to cells, which demonstrates a decrease in lifespan or tial Medium supplemented with 10% fetal bovine serum, 2 telomere length maintenance gene expression profiles. mM L-glutamine, 2 mM Glutamax I, and 1xMEM non-es 0490 Cell cultures: Two human skin fibroblast cell cul sential amino acids solution. Cells were washed in the same tures were obtained through the Coriell Cell Repository from medium, but without the fetal bovine serum. During the 24 the National Institute on Aging Cell Repository. The cultures hour experimental phase, cells were maintained in the same were established from biopsies of a Caucasian female, at 36 medium, but with only 1% fetal bovine serum. All cultures years and again at 50 years of age, AG7308 and AG14271 were incubated at 37°C. with 5% CO in a humidified cham respectively. ber. 0491 Culture media: Cells were grown in Minimal Essen tial Medium supplemented with 10% fetal bovine serum, 2 0482 Seeding of cells: On Day 1, each cell culture was mM L-glutamine, 2 mM Glutamax I, and 1xMEM non-es seeded into 6 well cluster dishes at ~1.5x10 cells in 4 mls sential amino acids solution. Cells were washed in the same medium per well. Three wells were seeded per test condition medium, but without the fetal bovine serum. During the 24 or control. hour experimental phase, cells were maintained in the same 0483 Experimental phase: On Day 3, wells were washed medium, but with only 1% fetal bovine serum. All cultures 1x with 2 mls medium, fed back 2 mls medium without fetal were incubated at 37°C. with 5% CO in a humidified cham bovine serum and preincubated for 30-60 minutes before ber. challenge with test or control conditions. After the preincu 0492 Seeding of cells: On Day 1, each cell culture was bation, medium was aspirated from the wells and 2.5 mls of seeded into 6 well cluster dishes at ~1.5x10 cells in 4 mls test or control condition in medium with 1% fetal bovine medium per well. Three wells were seeded per test condition serum was pipetted into the appropriate wells. or control. 0484 Nine conditions were tested: (1) cells were exposed 0493 Experimental phase: On Day 3, wells were washed to one of the test conditions (1 uM Idebenone, 0.001% coffee 1x with 2 mls medium, fed back 2 mls medium without fetal cherry, or 0.001% Green tea)4 hours before UV exposure, but bovine serum and preincubated for 30-60 minutes before not during or after UV exposure (2) cells were exposed to one challenge with test or control conditions. After the preincu of the test conditions 4 hours before, then during and after UV bation, medium was aspirated from the wells and 2.5 mls of exposure. (3) UVA1. UVB exposed and unexposed cells test or control condition in medium with 1% fetal bovine without test condition were used as controls. The experimen serum was pipetted into the appropriate wells. tal phase ended at 24 hours post UV exposure. Cells from 0494 Nine conditions were tested: (1) cells were exposed each test and control parameter were then prepped for RNA to one of the test conditions (1 uM Idebenone, 0.001% coffee isolation as described below. cherry, or 0.001% Green tea)4 hours before UV exposure, but 0485 UVB/UVA1 exposure: Cells exposed to UV not during or after UV exposure (2) cells were exposed to one received 200 ml/cm UVB/1 MED UVA1 using Thermo of the test conditions 4 hours before, then during and after UV Oriel Solar simulator model SP66923-3056. Cells were exposure. (3) UVA1. UVB exposed and unexposed cells exposed from the bottom of the culture dish. The UV dose without test condition were used as controls. The experimen delivered to the cells was adjusted for interference from the tal phase ended at 24 hours post UV exposure. Cells from plastic in the culture dish. each test and control parameter were then prepped for RNA 0486 Preparation of RNA: The RNA was isolated using isolation as described below. the Qiagen RNEasy Plus Mini Kit according to the manufac 0495 UVB/UVA1 exposure: Cells exposed to UV turer's protocol which can be found on the World WideWeb received 200 m.J/cm UVB/1 MED UVA1 using Thermo at qiagen.com/literature/Default. Oriel Solar simulator model SP66923-3056. Cells were aspx?Term=&Language=EN&LiteratureType=4&Product exposed from the bottom of the culture dish. The UV dose Category=10162. The RNA was quantified and examined for delivered to the cells was adjusted for interference from the purity using the 260/280 nm read method in a LQuant spec plastic in the culture dish. trophotometer before use in the array. 0496 Preparation of RNA: The RNA was isolated using 0487 Creation and Performance of the Custom Microar the Qiagen RNEasy Plus Mini Kit according to the manufac ray: Through the Custom Array design service of Superarray turer's protocol which can be found on the World WideWeb Bioscience Corporation, a 96 well RT-PCR Microarray was at qiagen.com/literature/Default. developed to illustrate genetic responses in cells treated as aspx?Term=&Language=EN&LiteratureType=4&Product above for specific genes involved in longevity as well as the Category=10162. The RNA was quantified and examined for relevant quality controls. The array was performed in com purity using the 260/280 nm read method in a LQuant spec pliance with manufacturer's guidelines and by recommended trophotometer before use in the array. US 2014/0079836A1 Mar. 20, 2014 57
0497 Creation and Performance of the Custom Microar 0502 PARP1, a genesignificantly downregulated in UVB ray: Through the Custom Array design service of Superarray exposed 36 year old cells and upregulated in cells exposed to Bioscience Corporation, a 96 well RT-PCR Microarray was antioxidant compounds, may play an intriguing role and be developed to illustrate genetic responses in cells treated as worth further study. The gene itselfis involved in DNA repair, above for specific genes involved in longevity as well as the apoptosis and maintenance of optimal niacin status in the relevant quality controls. The array was performed in com skin. pliance with manufacturer's guidelines and by recommended 0503 Green tea continuous incubation+UVB treated 36 manufacturer's protocol as can be found on the World Wide year old cells demonstrate a downregulation of TNF of -18. Web at superarray.com/Manual/perarrayplate.pdf. 44. TNF is a pro-inflammatory cytokine that plays a patho 0498 Custom Microarray Results: The results from the genic role in age related diseases. microarrays were determined using the delta CT method (0504 50 year old cells treated with UVA1 alone show a which uses comparisons of control genes and threshold significant downregulation in CYP19A1 of -10.5 fold. cycles of genes of interest to generate relative expression CYP19A1 is a variant of cytochrome P-450 and is involved in values. The full set of experimental conditions was cross Xenobiotic metabolism and detoxification. referenced and the data is provided herewith in DATA TABLE 6. Example 7 Summary of Results (Examples 3-6): An analysis of the results indicates: 0505. This example illustrates that application of modu 0499. The condition with the most statistically significant lating compounds (green tea polyphenols, coffee cherry genes affected is the 50 year old cells that were incubated in extract and idebenone) to cells demonstrates ability to alter coffee cherry continuously and exposed to UVB, with seven the gene expression of key components of the telomere length genes altered. The next highest significant gene total (with six maintenance complex. genes altered) is a tie with 36 year old cells given Green tea 0506 Cell cultures: A human skin fibroblast cell culture before UVB exposure both continuously as well as 4 hr was obtained through the Coriell Cell Repository from the beforehand, and the 4 hr incubation of 36 year old cells and National Institute on Aging Cell Repository. The culture, Idebenone. Both cell lines, when exposed only to the radia AG07999, was established from a biopsy of a 32 year old tion types tested, showed a significant and minimum of 4 fold Caucasian female. reduction in the TERT gene. 0507 Culture media: Cells were grown in Minimal Essen (0500. In the 36 year old cell line cells exposed to UVB tial Medium supplemented with 10% fetal bovine serum, 2 demonstrated a significant reduction in PARP1, NADSYN1, mM L-glutamine, 2 mM Glutamax I. During the 24 hour IFI44, TERT and NFKB1. In the same 36 year old cells experimental phase, cells were maintained in the same treated with the anti-oxidant compounds, those same genes medium, but with only 1% fetal bovine serum. All cultures demonstrate either no significant up or down regulation or are were incubated at 37°C. with 5% CO in a humidified cham significantly up regulated. This altered gene expression pat ber. tern is encouraging that the anti-oxidant compounds provide (0508) Seeding of cells: On Day 1, 1 ml of 5.0–6.5x10 some ability to “reverse or improve' the gene expression cells/ml was seeded into each of four 75 cm flasks containing triggered by the UVB exposure. 20 ml of culture medium.
36 year old Green tea Green tea. Idebenone Idebenone Coffee cherry Coffee cherry cell line UVB (4hr) (Continuous) (4hr) (Continuous) (4 hr inc.) (Cont. inc.) PARP1 -5.14 6.73 4.42 4.27 NA NA 4.92 NADSYN1 -3.06 3.61 3.35 2.21 NA 2.61 2.93 IFI44 -3.39 3.36 2.72 2.63 2.98 NA 3.86 TERT -4.63 NA 1.23 8.54 4.36 8.2O NA NFKB1 -2.32 3.36 NA 3.24 NA NA 2.73
0501. In the 50 year old cell line, the above statement also 05.09 Experimental phase: On Day 4, the medium was holds true. Interestingly, the only gene that showed statistical removed by aspiration and replaced with the test condition in significance in UVB exposed cells was TERT. It is downregu 20 ml culture medium but with only 1% fetal bovine serum. lated and to a larger degree than in the 36 year old cells. Again Test conditions (prepared and sourced as above) were 1) 1 uM treatment with anti-oxidants is either not significant or up idebenone 2) 0.001% coffee cherry 3) 0.001% green tea and regulates the TERT gene. 4) vehicle control. All four conditions had a final vehicle
Green tea Green tea 50 year old (4hr (Continuous Idebenone Idebenone Coffee cherry Coffee cherry cell line UVB incubation) inc.) (4 hr inc.) (Cont. inc.) (4 hr inc.) (Cont. inc.) TERT -7.01 NA NA NA 8.50 10.14 8.97 US 2014/0079836A1 Mar. 20, 2014
concentration of 0.01% DMSO. On Day 5 after 24 hours of medium, but with only 1% fetal bovine serum. All cultures exposure to test conditions, the cells were isolated and the were incubated at 37°C. with 5% CO in a humidified cham RNA isolated using the RT qPCR-grade RNA isolation kit ber. (Superarray Bioscience Corporation). 0520 Seeding of cells: On Day 1, 1 ml of 5.0–6.5x10 0510 Preparation of RNA: The RNA was isolated using cells/ml was seeded into each of four 75 cm flasks containing the RTc PCR-grade RNA isolation kit according to the 20 ml of culture medium. manufacturer's protocol which can be found on the World 0521 Experimental phase: On Day 4, the medium was Wide Web at superarray.com/Manual/qpcgrade.pdf. removed by aspiration and replaced with the test condition in 0511. The RNA was quantified and examined for purity 20 ml culture medium but with only 1% fetal bovine serum. using the 260/280 nm read method in a LOuant spectropho Test conditions were 1) 1 uM idebenone and 2) vehicle con tometer before use in the array. trol. All four conditions had a final vehicle concentration of 0512 Performance of the qPCRPrimer Assays: Assays for 0.01% DMSO. On Day 5 after 24 hours of exposure to test six exemplary genes of interest (TERT, TPP1, TERF1, conditions, the cells were isolated and the RNA isolated using TERF2, TINF2, POT1) as well as 18s ribosomal RNA as the the RT q PCR-grade RNA isolation kit (Superarray Bio housekeeping gene were purchased. The assay was per Science Corporation). 0522 Preparation of RNA: The RNA was isolated using formed in compliance with manufacturer's guidelines and by the Qiagen RNEasy Plus Mini Kit according to the manufac recommended manufacturer's protocolas can be found on the turer's protocol which can be found on the World WideWeb World Wide Web at superarray.com/Manual/realtimePCR. at qiagen.com/literature/Default. pdf. The assays were run for N=3 and analyzed using the aspx?Term=&Language=EN&LiteratureType=4&Product BioRad GeneX Software package. Any values out of range of Category=10162. The RNA was quantified and examined for the threshold cycles or inconsistent within the assay were purity using the 260/280 nm read method in a LQuant spec discarded from analysis. trophotometer before use in the array. 0513 qPCR Primer Assay results: The results from assays 0523 Analysis of Agilent Whole Human Genome Arrays: were determined using the delta CT method, which uses RNA taken from both samples was sent to Cogenics, Inc. comparisons of control genes and threshold cycles of genes of Morrisville, N.C. Cogenics, Inc. is a leading, state-of-the-art, interest to generate relative expression values (this process is microarray service provider that facilitates and accelerates handled by the GeneX package). Results (average of three transcriptome profiling and gene discovery processes for experiments) are shown in FIG. 7. The result with the lowest industrial and academic researchers. Cogenics procedure for percent error is the TERF2 gene. processing samples is as follows: RNA samples are received 0514. As illustrated in FIG.7, coffeecherry and idebenone and analyzed by Cogenics, Inc. using rigorous standardized affect the expression levels of two genes each (with coffee procedures that are designed to ensure quality and chain of cherry coming close to effecting a third). Idebenone effected custody. Each sample undergoes a thorough quality analysis TERF1 and TERF2, while coffee cherry effected TERF2 and using an Agilent Bioanalyzer microfluidics device, and is TINF2 (a negative regulator of telomere length). This may precisely quantified using a Nanodrop-1000 spectro-photom indicate slightly different mechanisms of action, or different eter. When the Agilent Oligonucleotide Microarray Platform timing/efficiency in the same mechanism, but further study is is utilized, samples are fluorescently labeled using the Agilent required. Low-Input Linear Amplification Kit. Upon completion of this 0515. The largest expression value for all anti-oxidant process, the labeled cFNA products are assessed using the compounds was seen in the TERF2 assay. TERF2 is said to same processes described above. These labeled samples are play a role in the protective activity of telomerase. then fragmented and hybridized to oligonucleotide microar 0516 Coffee cherry downregulates TINF2, which itself is rays. The microarrays are washed and then scanned using an a negative regulator of telomere length, indicating the poten Agilent DNA Microarray Scanner with Sure Scan technol tial that coffee cherry in the right concentration can aid in ogy. Data is extracted from the images produced by the scan maintaining the length of or possibly increasing the length of ner using Agilent's Feature Extraction Software. At this point, telomeres in cells. the scanned image is visually inspected for defects, and the extracted data is statistically analyzed to ensure quality of the Example 8 assay. Extracted data and images can be loaded into Rosetta Resolver Gene Expression Data Analysis System for in-depth 0517. This example illustrates that analysis of the whole analysis, both from a quality standpoint, as well as to develop human genome of cells exposed to the lifespan modulating biological understanding. agents (such as green tea polyphenols, idebenone and coffee 0524 Agilent Human Genome Array results: The results cherry extract, sourced as above) demonstrate longevity/ from the array show the gene expression profile of the entire lifespan extension effects in alternate pathways other than human genome for a sample treated with 1 uM Idebenone telomere length extension. relative to untreated control cells. 0518 Cell cultures: A human skin fibroblast cell culture 0525 Objective 1—Combine the gene expression data was obtained through the Coriell Cell Repository from the from the two fluorophore reversal hybridization replicates to National Institute on Aging Cell Repository. The culture, create a single data table representing the biological compari AG07999, was established from a biopsy of a 32 year old son of interest (Tx compared to UnTx). A table was generated Caucasian female. and provided as a tab-delimited text file. This file will contain 0519 Culture media: Cells were grown in Minimal Essen the log ratio, fold-change, log ratio p-value, etc. for every tial Medium supplemented with 10% fetal bovine serum, 2 transcript measured by the microarray. mM L-glutamine, 2 mM Glutamax I. During the 24 hour 0526. Using Rosetta Resolver, a single ratio comparison experimental phase, cells were maintained in the same was compiled based on the results of the two fluorophore US 2014/0079836A1 Mar. 20, 2014 59 reversal hybridization performed in the context of this CRY1, THC2397757, ZNF585A, ENST00000371408, project. The ratios were calculated such that the Tx sample NUDCD1, PGGT1B, DKFZp667E0512, CA13, AK094296, was in the numerator and the UnTX sample was in the AI051172, ZNF347, THC2324430, GPR132, BCHE, denominator. A table was generated, saved as tab-delimited ZNF785, THC2279.910, CR598370, LOC344887, text files, and is provided on the DVD that accompanied this ENST00000354343, SEC24A, C8orf61, A 32 P205522, report in the "Objective 1 subdirectory or the “Data CINP LRRIQ2, FLJ38973, AF086125, AI192327, ITGB2, Analysis’ directory. These files contain the log ratio, fold BP872463, IL1RAP, TAS2R44, LOC153457, CDCA7, change, log ratio p-value, etc. for every transcript present on RNF111, A 32 P45087, BC008476, AK023647, the microarray. AKO74181, C14orfA9, ZNF516, IL27RA, AA631975, 0527 Objective 2 Identify differentially expressed tran BX448200, THC2419011, IGSF9, PIGL, E2F8, HNRPLL, Scripts for the comparison generated in Objective 1 using C10orf27, NACA, TMEM154, TLK1, H43551, ANK1, standard criteria (specifically, an absolute fold change value A 23 P13202, AA725860, ENST00000366971, WO5707, >1.5, a log ratio p-value <0.001). A table for will be generated KIAA1432, SCD, KIAA 1377, THC241.1515, THC2339241, and provided as a tab-delimited text file. The file will contain TRIM23, GRIA1, LOC374491, THC2266906, C4orf32, the log ratio, fold-change, log ratio p-value, etc. for only the C11orf17, AL571926, A 32 P135790, AI263083, differentially expressed transcripts within the context of the AI925475, THC23.60810, NUPL1, FAM60A, AK001164, comparison. DCP 1 7, CSTA, PNPLA4, UTS2D, ATP8A2, and 0528. The criteria for identification of differentially C13orf1. expressed transcripts were an absolute fold change value D 1.5 0531. The following genes show a statically significant and a log ratio p-value <0.001. These criteria were applied to decrease in expression after treatment with idebenone and the comparison made in Objective 1. A table was generated, UVB: ZNF289, SDHC, HIST1H1A. A 23 P113762, saved as tab-delimited text files, and is provided on the DVD GOLGA2LY1, FLJ43692, EVL, PSAP, KLHDC8B, that accompanied this report in the "Objective 1” subdirec AKAP12, NFAT5, SPATA13, A 23 P 113263, A 32 tory or the “Data Analysis' directory. These files contain the P220567, SORD, LOC643668, ITSN1, HSP90AB1, log ratio, fold-change, log ratio p-value, etc. for every tran LTB4R2, WNT10A, FAM3A, AF212044, DENND4A, script that was identified as differentially expressed using the MDFI, THC2360912, FOXO4, F1P1L1, THC2290002, criteria detailed above HSP90AB3P, STARD3, NOL14, FAM73B, ZC3H13, VCP, 0529. The results of this experiment are shown in DATA DYNC2H1, EHBP1, C60rf204, FABP6, LOC285923, TABLE 7; this table lists all genes that show a statistically PHKG1, MYO15B, GRLF1, HAB1, ZNF792, PLEKHA2, significant change across the human genome. The genes NLRC5, NIPBL, OTUD7B, NPB, LARS, RASL10B, showing statistically significant expression changes can be SAFE2, MALL GRAMD1B, CDC2L1, UBE2E1, broken down into subsets, for instance based on the direction TMEM109, CGNL1, AK000053, DENND1B, ETV4, PKD2, ality of expression change under treatment, relationships BM455859, BQ772270, HNRNPU, A 24 P84719, RCP9, between the genes (e.g., pathway involvement), and so forth. TNRC15, ACTR2, KIAA0372, DDHD1, FER1 L3, RIFT, 0530. The following genes show a statistically significant KLHL17, MCAM, NPFFR1, C1orf144, PPFIBP1, ARF increase in expression after treatment with idebenone and GAP1, WDR31, KLC3, CEP290, TJP2, TCF 15, ANGPTL4, UVB: ARHGAP27, MGC34034, AI446524, LIN28B, PSG9, LRRC61, CLIP1, SKI, CCDC69, LOC650766, PIFI1, MPPED2, DCP 22 6, DCP 22 4, DCP 22 0, DCP AMAC1L2, RALBP1, NTRK3, ATF71P, KIF14, FLJ22659, 22 2, DCP 22 7, LOC440061, THC2319152, HRASLS, ZBED1, TNRC4, EP300, C1orf)6, LRIT1, ZDHHC22, BPI, LOC348174, CD1C, ZNF224, TTBK2, C12orfA2, A 23 P 170719, SMYD4, NOTCH2NL, GNAT1, PIK3R3, ABHD13, AW901755, A 24 P799680, THC2378994, LOC729392, AHCTF1, RETN, C7orf51, LOC440836, APOBEC3G, CDH7, A 24 P84738, EHF, PARP4, MIA3, A 23 P44053, TUB, PRPH, TTTY14, FLJ35379, C7orf29, THC2369020, A 24 P289973, THC2378839, TMC8, DIAPH3, LOC641999, SHC2, THC2278725, MFSD2, FAM40B, A 24 P7820, BC015334, KCNE3, KIAA1751, ZNF560, ZNF517, GSC2, and D31825. THC2312756, C60rf89, AQP10, AA918648, TUBB4, 0532. The following genes show a statically significant AK021467, L0051581, MSR1, THC23394.55, LOC389025, increase in expression after treatment with coffee cherry and A 24 P622375, THC2407737, C10orf59, A 24 P942151, UVB: ENST00000302942, ZNF224, DCP 22 0, DCP A 32 P157622, FAM84B, WWC2, ZNF597, TMEM162, 22 4, DCP 22 6, DCP 22 7, DCP 22 2, A 24 THAP5, CR605947, OTUD6A, THC2440027, WRNIP1, P799680, THC2319152, LOC348174, DKFZP434P211, PLA2G3, SOLH, MADCAM1, CPSF6, AK093508, C12orf42, CDRT15, WNT16, LOC38.9102, ENST00000356104, A 23 P 10605, THC23783.78, A 24 MGC39584, ENST00000356104, MTL5, WNK1, P524164, DCP 22 9, C6Orf5, AI652920, KCNMB2, CA503034, LGI2, KRIT1, DCP 22 9, AF334588, CD34, THC2397757, TXNDC4, THC2448178, CV326037, H40632, ADAM32, LOC645561, KCNE1, SLC16A12, DAGLA, BANK1, KATNAL2, THC2382717, ITGA2, CDC2L6, DUSP13, PCSK1, BE766438, KIAA 1333, TDO2, LOC645561, AA581414, SLC7A11, AL547361, AB01 1149, AF146694, FLJ39653, LOC90586, THC2408033, ZNF516, THC2343350, PCSK9, KCNH2, C17orf67, CNOT6L, FLJ40330, THC2437.177, T70285, FLJ22662, A 24 THC2375853, THC2342473, THC2368209, AI70.9405, P524164, THC24.07334, CR605947, THC2375512, THC2322443, AA344632, WNT16, CNPY3, AI161396, SEC24A, THC2406779, AA586832, STYX, BX433326, DKFZP434P211, BE719776, TIGD3, THC2280741, SCFV, RAB9B, CA843452, PLEKHK1, LOC728499, BQ000605, AI873070, THC2408828, THC2235542, SC4MOL, PTPRO, ZNF681, AF086125, GDF6, ENST00000306515, MYH8, DHCR24, AF086205, STK4, DOCKS, CACNA2D1, T62549, AL040873. A 24 P622375, THC2397757, THC2316768, SLC16A14, OR10A5, THC2437.177, CELSR1, AB01 1149, DAGLA, THC2440027, FAM133A AKO94571, A 24 P3993.41, CELSR1, AKO26984, AA019203, AW885990, TRIM59, THC2337493, PTPN11, AF086329, THC2400593, IL2, CECR2, KLF8, ENST00000379108, AI559980, FLJ21777, LOC646371,
US 2014/0079836A1 Mar. 20, 2014 61
LOC285923, PGS1, LOC44224.5, MAN1B1, DGKA, tion of endogenous antigen (6 genes); antigen processing and LTB4R2, PTPRU, RRBP1, LOC643668, IDH3B, ENO2, presentation of endogenous peptide antigen via MHC class I CAMK1, ADORA1, PPM1G, DBNL, TJP2, BCAP31, (7 genes); apoptosis (49 genes); cellular aromatic compound HARS, WDR82, MVK, EWSR1, SDHAL2, GAL3ST4, metabolic process (9 genes); bile acid and bile salt transport NFATC3, KRT16, RANGAP1, ZCCHC3, C12orfA1, (2 genes); blood coagulation (18 genes); cAMP metabolic ANXA2P1, GBA2, NDE1, AGPAT6, PIK3IP1, TRAFD1, process (4 genes); calcium-mediated signaling (7 genes); BAP1, RBM23, FLJ401 13, P2RX5, MMP14, PAK1, canalicular bile acid transport (2 genes); carbohydrate trans AURKB, LOC220077, ACADS, LOC441455, PAFAH2, port (6 genes); cell cycle (67 genes); cell cycle arrest (32 EWSR1, FUS, C1orf216, APOL2, BE379389, SERPINB6, genes); cell cycle checkpoint (6 genes); cell differentiation PIAS3, UNC84B, ACP2, C20orf3, YKT6, WDR31, PVR, (54 genes); cell division (38 genes); cell motion (29 genes); BRD4. NOC2L, ORC1L, COIL. A 24 P41483, PSMC3, cellular component organization (6 genes); cell proliferation DHTKD1, PPDX, HADHA, ST6GALNAC6, RAPGEF1, (57 genes); cell-cell signaling (50 genes); cellular response to ZNF768, PSMD2, FKBP4, PQLC2, U87972, IFIT3, SELO, starvation (2 genes); citrate metabolic process (2 genes); RFC3, KLHL17, PACSIN3, MAGI1, MAPK12, SMARCA4, coenzyme A metabolic process (2 genes); cyclooxygenase OPRL1, TCF25, HSPA6, A 24 P195621, KIAAO913, pathway (2 genes); cytokine-mediated signaling pathway (5 KLHDC8B, GBA2, C1orf144, NFKBIB, ACAD10, TNIP2, genes); cytoskeletal anchoring at plasma membrane (6 PHKG1, SNX17, SNRP70, CCDC69, A 23 P44053, genes); determination of left/right symmetry (2 genes); mul ETV4, CCNF, LGALS9, SEC24C, FLOT1, VCP, ticellular organismal development (72 genes); double-strand ST6GALNAC2, F2RL3, MALL, PAF1, PSAP, PSAP, break repair (5 genes); epidermis development (13 genes); TMEM109, SUV39H1, ENO, B, DNAJB2, TOM1, GRM4, erythrocyte differentiation (3 genes); folic acid transport (4 ARFGAP1, VPRBP, POLE, C10orf10, AGPAT6. A 24 genes); glucose transport (6 genes); glutamine metabolic pro P315674, C20orf165, KLHDC4. A 24 P229766, TSR2, cess (4 genes); glycerol-3-phosphate metabolic process (4 SFXN5, KIAA1715, DYSF, NBPF10, ZNF289, A 24 genes); glycosaminoglycan biosynthetic process (9 genes); P928031, AAO85955, FLJ35379, TUB, MAP2K7, ACE, glyoxylate cycle (2 genes); growth (6 genes); hemoglobin TTTY14, CCDC19, IGHG1, E4F1, PPME1, KIAA1751, biosynthetic process (3 genes); heparan Sulfate proteoglycan A 24 P913855, A 24 P247303, SMC4, MSH4, GPR120, biosynthetic process (5 genes); hindbrain development (3 NKPD1, GRM5, DCLK3, D90075, ADRA2B, genes); histone acetylation (4 genes); cellular iron ion THC2374442. A 32 P11 1919, ANKRD44, and MYCL1. homeostasis (7 genes); isocitrate metabolic process (2 genes); 0536 The data from this experiment is further broken lactation (5 genes); megakaryocyte differentiation (2 genes); down into additional subsets of genes, provided herewith in mevalonate transport (3 genes); mitosis (24 genes); mitotic DATATABLE8(Telomere complex genes); DATATABLE9 chromosome movement towards spindle pole (2 genes); (DNA Damage and Repair genes); DATA TABLE 10 (Cus monocarboxylic acid transport (3 genes); muscle organ tom Array I Genes); DATA TABLE 11 (Custom Array II development (23 genes); negative regulation of B cell differ Genes); DATA TABLE 12 (Anti Aging Genes); DATA entiation (3 genes); negative regulation of DNA replication (2 TABLE 13 (Inflammation Genes); DATA TABLE 14 (Mito genes); negative regulation of JAK-STAT cascade (2 genes); chondrial/Cellular respiration/Mitochondrial biogenesis negative regulation of Ras protein signal transduction (4 genes); and DATA TABLE 15 (Nitric Oxide Synthase genes). genes); negative regulation of cell differentiation (1 genes); 0537. The genes showing a statistically significant change negative regulation of cell proliferation (37 genes); negative in expression treatment with coffee cherry extract can also be regulation of follicle-stimulating hormone secretion (5 grouped by art recognized pathways, using for instance genes); negative regulation of interferon-gammabiosynthetic Rosetta Resolver Gene Expression Data Analysis System. process (3 genes); negative regulation of lipoprotein lipase Genes can also be divided by Rosetta Resolver into “path activity (2 genes); negative regulation of macrophage differ ways” as they are defined by the Gene Ontology (see, for entiation (3 genes); negative regulation of phosphorylation (3 instance, AmiGO, available on-line at amigo.genontology. genes); negative regulation of programmed cell death (1 org, and particularly amigo.geneontology.org/cgi-bin/amigo/ genes); negative regulation of transcription (16 genes); nega go.cgi). Definitions of the pathways that correspond to the tive regulation of transcription from RNA polymerase II pro following “pathway” designations also can be found on-line, moter (19 genes); nervous system development (47 genes); for instance at the Gene Ontology website. neural tube closure (2 genes); neutrophil activation (2 genes); 0538 For the sample analyzed 8 hours after treatment with nitric oxide mediated signal transduction (3 genes); nucleo coffee cherry, statistically significant expression changes Some assembly (14 genes); nucleotide catabolic process (2 were seen genes in the following Primary GO (Gene Ontol genes); ovarian follicle development (3 genes); parturition (4 ogy) Term Name “pathways: ATP catabolic process (2 genes); peptide cross-linking (3 genes); activation of phos genes); DNA metabolic process (5 genes); DNA repair (29 pholipase C activity (6 genes); positive regulation of 1-kap genes); DNA replication (33 genes); DNA replication check paB kinase/NF-kappaB cascade (17 genes); positive regula point (4 genes); DNA replication initiation (11 genes); G1 tion of angiogenesis (5 genes); positive regulation of cell phase of mitotic cell cycle (6 genes); JAK-STAT cascade (7 adhesion (4 genes); positive regulation of cell growth (2 genes); RNA elongation from RNA polymerase II promoter genes); positive regulation of cell proliferation (23 genes); (4 genes); UDP-N-acetylglucosamine biosynthetic process positive regulation of fibroblast proliferation (2 genes); posi (1 genes); actin cytoskeleton organization (24 genes); actin tive regulation of follicle-stimulating hormone secretion (3 filament bundle formation (3 genes); activation of MAPKKK genes); positive regulation of lipid metabolic process (2 activity (4 genes); activation of NF-kappaB-inducing kinase genes); positive regulation of neurogenesis (1 genes); positive activity (5 genes); androgen receptor signaling pathway (11 regulation of mitotic cell cycle (1 genes); positive regulation genes); angiogenesis (24 genes); anion transport (5 genes): of protein kinase activity (3 genes); protein amino acid anti-apoptosis (23 genes); antigen processing and presenta dephosphorylation (35 genes); protein complex assembly (27 US 2014/0079836A1 Mar. 20, 2014 62 genes); purine base biosynthetic process (4 genes); purine ment of circadian clock (3 genes); epithelial to mesenchymal nucleotide biosynthetic process (4 genes); pyrimidine nucle transition (2 genes); erythrocyte differentiation (3 genes); otide metabolic process (3 genes); rRNA processing (13 establishment or maintenance of chromatin architecture (13 genes); receptor-mediated endocytosis (13 genes); regulation genes); establishment of mitotic spindle localization (2 of MAP kinase activity (2 genes); regulation of bone miner genes); ether lipid biosynthetic process (2 genes); germ cell alization (3 genes); regulation of cholesterol biosynthetic migration (7 genes); glial cell migration (2 genes); glycerol process (2 genes); regulation of cyclin-dependent protein 3-phosphate metabolic process (4 genes); glycoprotein bio kinase activity (19 genes); regulation of inflammatory synthetic process (3 genes); glycosaminoglycan biosynthetic response (2 genes); regulation of lipid metabolic process (2 process (8 genes); gonad development (3 genes); growth (7 genes); regulation of mitosis (5 genes); regulation of ossifi genes); growth hormone secretion (3 genes); hemoglobin cation (1 genes); regulation of retroviral genome replication biosynthetic process (3 genes); histone acetylation (4 genes); (2 genes); regulation of transcription, DNA-dependent (291 hydrogen peroxide catabolic process (3 genes); induction of genes); regulation of transcriptional preinitiation complex apoptosis by intracellular signals (9 genes); induction of apo assembly (3 genes); regulation of transforming growth factor ptosis via death domain receptors (6 genes); induction of beta receptor signaling pathway (3 genes); response to drug (5 negative chemotaxis (2 genes); induction of positive chemo genes); response to external stimulus (3 genes); response to taxis (4 genes); insulin secretion (3 genes); internal protein stress (16 genes); skeletal system development (22 genes); amino acid acetylation (2 genes); intra-S DNA damage sphingoid catabolic process (1 genes); sphingosine metabolic checkpoint (2 genes); lactation (4 genes); lysine catabolic process (1 genes); tRNA processing (8 genes); thiamin trans process (2 genes); megakaryocyte differentiation (2 genes); port (2 genes); tissue development (2 genes); transcription mesoderm migration (2 genes); mevalonate transport (4 (226 genes); transcription from RNA polymerase II promoter genes); mitosis (52 genes); mitotic chromosome condensa (36 genes); traversing start control point of mitotic cell cycle tion (5 genes); mitotic chromosome movement towards (4 genes); and Valine metabolic process (3 genes). spindle pole (2 genes); mitotic metaphase plate congression 0539 For the sample analyzed 24 hours after treatment (2 genes); mitotic recombination (4 genes); mitotic sister with coffee cherry, statistically significant expression chromatid segregation (4 genes); mitotic spindle elongation changes were seen in genes in the following Primary GO Term (2 genes); monocarboxylic acid transport (4 genes); motor Name pathways: DNA damage checkpoint (8 genes); DNA axon guidance (2 genes); muscle organ development (43 damage response, signal transduction by p53 class mediator genes); negative regulation of B cell differentiation (3 genes); resulting in cell cycle arrest (3 genes); DNA metabolic pro negative regulation of DNA replication (3 genes); negative cess (11 genes); DNA recombination (18 genes); DNA repair regulation of T-helper 2 cell differentiation (3 genes); nega (61 genes); genes DNA replication (66 genes); DNA replica tive regulation of follicle-stimulating hormone secretion (7 tion checkpoint (6 genes); DNA replication initiation (12 genes); negative regulation of helicase activity (2 genes); genes); DNA replication-dependent nucleosome assembly (3 negative regulation of interferon-gamma biosynthetic pro genes); DNA strand elongation during DNA replication (4 cess (3 genes); negative regulation of lipoprotein lipase activ genes); G0 to G1 transition (3 genes); G2/M transition of ity (2 genes); negative regulation of macrophage differentia mitotic cell cycle (9 genes); L-glutamate transport (5 genes); tion (3 genes); negative regulation of phosphorylation (3 MAPK export from nucleus (3 genes): MAPK phosphatase genes); negative regulation of cell cycle (37 genes); negative export from nucleus, leptomycin B sensitive (3 genes); NAD regulation of protein catabolic process (3 genes); negative biosynthetic process (5 genes); Rho protein signal transduc regulation of sister chromatid cohesion (2 genes); negative tion (10 genes); UDP-N-acetylglucosamine transport (2 regulation of transcription (25 genes); negative regulation of genes); UV protection (2 genes); actin modification (3 transcription from RNA polymerase II promoter (30 genes); genes); activation of MAPKK activity (6 genes); activation of neuron recognition (5 genes); neurotransmitter uptake (4 MAPKKK activity (4 genes); activation of NF-kappaB-in genes); nitric oxide biosynthetic process (5 genes); nitric ducing kinase activity (6 genes); age-dependent response to oxide mediated signal transduction (5 genes); nucleoside reactive oxygen species (3 genes); cellular aldehyde meta metabolic process (6 genes); nucleosome assembly (32 bolic process (5 genes); angiogenesis (18 genes); apoptosis genes); nucleotide biosynthetic process (7 genes); nucleotide (99 genes); arginine catabolic process (6 genes); blood catabolic process (3 genes); nucleotide-excision repair (7 coagulation (29 genes); cAMP metabolic process (4 genes); genes); organ morphogenesis (31 genes); organic anion trans calcium-mediated signaling (8 genes); canalicular bile acid port (9 genes); ovarian follicle development (4 genes); partu transport (3 genes); cardiac cell differentiation (2 genes); cell rition (4 genes); pentose-phosphate shunt (5 genes); peptidyl cycle (133 genes); cell cycle arrest (30 genes); cell cycle amino acid modification (3 genes); phosphoinositide checkpoint (9 genes); cell death (12 genes); cell differentia mediated signaling (15 genes); positive regulation of JNK tion (81 genes); cell division (68 genes); cell growth (15 cascade (6 genes); positive regulation of T-helper 1 cell dif genes); cell migration (11 genes); cell proliferation (105 ferentiation (3 genes); positive regulation of angiogenesis (5 genes); cell recognition (6 genes); cell-cell signaling (72 genes); positive regulation of axonogenesis (4 genes); posi genes); cell-matrix adhesion (28 genes); cell-substrate junc tive regulation of cell adhesion (3 genes); positive regulation tion assembly (2 genes); chemotaxis (28 genes); collagen of fibroblast proliferation (2 genes); positive regulation of fibril organization (4 genes); complement activation (4 follicle-stimulating hormone secretion (4 genes); positive genes); cortical actin cytoskeleton organization (6 genes); regulation of glucose import (3 genes); positive regulation of cyclin catabolic process (2 genes); cytokine-mediated signal mitotic metaphase/anaphase transition (3 genes); positive ing pathway (8 genes); cytokinesis (10 genes); dTDP biosyn regulation of transforming growth factor beta receptor signal thetic process (3 genes); dTTP biosynthetic process (3 ing pathway (6 genes); protein amino acid acetylation (4 genes); deoxyribonucleoside diphosphate metabolic process genes); protein amino acid dephosphorylation (52 genes); (2 genes); in utero embryonic development (9 genes); entrain protein folding (64 genes); protein hetero-oligomerization (6 US 2014/0079836A1 Mar. 20, 2014
genes); protein import into mitochondrial inner membrane (4 antioxidant media for 12 days. The flies are then transferred to genes); protein import into mitochondrial matrix (3 genes); a stressor media (either 3% H2O, or 20% sucrose solution) protein localization (7 genes); protein tetramerization (4 which has been shown to shorten lifespan. All the flies are genes); proteolysis (109 genes); purine ribonucleoside mono examined daily and media changed as required until all the phosphate biosynthetic process (4 genes); pyrimidine nucle flies have died. The date (post 12 day antioxidant incubation) otide metabolic process (5 genes); pyruvate transport (3 is recorded. genes); rRNA processing (19 genes); regulation of 1-kappaB 0548 Lifespan results: The cumulative average lifespan of kinase/NF-kappaB cascade (3 genes); regulation of MAP all flies for each group are then computed and compared kinase activity (3 genes); regulation of Wnt receptor signaling against the cumulative average of the control (untreated) flies pathway (7 genes); regulation of cyclin-dependent protein to determine if the Antioxidants increase lifespan. The aver kinase activity (18 genes); regulation of mitochondrial mem age lifespan increase is separated by sex as well as combined brane permeability (2 genes); regulation of neuron differen for both sexes. This lifespan extension is expressed as a per tiation (5 genes); regulation of protein stability (5 genes); cent increase or decrease over control values. regulation of proteolysis (6 genes); regulation of retroviral 0549 Drosophila cultures: A wingless variant of Droso genome replication (2 genes); regulation of transcription phila Melanogaster was purchased from Carolina Biological from RNA polymerase II promoter (71 genes); regulation of Supply. These flies were fed nutrient media (Formula 4-24) transcription, DNA-dependent (447 genes); regulation of and experimental flies were collected within 18 hours after transforming growth factor beta receptor signaling pathway hatching to ensure the females were Virgin. (4 genes); response to drug (7 genes); response to external stimulus (4 genes); response to nutrient (10 genes); response 0550 Culture media: Flies were given a combination of to radiation (7 genes); response to Superoxide (3 genes); the following: 1) Formula 4-24 without blue coloring for response to unfolded protein (16 genes); sensory organ devel breeding and post exposure to antioxidants, 2) Formula 4-24 opment (2 genes); skeletal system development (35 genes); with the addition of 1% idebenone or Coffee Cherry extract spindle organization (8 genes); Superoxide metabolic process for 12 days following hatching, 3) Formula 4-24 with the (5 genes); synapse organization (2 genes); tRNA processing addition of 3% H2O2 to oxidatively stress the flies (and (12 genes); transcription (355 genes); transforming growth decrease the lifespan) post AOX incubation, and 4) 20% factor beta receptor signaling pathway (13 genes); transport sucrose solution on filter paper following AOX incubation to (126 genes); traversing start control point of mitotic cell cycle serve as a low nutrient media and known lifespan shortening (10 genes); ureteric bud development (3 genes); vacuolar agent. transport (2 genes); Valine metabolic process (6 genes); and 0551 Experimental phase: The experiments performed on the flies generally followed the following pattern: 1) The flies virus-host interaction (6 genes). are hatched in normal media and sexed and transferred to Example 9 vials containing varying levels (1%, 0.1% or 0.01% of either Idebenone or Coffee Cherry extract) or normal media to serve 0540. This example describes a whole animal analysis of as a control group. The flies remain in the antioxidant media survival/longevity in fruit flies, after treatment with various for 12 days. 2) The flies are then transferred to a stressor antioxidants. media (either 3% H2O2 or 20% sucrose solution) which has 0541 Drosophila cultures: A wingless variant of Droso been shown to shorten lifespan. 3) All the flies are examined phila Melanogaster was purchased from Carolina Biological daily and media changed as required until all the flies have Supply. These flies were fed nutrient media (Formula 4-24) died. 4) The date (post 12 day antioxidant incubation) is and experimental flies were collected within 18 hours after recorded. hatching to ensure the females were virgin. 0552 Lifespan results: The cumulative average lifespan of 0542 Culture media: Flies were given a combination of all flies for each group are then computed and compared the following: against the cumulative average of the control (untreated) flies 0543. 1) Formula 4-24 without blue coloring for breeding to determine if the antioxidants increase lifespan. The average and post exposure to antioxidants. lifespan increase is separated by sex as well as combined for 0544, 2) Formula 4-24 with the addition of 1% idebenone both sexes. This lifespan extension is expressed as a percent or coffee cherry extract for 12 days following hatching. The increase or decrease over control values and is displayed preparation of the 4-24 media was either w/w ratio with all the below. dry components (for example 1% idebenone and 99% 4-24) hydrated with sterile water, or the full amount of 4-24 medium was hydrated with an appropriately diluted (coffee berry was diluted in sterile HO, and idebenone was diluted PARAMETER MALE FEMALE Average of Both initially in sterile alcohol, and then serially diluted using H2O2 5.67 days 5.11 days 5.32 days H2O) testing compound. 1% Idebenone + H2O, 5.32 days 5.50 days 5.75 days (0545 3) Formula 4-24 with the addition of 3% HO, to 1% CoffeeCherry + 6.53 days 7.71 days 7.10 days oxidatively stress the flies (and decrease the lifespan) post H2O2 AOX incubation. % Change Idebenone --8% -7% --8% % Change Coffeecherry --15% --51% +33% 0546 4) 20% sucrose solution on filter paper following 20% Sucrose 9.73 days 12.2 days 11.32 days AOX incubation to serve as a low nutrient media and known 1% Idebenone + Sucrose 12.11 days 11.7 days 11.89 days lifespan shortening agent. 1% CoffeeCherry + 9.25 days 13.1 days 11.09 days Sucrose 0547. The flies are hatched in normal media and sexed and % Change Idebenone +24% -4% --5% transferred to vials containing varying levels (1%, 0.1% or % Change CoffeeCherry -5% +7% -2% 0.01% of either Idebenone or Coffeecherry extract) or normal media to serve as a control group. The flies remain in the US 2014/0079836A1 Mar. 20, 2014 64
0553. The above table shows the change in lifespan for contain the log ratio, fold-change, log ratio p-value, etc. for either coffee cherry or idebenone for Drosophila placed on every transcript measured by the microarray. known longevity decreasing media following 12 days incu 0562) Objective 2 Identify differentially expressed tran bation with the antioxidant. Statistical significance was Scripts for the comparison generated in Objective 1 using reached on the coffee cherry pretreatment before H.O. standard criteria (specifically, an absolute fold change value >1.5, a log ratio p-value <0.001). A table for will be generated and provided as a tab-delimited text file. The file will contain the log ratio, fold-change, log ratio p-value, etc. for only the PARAMETER MALE FEMALE Average of Both differentially expressed transcripts within the context of the comparison. 0.01% Idebenone Not Completed 23.4 days 23.4 days 0.01% CoffeeCherry Not Completed 11 days 11 days 0563. The criteria for identification of differentially 0.1% Idebenone Not Completed 10.2 Days 10.2 days expressed transcripts will be an absolute fold change 0.1% CoffeeCherry 4.1 days Not Completed 4.1 days valued 1.5 and a log ratio p-values0.005. 1% Idebenone 8.1 days 12.4 days 10 days 1% CoffeeCherry 10.25 days 14.7 days 12.6 days Example 11 Untreated Control 26.5 days 38.4 days 33.4 days 0564. This example provides a system to capture a repre sentation of the gene expression profiles of cultured human fibroblasts following antioxidant Supplementation and expo 0554. The above table shows change in average lifespan sure to a second form of oxidative stress (in the form of when Drosophila flies are first hatched and placed directly hydrogen peroxide). This provides a sampling of genes that onto media containing the antioxidant alone. are significantly altered when given antioxidants as a method for protection against H2O induced oxidative stress. These Example 10 genes are indicative of any protective or harmful effects anti oxidants have on cells oxidatively stressed with H2O. It will 0555. This example provides a system to capture a repre also demonstrate any differences between the mechanisms of sentation of the gene expression profiles of cultured human action of H2O induced oxidative stress when compared to fibroblasts following antioxidant Supplementation and expo UV induced oxidative stress describing possible targets for sure to oxidative stress (in the form of UV radiation). This restorative agents for both types of stress. provides a sampling of genes that are significantly altered Cell cultures: A human skin fibroblast cell culture (or cul when given antioxidants and treated with UV when compared tures) will be obtained through the Coriell to cells only damaged with UV radiation. These genes are 0565 Cell Repository from the National Institute on indicative of the pathways of repair or protection that are Aging Cell Repository. The initial culture, AG07999, was involved with antioxidant Supplementation and UV damage. established from a biopsy of a 32 year old Caucasian female. 0566 Culture media: Cells will be grown in Minimal 0556 Cell cultures: A human skin fibroblast cell culture Essential Medium supplemented with 10% fetal bovine (or cultures) will be obtained through the Coriell Cell Reposi serum, 2 mM L-glutamine, 2 mM Glutamax I. During the 24 tory from the National Institute on Aging Cell Repository. hour experimental phase, cells will be maintained in the same The initial culture, AG07999, was established from a biopsy medium, but with only 1% fetal bovine serum. All cultures of a 32 year old Caucasian female. will be incubated at 37° C. with 5% CO, in a humidified 0557. Culture media: Cells will be grown in Minimal chamber. Essential Medium supplemented with 10% fetal bovine 0567 Experimental phase: On Day 1, cells will be seeded serum, 2 mM L-glutamine, 2 mM Glutamax I. During the 24 into 6 well dishes in equivalent cell numbers. On Day 2, the hour experimental phase, cells will be maintained in the same medium will be removed by aspiration and replaced with 600 medium, but with only 1% fetal bovine serum. All cultures uM H2O2 in medium. Test conditions will be: 1) 0.0001% will be incubated at 37° C. with 5% CO, in a humidified coffee cherry extract and 2) H2O2 alone controls. chamber. 0568. These conditions may be expanded at a later date to 0558 Experimental phase: On Day 1, cells will be seeded include new extracts/compounds. After the determined time into each of four 75 cm flasks containing 20 ml of culture points (e.g., 24 hours, 8 hours, and longer—e.g., 144 hours), medium. On Day 4, the medium will be removed by aspira the cells will be lysed and the RNA will be extracted. The tion and replaced with the test condition (Antioxidant Supple RNA will then be run on Affymetrix whole human genome mentation) in 20 ml culture medium but with only 1% fetal microarrays and the results compiled and analyzed. bovine serum. Test conditions will be: 1) 1 uMidebenone; 2) 0569. Affymetrix Human Genome Array results: Basic Green Tea; 3) Coffeecherry extract; and 4) untreated control. analysis of the data will involve determining significant fold 0559 These conditions will also be duplicated for the changes, p-values and basic statistics. Data will be sorted by same conditions but after stress with UVA, UVB or a combi significance, statistical accuracy and delta value. nation of UVA and UVB light from a solar simulator/mono chrometer. After the determined time points (e.g., 24 hours, 8 Example 12 hours, and longer—e.g., 144 hours), the cells will be lysed 0570. This experiment was designed to determine if the and the RNA will be extracted. The RNA will then be run on tested antioxidant compounds had any effect on mitochon Agilent whole human genome microarrays (Kronick, Expert drial biogenesis in cultured human fibroblasts when given Rev. Proteomics 1(1):19-28, 2004) and the results compiled antioxidant compounds or stressed with H2O, or a combina and analyzed. tion of both. Any increase in staining should correlate to an 0560. During the analysis two objectives will be exam increase in the numbers of mitochondria present since the ined: cells were all seeded at the same confluency and cell numbers. 0561. Objective 1—Combine the gene expression data 0571 Cell cultures: A human skin fibroblast cell culture from the two fluorophore reversal hybridization replicates to obtained through the Coriell Cell Repository from the create a single data table representing the biological compari National Institute on Aging Cell Repository was used. The son of interest (Tx compared to UnTx). A table will be gen culture, AG07999, was established from a biopsy of a 32 year erated and provided as a tab-delimited text file. This file will old Caucasian female. US 2014/0079836A1 Mar. 20, 2014
0572 Culture media: Fibroblast cells were grown in Mini Example 13 mal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 2 mM Glutamax I. 0577. This experiment was designed to determine if the During the experimental phase, cells were maintained in the tested antioxidant compounds had any effect on mitochon same medium, but with only 1% FBS. All cultures were drial biogenesis in cultured human cardiac myocytes when incubated at 37°C. with 5% CO in a humidified chamber. given antioxidant compounds or stressed with H2O, (or a 0573 Experimental phase: On Day 1, cells were seeded at combination of both). An increase in staining should correlate near confluency into 24 well cluster dishes in 500 ul appro to an increase in the numbers of mitochondria present since priate medium. On Day 2, all wells were aspirated. Test wells the cells were all seeded at the same confluency and cell received 500 ul of an antioxidant supplement (coffee cherry numbers. extract or idebenone) in medium. Test control wells received 500 ul of medium only. After 24 hours, 48 hours, or 72 hours, 0578 Cell culture: Human cardiac myocytes were wells were aspirated and 300 ul MitoTracker Green in obtained through Promocell (Germany). The myocytes were medium was added to each well and incubated for 1 hour. established from a 52 year old Caucasian female. After 1 hour, wells were aspirated and washed 2x with 300 ul appropriate medium and read using a fluorometer. 0579 Culture media: Cardiac myocytes were grown in 0574. The percent change (in RFU's or Relative Fluores myocete cell growth medium with Supplements recom cence Units) of test wells over controls indicated an increase mended by and purchased from Promocell. This medium was or decrease in the numbers of mitochondria present in the used for all phases of growth and experimentation using the cells following the antioxidant Supplement treatment. myocytes. (0575 Coffee cherry extract dilutions tested were: 0.01%, 0580 Experimental phase: On Day 1, myocytes were 0.001%, 0.0001%, 0.00001%, and 0.000001%. Idebenone dilutions tested were: 10 uM, 1 uM, 0.1 uM, 0.01 uM, 0.001 seeded at near confluency into 24 well cluster dishes in 500 ul uM appropriate medium. On Day 2, all wells were aspirated. Test 0576 Results: Using 600 uMH0, to induce cellular stress wells received 500 ul of an antioxidant supplement (coffee for 30 or 60 minutes and coffeeberry (CB) to induce cellular cherry extract COFFEEBERRYR) in medium. Test control recovery, the following results were found at 24 and 48 hours: wells received 500 ul of medium only. After 24 hours or 48 hours, wells were aspirated and 300 ul MitoTracker Green in medium added to each well and incubated for 1 hour. After 1 Results: calculate % over control hour, wells were aspirated and washed 2x with 300 ul medium and read using a fluorometer. H5O2 TX: H2O, TX: 0581. The percent change (in RFU's or Relative Fluores 30 min 60 min 30 min 60 min cence Units) of test wells over controls indicated an increase at 24 hours at 48 hours or decrease in the numbers of mitochondria present in the --OOO1% CB -1.3% -19% O.4% -0.9% cells following the antioxidant Supplement treatment. --OOOO1% CB -8% -1.3% O.2% 1.6% 0582 Coffee cherry extract dilutions tested were: 0.01%, 0.001%, 0.0001%, 0.00001% and 0.000001%.
MYOCYTES with COFFEEBERRY (R) results Results: calculate % RFU over control
24 hours 48 hours
--O.O1% CB -7.6 10.3 --OOO1% CB -9.7 -135 --OOOO1% CB -11.1 -0.8 --OOOOO1% CB -11.8 17.9 --OOOOOO1% CB -0.8 -6.2
24 hr Raw data: 24hr Mean: 48 hr Raw data: 48 hr Mean:
--0.01% CB 955 1022 968 982 886 907 1029 941 --OOO1% CB 874 1024 981 960 554 569 1089 737 --OOOO1% CB 776 1051 1009 945 964 535 1039 846 --OOOOO1% CB 834 1045 935 938 946 952 1120 1006 --OOOOOO1% CB 957 1177 1028. 1054 1091 77O 538 800 987 864 1184 1217 1063 Controls 939 1035 586 853 US 2014/0079836A1 Mar. 20, 2014 66
This data is also shown in FIG. 8. Example 16 0583. It appears there is biphasic response of the mito chondria to coffee cherry extract. Without wishing to be lim COFFEEBERRYR) Challenge Experiment for RNA ited to any particular mechanism, depending on the dosage Isolation for Microarray Analysis coffee cherry can function to either increase or decrease the 0593. Humandermal fibroblasts (ag.07999) were seeded at number of mitochondria. near confluency in 6-well dishes in 4 ml MEM, 10% FBS/ well. 24 hours after seeding, wells were aspirated and 3 ml Example 14 coffeeberry dilution in MEM, 1% FBS added, or MEM, 1% 0584) This example provides a method that can be used to FBS only for control wells. COFFEEBERRYR) dilutions determine the rate of mitochondrial respiration or efficiency used were: 0.1%, 0.01%, 0.005%, 0.001%, 0.0005%, following antioxidant Supplementation. 0.0001%, 0.00005%, 0.00001%, and 0.000001%. 24 hours 0585 Cell culture: Human cardiac myocytes were after coffeeberry challenge, wells were aspirated, 1 ml obtained through Promocell (Germany). The myocytes were trypsin-EDTA added to each well, swirled and aspirated. established from a 52 year old Caucasian female. Trypsin-EDTA was again added to each well and cells 0586 Culture media: Cardiac myocytes will be grown in retrieved with MEM 10% FBS when released from the Sub myocete cell growth medium with Supplements recom stratum and centrifuged to a pellet. mended by and purchased from Promocell. This medium will 0594 RNA was collected from the harvested cells accord be used for all phases of growth and experimentation using ing to protocol using RT2 qPCR-Grade RNA. Isolation Kit the myocytes. from SABiosciences Co. The RNA was then used to create cDNA (using a First Strand Synthesis Kit, SABiosciences) 0587 Experimental phase: On Day 1, myocytes will be and run on the BioRadicycler RT-PCR machine using Cus seeded at near confluency into 24 well cluster dishes in 500 ul tom RT-PCR Microarray (CAPHO9464A, SABiosciences) appropriate medium. On Day 2, all wells will be aspirated. (Array 2) and SYBR Green Reaction Mix (SABiosciences). Test wells will receive 500 ul of an antioxidant supplement Data from Example 15 are shown in FIG. 20; additional (coffee cherry extract) in medium. Test control wells will results are described and analyzed below. receive 500 ul of medium only. After the determined time points (using the same time points as previous experimenta Results and Discussion for Examples 15 and 16 tion 24 hours, 8 hours, and if desired at 144 hours to mimic apple stem cell paper/patent of exposure to test conditions), 0595 FIGS. 9-18 show changes in the expression of spe the cells will have mitochondrial efficiency measured using cific genes (VEGFA, HMOX1, CCL4L1, DDC, NOS2A, the Clark electrode or Seahorse XF24 Flux Analyzer accord SIRT1, SIRT2, SIRT3, SIRT4, TERT, PTGS2, and IFI44) in ing to the recommended protocols human fibroblasts treated with coffee cherry extract 0588 Coffee cherry extract dilutions to be tested: 0.01%, (0.000001%, 0.0001% or 0.01%) or chlorogenic acid 0.001%, 0.0001%, 0.00001% and 0.000001%. (0.000005%, 0.00005%, or 0.005%). 0589 Mitochondrial Efficiency results: Basic analysis of 0596 FIG. 9 (VEGFA; VEGF; Vascular Endothelial the data will involve determining significant changes, p-val Growth Factor) shows the change in relative expression of ues and basic statistics. VEGFA with decreasing concentrations of coffee cherry extract. A relatively high concentration of coffee cherry Example 15 extract (0.01%) induces the expression of this protein, while lower levels (0.0001% and 0.000001%) actually repress 0590 Chlorogenic acid challenge experiment for RNA expression of this protein. VEGFA is a homodimeric glyco isolation for microarray analysis protein of relative molecular mass 45,000, is the only mitogen 0591 Humandermal fibroblasts (ag.07999) were seeded at that specifically acts on endothelial cells. It may be a major near confluency in 6-well dishes in 4 ml MEM, 10% FBS/ regulator of tumor angiogenesis in vivo. VEGFA is a candi well. 24 hours after seeding, wells were aspirated and 3 ml date hormone for facilitating glucose passage across the chlorogenic acid dilution in MEM, 1% FBS added, or MEM, blood-brain barrier under critical conditions, tumor angio 1% FBS only for control wells. Chlorogenic acid (Acros genesis, VEGF and IL6 are produced together in the intraocu Organics, Geel, Belgium) dilutions used were: 0.005%, lar tissues and that both are involved in the pathogenesis of 0.0005%, 0.00005%, 0.000005% and 0.0000005%. 24 hours diabetic macular edema. Increasing expression of VEGF can after chlorogenic acid challenge, wells were aspirated, 1 ml be useful for improving wound healing, while decreasing trypsin-EDTA added to each well, swirled and aspirated. expression can be important for treating macular degenera Trypsin-EDTA was again added to each well and cells tion of the retina. It is noted that TGFB behaves similarly with retrieved with MEM 10% FBS when released from the Sub regard to expression responses to antixoidants. stratum and centrifuged to a pellet. 0597 VEGF is involved in stimulating and inhibiting 0592 RNA was collected from the harvested cells accord growth of new blood vessels, which makes this a particularly ing to protocol using RT2 qPCR-Grade RNA. Isolation Kit important gene in wound healing and cancer, as well as macu from SABiosciences Co. The RNA was then used to create lar degeneration of the retina and other diseases. Thus, the cDNA (using a First Strand Synthesis Kit, SABiosciences, discovery herein that antioxidant compositions such as coffee and following the manufacturers instructions) and run on the cherry and chlorogenic acid can be used to either induce or BioRad iCycler RT-PCR machine using Custom RT-PCR repress VEGF expression enables methods of treating each of Microarray (CAPHO9464A, SABiosciences) (Array 1) and these conditions. SYBR Green Reaction Mix (SABiosciences). Data from 0598 Collagen 1A1 (the dominant from of collagen in Example 16 are shown in FIG. 19; additional results are skin) exhibits a similar dosage response to VEGF, so it is described and analyzed below. believed that (relatively) higher concentrations of antioxi US 2014/0079836A1 Mar. 20, 2014 67 dants could be used to improve fine lines, wrinkles, and other messenger molecule with diverse and very important func aspects of skin. It may be particularly beneficial to reduce tions throughout the body. In the brain and peripheral nervous expression or activity of MMP-1 collagenase concurrently, so system, NO displays many properties of a neurotransmitter; it it is particularly useful that MMP1 is downregulated (at about is implicated in neurotoxicity associated with stroke and neu the same degree for all concentrations). This is particularly rodegenerative diseases, neural regulation of Smooth muscle, useful for anti aging in skin or repairing aging, and may be including peristalsis, and penile erection. NO is also respon useful to reverse, inhibit, delay, or offset defects of the skins sible for endothelium-derived relaxing factor (EDRF) activ dermal matrix, including for instance fine lines, wrinkles, ity regulating blood pressure. In macrophages, NO mediates sagging, tone, and so forth. tumoricidal and bactericidal actions, as indicated by the fact 0599 FIG. 10 (HMOX1; Heme Oxygenase 1) shows the that inhibitors of NO synthase (NOS) block these effects. NO change in relative expression of HMOX1 with decreasing plays a significant role in mitochondrial biogenesis as well. concentrations of coffee cherry extract. A relatively high con The ability to modulate NO expression either up or down (as centration of coffee cherry extract (0.01%) induces the illustrated in this figure) can have important role in lifespan, expression of this protein, while lower levels (0.0001% and mitochondrial biogenesis, healthy longevity and good health 0.000001%) actually repress expression of this protein in general. Coffee cherry displays a dose dependent increase similar to the pattern observed for VEGFA. HMOX1 cata in expression, whereas chlorogenic acid showed biphasic lyzes the rate limiting step in the catabolism of heme to form response with up or down regulation of expression being biliverdin, which is subsequently converted to bilirubin by dependent upon the dose. biliverdin reductase, free iron, and carbon monoxide. Heme 0603 FIG. 14 (SIRT1, Sirtuin1) shows the change in rela oxygenase shows antioxidative effects and induced HmoX1 tive expression of SIRT1 with increasing concentrations of may protect against lipopolysaccharide-induced septic coffee cherry extract and chlorogenic acid (a component of shock. coffee cherry extract). SIRT1 is a stress-response and chro 0600 FIG. 11 (CCL4L1; a.k.a. LAG 1) shows the change matin-silencing factor. It is an NAD(+)-dependent histone in relative expression of HMOX1 with increasing concentra deacetylase involved in various nuclear events such as tran tions of coffee cherry extract and chlorogenic acid (a compo scription, DNA replication, and DNA repair. SIRT1 protein nent of coffee cherry extract). CCL4L1 is one of several binds and deacetylates the p53 protein the deacetylase activ cytokine genes clustered on the q-arm of chromosome 17. ity accounts for silencing, recombination Suppression, and Cytokines area family of secreted proteins involved in immu extension of life span in vivo. Furthermore, SIRT1 repressed noregulatory and inflammatory processes. This protein is p53-dependent apoptosis in response to DNA damage and similar to CCL4 which inhibits HIV entry by binding to the oxidative stress. The SIRT1 gene is turned on by a caloric cellular receptor CCR5.The copy number of this gene varies restriction diet, and this protects cells from dying under stress among individuals; most individuals have 1-5 copies in the and may extend lifespan. Coffee cherry and chlorogenic acid diploid genome, although rare individuals do not contain this each demonstrated a non linear dose response curve for gene. It has been suggested that the most effective anti-HIV increasing or decreasing expression of SIRT1 with chloro drugs would be those that increase expression of whichever genic acid being entirely decreasing expression but coffee CCL4 protein, i.e., ACT2 or LAG1 has the highest affinity for cherry could either increase or decrease (and thus modulate) CCR5 identified B-cell lines that express predominantly the expression of SIRT1. LAG 1. Coffee cherry exposure results in significantly 0604 FIG. 15 (TERT:Telomerase Reverse Transcriptase) increased expression of CCL4L1 in a dose dependent manner, shows the change in relative expression of TERT with which can now be exploited for altering immune response and increasing concentrations of coffee cherry extract and chlo particularly for treatment of HIV infection. In contrast, chlo rogenic acid (a component of coffee cherry extract). Coffee rogenic acid displays a non-linear dosage response, with the cherry showed a linear dose response whereas chlorogenic middle dosage (0.00005%) yielding a marked repression of acid showed a non linear response; the relative TERT gene CCL4L1. expression increased with increasing coffee cherry concen 0601 FIG. 12 (DDC; Dopa Decarboxylase) shows the tration, but could be either induced or reduced with different change in relative expression of DDC with increasing con amounts of chlorogenic acid. The ability to enhance telomere centrations of coffee cherry extract and chlorogenic acid (a maintenance with coffee cherry may increase lifespan. component of coffee cherry extract). DDC is an enzyme 0605 FIG. 16 (PTGS2: Prostaglandin-Endoperoxide implicated in two metabolic pathways (biosynthesis for bio Synthase 2) shows the change in relative expression of amines and catecholamines), synthesizing two important PTGS2 with increasing concentrations of coffee cherry neurotransmitters: dopamine and serotonin. A polymorphism extract and chlorogenic acid (a component of coffee cherry in tyrosine hydroxylase (TH; OMIM 191290), the rate-limit extract). A major mechanism for the regulation of prostaglan ing enzyme in the synthesis of catecholamines, is associated din synthesis occurs at the level of cyclooxygenase, also with variation in human longevity. The ability to increase known as prostaglandin-endoperoxide synthase. PTGS1 is expression with coffee cherry illustrated in this figure pro involved in production of prostaglandins for cellular house vides a method for altering the production of neurotransmit keeping functions, whereas PTGS2 is associated with bio ters and may be exploited in the treatment of depression, logic events such as injury, inflammation, and proliferation. Parkinson's disease, lifespan extension and a host of other PTGS2 encodes the pro-inflammatory cyclooxygenase 2 clinical diseases and metabolic functions associated with enzyme believed to be the rate-limiting step in the synthesis aging. of prostaglandins, important mediators of inflammation. 0602 FIG. 13 (NOS2A; Nitric Oxide Synthase 2A) shows Increasing amounts of coffee cherry extract induced an the change in relative expression of NOS2A with increasing increase in PTGS2 gene expression until a plateau was concentrations of coffee cherry extract and chlorogenic acid reached at the doses tested. In contrast, chlorogenic acid (a component of coffee cherry extract). Nitric oxide (NO) is a showed a more linear type of dose response that produced at US 2014/0079836A1 Mar. 20, 2014
higher concentrations a significant decrease in PTGS2 gene 0611 FIG. 23 shows the relative expression of select expression. Inflammation, especially chronic inflammation, genes in the telomere maintenance pathway after exposure to is associated with many diseases and directly or indirectly coffee cherry extract. Coffee cherry produces an increase in with reduced lifespan (or particularly reduced healthy TERT gene expression and a decrease in POT1, both of which lifespan), so the ability to significantly decrease the expres are associated with enhanced telomere maintenance and pos sion of PTGS2 as illustrated in this figure is an important anti sible increased longevity, whereas a decrease in TERF2 is inflammatory option. Also it is an option to decrease inflam contraindicated for this goal. There is an increase in POT1 mation without the use of steroids and their attendant adverse downregulation with higher dose or concentration but a non side effects, as chlorogenic acid may function partially as a linear response of TERT as concentration increases; however, non steroidal anti inflammatory compound that is derived both remain in a favorable gene expression pattern for from botanical sources. increasing longevity. TERF2 however at lower concentra 0606 FIG. 17 (IFI44 (a.k.a. p44); Interferon Induced Pro tions is unfavorable but changes to favorable for increasing tein 44) shows the change in relative expression of IFI44 with longevity at higher concentrations. TPP1 is variable. The increasing concentrations of coffee cherry extract and chlo POT1 (Protection Of Telomere) gene forms an important rogenic acid (a component of coffee cherry extract). IFI44 is POT1-TPP1 telomere complex which is a telomerase proces induced in the liver of chimpanzees infected with hepatitis C sivity complex. TPP1 expression is dose response variable. At or hepatitis D virus, but not in the liver of those infected with highest concentrations TERF2, POT1, TPP1 and TERT gene hepatitis B virus. Others have suggested that IFI44 induction expression all favor enhanced telomere maintenance and is the result of interferons produced in response to viral infec increased longevity. tion. IFI44 is inducible by interferon (IFN)-alpha (OMIM: 0612. It is also noted that KL (Klotho) expression 147660) and IFN-beta (OMIM: 147640), but not by IFN increases with increasing levels of coffee cherry (similar to gamma. Chlorogenic acid may significantly decrease expres TERT), and as with TERT, more KL is good for longevity and sion and reduce interferon production whereas coffee cherry healthy lifespan. may increase expression thus allowing the ability to modulate 0613 FIG. 24 shows the relative expression of the up or down the gene expression of this interferon. PARP1-4 genes after exposure to coffee cherry extract. 0607 FIG. 18. Relative expression of SIRT1-4 in human Among other things, PARP activates signalling to release skin fibroblasts 24 hours after exposure to coffee cherry Apoptosis Inducing Factor (AIF) from mitochondria result extract. The SIRT genes code for proteins which are enzymes ing in caspase independent pathways for apoptosis/pro which deacetylate proteins that contribute to cellular regula grammed cell death and may have a role related to DNA tion Such as reaction to stressors or regulating longevity. repair and PARG gene function. Members of the PARP fam Coffee cherry down regulates SIRT1 and SIRT4 at all the ily typically interact with each other. Decreased expression of tested concentrations but can either down or up regulate PARP genes may be beneficial in extending cell lifespan expression of SIRT2 and SIRT3 depending on the concentra which is of value for healthy cells, but in contrast for diseased tion. or cancerous cells the ability to increase PARP expression and 0608. The genes from Examples 15 and 16 were also bro promote apoptosis for the more rapid death of these unhealthy ken out into various Smaller sets for comparative analysis: cells may also be desirable. Thus, modulation either to 0609 FIG. 21 shows the relative expression of select decrease or increase PARP expression can be useful for over genes in the mitochondrial function/biogenesis pathway after all longevity of a tissue, organ or organism. exposure to chlorogenic acid (FIG. 21a) or coffee cherry 0614 FIG.25 is a graph illustrating the relative expression extract (FIG. 21b). The response for these genes involved in of specific genes in human skin fibroblasts 24 hours after mitochondrial pathways and biogenesis are essentially oppo exposure to chlorogenic acid which demonstrate a classic bell site for coffee cherry (which primarily increases gene expres shaped pattern for dose response that indicates a single direc sion) and chlorogenic acid (which primarily decreases gene tional change and then return to baseline after a peak expres expression). Not only are the directions of the change in gene sion level. As the doses increase, the gene response either expression essentially opposite, but in examining individual increases or decreases until a peak expression level is gene expression patterns at different doses or concentrations, reached. Beyond that dosage any increases in concentration the chlorogenic acid shows a consistent pattern of a bell of the compound gives "diminishing returns' or a lessening of shaped dose response curve with the greatest expression at the effect. This effect is either an upregulation or a downregu middle range of the tested doses while the coffee cherry show lation, not bidirectional. more variation in dose response and is nonlinear for many but 0615 FIG. 26 is a graph illustrating the relative expression not all the genes illustrated. of specific genes in human skin fibroblasts 24 hours after 0610 FIG. 22 shows the relative expression of select exposure to chlorogenic acid which demonstrate a classic bell genes in the DNA repair pathway after exposure to chloro shaped pattern for dose response that begins as a negative genic acid (FIG. 22a) or coffee cherry extract (FIG. 22b). expression value and as the dosage increases it passes through With the exception of TERT the coffee cherry overall pattern the Zero expression value and has an positive expression value is essentially one of decreasing gene expression at lower until a threshold dose is reached and then returns to the other concentrations but the amount of decrease becomes less or side of the axis similar to the starting dose. This is the first may even become positive increase in gene expression with type of bi-directional dose response noted. increasing dose. TERT shows a linear increase in gene 0616 FIG.27 is a graph illustrating the relative expression expression with increasing dose or concentration which is of specific genes in human skin fibroblasts 24 hours after favorable for DNA and telomere function repair. In contrast, exposure to chlorogenic acid which demonstrate a classic bell chlorogenic acid shows a nonlinear dose response for all the shaped pattern for dose response that begins as a positive illustrated genes and in the case of TERT actually decreases expression value and as the dosage increases it passes through gene expression which is opposite of the coffee cherry effect. the Zero expression value and has an negative expression US 2014/0079836A1 Mar. 20, 2014 69 value until a threshold dose is reached and then returns to the 0624. It is also believed that some of the effects observed other side of the axis similar to the starting dose. This is the are due to offsetting penalties between different genes, such second type of bi-directional dose response noted. that when the inducing compound concentration changes the 0.617 Additional general conclusions and connections can net effect on a gene goes from up to down, or down to up, be drawn based on the data provided by Examples 15 and 16. either as direct effect or some more distant effect in the 0618. In generally, coffee cherry seems to have a linear pathway. dose response curve, whether that is headed towards upregu 0625. It is also understood that there may be differential lation or downregulation. A few of the observed linear effects observed due to inherent differences between the chlo responses go from down to up, or up to down regulation, Such rogenic acid and coffee cherry extract used. For instance, the that opposite effects at observed different doses of the same cultured cells might preferentially absorb compounds from coffee cherry. The genes that show linear expression changes COFFEEBERRYR) extract, or might preferential absorb with dosage that are always induced (upregulated) or always Some complex of compounds from that mixed extract that are repressed (downregulated) are basic traditional drug dose missing from the purified chlorogenic acid preparation; there response curves—in general the higher the dose the greater even may be a synergistic impact from the mixed extract the response (though it is noted that side effects may or may preparation. In addition, the amount of chlorogenic acid used not mirror dose). However, there are also genes that show directly is considerably higher than the amount of chloro clearly responses that are not simply linear—and these high genic acid present in the coffee cherry extract; as Such, the light that it can be very important to carefully regulate the enriched chlorogenic acid may be trigger effects that are only dosage of the lifespan influencing agent. seen at levels well beyond the levels of coffee cherry extract 0619. Overall, chlorogenic acid also has a very consistent assayed here. pattern in that there are almost no linear responses to chang 0626. At the two most active concentrations, more thana ing concentration. The dosage response curves for chloro third of the assayed genes (using Array 1) show significant genic acid are essentially all bell shaped curves, either all responses. There is variation in good/bad with the overall above the baseline (so all dosages result in upregulation in a dose, as well as the magnitude of effects on specific genes bell-shaped response), or they are all below baseline (so all 0627. On the larger microarray (Array 2.0), with coffee dosages result in downregulation, but in a bell-shaped cherry extract, more significant changes were observed at the response), though some straddle the baseline and like the highest concentrations, though there are similar changes at all coffee cherry abovego from up to down regulation or downto three concentrations (though the magnitude is different and, up. There are only a few genes for which the dosage response as noted, a few genes change from up to down or down to up). to chlorogenic acid is linear. This is a startling result. It appears the effects on gene expression generally increase in 0620. By (generally) comparing the curves seen with chlo whatever direction they were headed with higher dosages of rogenic acid versus those from coffee cherry, it is apparent coffee cherry. Included in this is that gene expression that is that something is quite different is occurring in the coffee becoming less good gets more so as the concentration cherry. The chlorogenic acid appears in many cases not to be increases. Again, this highlights that the dosage is particularly the dominant effect on gene expression. However, there are important. cases where the chlorogenic is the dominant effect. Of special interest is that as the concentration/dose changes, the Example 17 response balance shifts and sometimes the chlorogenic acid 0628 Dosage Analysis in Human Tissue Samples effect alone is altered to an often opposite effect compared 0629. This example provides representative methods that to the coffee cherry (see, e.g., FIGS. 11-13, 15, for instance). can be used to analyze the effects of different dosages of 0621. In general, with chlorogenic acid, there is overall lifespan influencing compounds in a human test system. more activity at the 0.000005% and the 0.0005%, thus high 0630. In a first embodiment, a formulation containing the lighting a beneficial dose—and more generally, that less in test compound (e.g., a composition comprising one or more this case may be more beneficial (or at least more effective) antioxidant compounds) is applied topically in a serum for than more. instance twice daily in the AM and PM to skin (such as facial 0622. Without intending to be limited to any one explana skin). Optionally, different subjects in the study are given tion for what is observed in these dosage response analyses, different dosages of the test compound, and/or different dos some of the observed effects are likely to be antioxidants that age regimens. This elected regimen is followed for 12 weeks, behave as pro-oxidants under certain circumstances, such as for instance. No other changes in skin care routine are low or high concentrations. In addition, in Some instances allowed, though daily use of SPF 30 Zinc oxide sunscreen is chlorogenic acid (alone, or as a component of the coffee optionally required to enable clear differentiation and recog cherry extract) or another component of the coffee cherry nition of the impact of the text compound. extract may be being converted into other related chemical 0631 Biopsies of the subjects skin are taken using a 3.0 (s)—either by a component of the test biological system, or mm punch at pre treatment (to obtain an initiation baseline) through equilibrium interconversions (which can be influ and also at 12 weeks after commencing treatment. Biopsies enced strongly by relative concentration). There could also be are taken on the upper cheek area both pre and post treatment. other chemical reactions going on. Thus, what is observed is A third biopsy is taken at the baseline pretreatment visit from the end result of impact on a gene (or set of genes), including behind the ear in a non Sun exposed area. The biopsies are any actions that take place somewhere else upstream in a then analyzed to determine changes in gene expression, for pathway that impacts the specific gene being assayed. instance using one of the custom microarrays described 0623. In some instance, receptor sites blocked may be herein. By comparing the different biopsy samples, one can blocked or competitively inhibited (or stimulated) by one or assess changes in gene expression that result from the test the other of chlorogenic acid or a component in the coffee compound therapy, as well as changes from environmental/ cherry extract—which can result in complex interactions. UV light damage (by comparing light exposed to unexposed US 2014/0079836A1 Mar. 20, 2014 70 skin). With multiple subjects to which different dosages of a so severely injured that they will proceed to undergo apopto test compound are applied, dosage response curves can be sis and Subsequently these cells will die. generated and optimized dosages determined. 0636. The cells which have been exposed to a sufficient 0632. In a second embodiment a formulation containing a amount of the coffee cherry extractor modulating compounds test compound is taken orally once daily in the AM before will have DNA damage either prevented or repaired by meal for 24 weeks. Prior to initiating treatment baseline blood mechanisms discussed previously. One or more of the telom samples and skin biopsies are taken for analysis with a ere maintenance genes will have their activity modulated to focused microarray, Such as one of the custom microarrays protect and defend or even repair the structural integrity of the provided herein. These samples are repeated at 24 weeks and telomere on DNA within the nucleus and/or the mitochon also analyzed with the same methodology. By comparing drial DNA. The gene expression changes produced by the UV gene expression patters from the different samples, one can injury may be neutralized or countermanded or alternative assess changes in gene expression that result from the test repair pathways as described earlier may also be activated or compound therapy. With multiple subjects to which different a combination of both activities. dosages of a test compound are applied, dosage response 0637 ROS within the mitochondria may also be neutral curves can be generated and optimized dosages determined. ized or diminished in activity so that the cell injury is either 0633 With these and similar methods (an optionally in prevented or diminished or repaired. In particular hydroxyl combination with or following cell-based microarray analy radicals, hydrogen peroxide and reactive nitrogen species ses), one can characterize the biological effect and effective may be so affected. ness of for instance different plant preparations (peel vs. bean 0638. The mitochondrial DNA polymerase enzyme Pol or seed VS. pulp VS. stem vs. bark VS. leaves and so forth), gamma may have its expression level modulated to facilitate preparations form different plants (such as plants listed repair to mitochondrial DNA. herein), various concentrations or mixtures or methods of 0639. The modulating compounds may also signal for the preparing plant extracts, specific components from naturally biogenesis and production of new mitochondria as well as occurring extracts, and so forth. improving or protecting the respiratory efficiency of the mito chondria by quenching ROS. Example 18 0640 The apoptosis process which was being initiated may also be stopped thus preventing cell death. Mechanism of Action for the Application of 0641 Thus the lifespan of various cells types and specific Sufficient Quantities of Idebenone to Alter the cells within the skin may have their lifespan prolonged by Longevity of Cells protecting the telomere structure or by preventing oxidative 0634 Cells under oxidative stress have a tendency to stress damage by the ROS or even by preventing cell death via “stall electrons around Complex I in the electron transport apoptosis. The functional capacity or efficiency of the mito system which in turn causes damage to the cell. If the electron chondria may also be improved either directly or indirectly transport system cannot move the electrons past Complex I, a through increasing the actual number of mitochondria. Mito feedback loop of further ROS generation may occur causing chondrial biogenesis or increase in number of mitochondria further damage. One of the mechanisms of action of the may be produced when the modulating compound activates idebenone compound and its electron derivatives which or increases the activity or expression level of genes which transfer electrons is the ability to take the electrons and to increase mitochondrial numbers such as the gene PGC-1al bypass Complex I transferring them into Complex III further pha. “downstream” and eliminating the tendency for a “bottle 0642 Alternate pathways exist for maintaining the telom neck” at Complex I and an increase in ROS production. The ere structure and these may be activated instead of or in circumnavigation of Complex I increases the efficiency of the addition to the traditional maintenance pathways. mitochondrial respiration and decreases the production/accu 0643. The net effect of these various pathways and mecha mulation of ROS and modulating the cell toward an increased nisms of action by the modulating compounds is to allow the lifespan. structure and function of the skin to maintain a healthier and younger State which in turn allows the skinto maintain a more Example 19 youthful appearance and delay or minimize premature UV photoaging which is typically manifest by the appearance of Use of a Modulating Compound in a Topical fine lines, wrinkles, uneven pigmentation, loss of skin radi Composition for Anti-Aging ance, loss of skin elasticity and tone, skin sagging, reduced 0635 A stable topical cream formulation containing a set blood circulation and often slower wound healing as well as amount of coffee cherry extract (e.g., 0.05%, 0.1%, 0.15%, various other signs of premature photoaging. Thus, the topi 0.2%, 0.25%, 0.3%, 0.5%, 0.75%, 1%, 2%) may be applied cal formulation helps maintain the youthful appearance and twice daily to the skin of the face, chest, forearms and hands functions as an anti-aging topical formulation. which are exposed to UV sunlight. The active modulating compounds penetrate the outer epidermal layer of the skin Example 20 where keratinocytes reside and enter the dermal layer where fibroblasts and many other skin cells reside and the UV light Combination of Modulating Agents with a Sunscreen is absorbed into these cells. The cells are environmentally Formulation into a Topical Skin Care Lotion injured by UV light to which these skin areas are exposed. 0644. A formulation of 0.1% idebenone in combination Some of the cells are mildly to moderately injured and various with a physical Sunscreen Zinc oxide and the coffee cherry degrees of direct DNA damage is produced in these cells as extract acids at 0.05% in a stable topical lotion is applied to well as increase in ROS in the cell as well as mitochondrial the skin prior to engaging in outdoor sports activities on a and membrane injury. Some of the cells are sunburned and bright Sunny day. This formulation is used once to twice daily US 2014/0079836A1 Mar. 20, 2014
on an regular basis to allow the accumulation of the modu caloric restriction pathways which are known to extend lating agents into the skin in a more or less steady state or lifespan as well as the modulating compound effects are both reservoir effect. The acute sun exposure and activities allow a activated in such as way as to further extend the lifespan of the certain portion of the UV light of all wavelengths to enter the pet than would have occurred using only resveratrol to skin. The modulating compounds help protect and defend the Supplement the pet's diet. skin cells from the UV light injury from the environment. The gene expression changes describe in prior examples illustrate Example 22 that until the UV exposure occurs that these modulating agents have no effect on the expression levels of many genes The Use of Modulating Compounds to Enhance or and it is only after acute exposure to the UV light that various Improve the Efficacy of Cancer Chemotherapeutic gene expression modulations begin to occur setting in motion Agents the various protective and repair mechanisms within the skin 0648. A formulation of lifespan modulating compounds is cells that preserve normal healthy cell function and protect selected which produces inhibition of the telomere mainte and extend the lifespan of at least some of the cells relative to nance genes and it is administered in conjunction but not what would have occurred had the modulating agents not necessarily in direct combination with a chemotherapeutic been included in the skin care lotion. The response to the UV agent targeting the cancer cells. The ability of the cancerous light depends on the amount of UV light injury and also to cells to reverse apoptosis and acute cell death and/or the some degree to the proportion of UVB versus UVA1 light disruption of telomerase activity which helps to preserve or which contact the cells since the gene response and DNA immortalize the cancer cells creates a significantly higher damage pattern is different for these different wavelengths of death rate of the cancer cells thus improving the clinical result UV light. This mixture of compounds includes an antioxidant of the chemotherapy and also potentially increasing the prob idebenone which more specifically targets and protects mito ability of curing the cancer. chondria since it is a derivative of the naturally occurring 0649. Such a formulation may also be utilized to reduce ubiquinone which serve a vital role in mitochondrial function the amount or concentration of chemotherapeutic agent and electron transport. As described earlier idebenone is a needed to effectively treat the cancer thus reducing the risk of smaller molecule thus allowing better penetration into the the side effects and adverse events produced by the chemo skin and also it has more potent antioxidant activity than therapeutic agent. ubiquinone as well. 0650 Yet another possibility is to utilize a combination of 0645. The antioxidants in the coffee cherry extract and its lifespan modulating agents so that the above described events effects impact the mitochondria less specifically and target occur, but also so that healthy non cancerous cells can be various cellular pathways in the cell in general. Thus the additionally protected from lifespan shortening effects of the coffee cherry extract helps to quench a different mix of ROS chemotherapy or radiation. This is differential modulation and their pathways as well as having a different pattern of wherein the immortalized cancer cells lines are basically gene expression modulations for various protective and repair made more mortal and Susceptible to the cancertherapy while processes. The differential expression of genes between the the non cancerous cells have their ability to protect, defend idebenone and the coffee cherry extract are seen in prior and repair damage from the cancer therapy enhanced. examples. Also it is noteworthy that some of these gene expression patterns only show a modulation effect on gene Example 23 expression after the UV exposure occurs and thus show the ability of the modulating compounds to behave in a quiescent Use of Lifespan Modulating Agents to Protect or manner until oxidative stress or DNA damage or other cellu Extend the Lifespan of Acutely Injured Cells lar injury occurs. 0651 Idebenone 0.05% incorporated into an aerosol Example 21 inhaler may be used to treat acute pulmonary injuries. For example a fireman who is suffering from acute Smoke inha Use of Oral Supplements Containing the Modulating lation injury to his or her lungs or who has an acute injury Compounds to Extend the Lifespan of Companion from exposure to an environmental hazardous material Such as inhaling a toxic gas such as chlorine may repeatedly use an Animals Such as Cats and Dogs inhaler or nebulizer to help to prevent apoptosis and cell death 0646. A sufficient amount of idebenone and/or coffee of vital pulmonary tissues by modulating the gene expression cherry extract may be included in pet food for long term of cells which controls apoptosis. Continued use can help to ingestion or in pet vitamin or nutritional Supplement tablets or protect or repair telomere structure so that cells do not have other formulations. As the animals are exposed to various their lifespan shortened so that the lungs as an organ do not as environmental stresses, diseases, and oxidative stress various the patient ages Suffer premature aging and contribute or diverse cells within their tissues and organs will retain their directly cause reduced lifespan of the entire organism or mitochondrial respiratory efficiency and/or telomeric struc patient. The inflammatory pathways which if activated in ture longer than if they had not received the modulating eitheran acute or chronic manner may produce DNA damage, compounds. This is not the same process as caloric restriction mitochondrial inefficiency may also be modulated extending or the use of compounds which mimic caloric restriction but the lifespan of the cells and organ. Stimulation of the biogen they result in extension of what would otherwise have been esis of additional mitochondria may also help to offset the the lifespan of the pet and typically would extend the healthy cells which die thus extending the functional lifespan or the lifespan of the animal. efficiency of the Surviving cells. Another example is the expo 0647. Such a supplement or food containing these com Sure of a complex mix of toxic chemicals to rescue workers at pounds may also be combined with a caloric restriction the World Trade Center disaster of September 11 and the mimic such as resveratrol in sufficient amount so that both the development a few years later of severe disability from US 2014/0079836A1 Mar. 20, 2014 72 delayed onset of pulmonary fibrosis and other pulmonary naturally occurred thus delaying or preventing the onset of problems caused by the inhaled exposure which may have gray or white hair. Various mechanisms of action may occur been mitigated by the use of inhaled modulating agents. since the hairis Subjected to a variety of environmental insults and injuries ranging from UV light, hair care products, hair Example 24 dye processes, straightening processes, heat fromhair dryers, etc. These may contribute to damage to the DNA and telomere Extending the Lifespan of Plants maintenance genes or to the mitochondria or both. Either or 0652 Commercially poinsettias and orchids are among both the melanocytes and/or the stem cells may be affected. the major commercial pot plant and flower crops. It is useful Hair thinning or hair loss allows the scalp to have increased to have uniformity of plant size, flower color, and other physi UV exposure which also may accelerate premature aging and cal characteristics as well as flowering time and plant vigor or hair color change or loss of hair color. health so that a uniform crop may be produced and so that the plants may all be given the same culture. Cloning plants has Example 27 become a major commercial enterprise so that all the plants Organ Transplant Applications produced are identical copies of each other. These plants are originally generated from Sterile tissue culture in laboratories 0656 Organs being prepared for transport and subsequent and eventually the cells lines become senescent and the com transplantation may before, during or after or combination of mercial production may be halted or mutations may enter the these time periods be perfused with a solution containing cloning process which would produce deformed plants or coffee cherry extract alone or in combination with green tea flowers of no commercial value. Coffee cherry extracts in the polyphenols to modulate the gene expression for telomere coffee plant itself function to protect the plants from various structure maintenance and/or for modulating the gene expres environmental injuries and stress and disease injury. The tis sion which controls mitochondrial biogenesis. While there is Sue culture media for this plant cloning process may have one an important benefit for preventing or diminishing or delay or a combination of modulating agents incorporated into the ing the onset of apoptosis and viability of the cells and the media to help to extend the lifespan of the cell culture and thus organ itself thus prolonging the time available from organ allow longer commercial production of the cloned plant. harvest to transplantation as well as possibly improving the Reducing the possibility of DNA damage and mutations in ability of the organ to Survive transplant and/or Subsequent the plants is also a potential benefit. Another potential benefit anti rejection therapy, an important function is to modulate is to help to prevent or repair damage to the plants which the telomere structure maintenance and/or mitochondria bio might be produced by pesticides or fungicides which are used genesis. This may be particularly important when an organis to treat diseases in these plants once grown out of tissue being transplanted from an older donor into a significantly culture since flower deformities may result from DNA dam younger donor so that the lifespan of the organ itself is age to the plants. extended beyond what it would be if it were untreated. Example 25 Example 28 Use of Modulating Agent in Ophthalmic Eye Drop Extending the Lifespan of Autologous Grafts Solution 0657. Autologous human skin fibroblasts from tissue cell 0653. An ophthalmic preparation containing 0.01% coffee culture are injected into wrinkles and acne Scars on the face of cherry extract is utilized as a preventive therapy for cataracts. the person from which the skin cells were harvested weeks Cataracts are thought to be caused in part by environmental earlier and cultured in vitro before being returned to the damage such as UV light and/or free radical/ROS damage. person’s body. The cell culture had 0.005% idebenone added Protecting or repairing Such damage before permanent struc to their final culture transport media before being frozen for tural protein changes that lead to cataracts occur may delay transport to the doctors office for re-implantation into the the onset or even prevent cataracts. person’s face. The freeze/thaw cycle and transport as well as the injection process trauma injure or kill a percentage of Example 26 these autologous fibroblast cells. By utilizing the modulating compound the cell death rate due to apoptosis and also the Use of Modulating Compounds to Delay the Onset other injuries is reduced allowing a better transplant Success of Gray or White Hair rate. After injection the improved status of the mitochondria 0654 The graying or turning white hair is a sign of aging. allows better cell function in producing structural skin pro There is widespread variation in the age of onset of gray or teins such as collagen which in turn produces a greater reduc white hair. The pigment in hair is produced by pigment pro tion in the severity of wrinkles and/or acne scars. ducing cells called melanocytes. The melanocytes are replen ished by stem cells. It is believed that the death of melanocyte Example 29 stem cells associated with the hair follicle and hair bulge lead to the development of gray or white hair. A topical formula Demonstrating the Polyphenol Protective Effect on tion to apply to the scalp composed of 1.5% coffee cherry Telomerase Expression Versus UVB Radiation extract and 0.5% idebenone (with optional other ingredients) 0658. The antioxidant effect of polyphenols (found in may be utilized to extend the lifespan of either the melanocyte green tea and other sources) is well illustrated in the literature cells or the stem cells which produce new melanocyte cells. and shows impressive anti-sunburn cell capabilities. What 0655 By prolonging their lifespan the natural color of the has not been described is the effect addition of polyphenols to hair is preserved until an older age than would have otherwise UV irradiated cells has on the gene expression profiles of the US 2014/0079836A1 Mar. 20, 2014
cells. Specifically the cells in our experimentation (36 y.o. referenced herein), but with sets of genes that are defined human skin fibroblasts) have demonstrated a 1.6 fold increase based on the research described herein. Thus, additional in the gene responsible for telomerase activity in non irradi arrays are contemplated that contain at least some of the genes ated cells given the polyphenol compounds, and a 1.7 fold listed herein in DATA TABLE 7 and/or custom Array 2. increase in irradiated cells given polyphenols when compared 0662 Arrays can be manufactured using art-recognized to irradiated control cells. This demonstrates that either techniques, including for instance custom array services that through direct binding to gene promoter sites, or through are available commercially. second messenger systems in the cellular environment trig gered by the ROS reduction/antioxidant effect or some other Example 33 mechanism(s). These polyphenol compounds trigger the cell to produce more telomerase which protects the DNA and Use of Lifespan-Influencing Gene Arrays telomere structural integrity or telomere length resulting in a 0663 With the provision herein of specific life-span influ potentially increased lifespan for the cells. encing gene arrays (that is, sets of genes that can be used on arrays), as well as guidance for selecting genes from those Example 30 discussed herein to form additional arrays, there are now enabled myriad methods of using these arrays. Lifespan Regulating Compounds Demonstrate 0664 Merely by way of example, the arrays provided Increased Production of PARP1 and TERT Gene herein can be used to: characterize the lifespan influencing Signals in Cells Exposed to UVB Radiation characteristics of test compounds, experimental or known 0659 UVB radiation has been shown to affect a down drugs, extracts or enriched fractions thereof or individual regulation of TERT and PARP1 in cells so exposed, and this components found therein, specific concentrations of Such was evidenced in the experiments contained in prior (applied to cells, tissues, organs, or whole organisms—from examples. In these experiments the cells exposed to UVB and which a genetic sample is then obtained for the array analy not treated with any lifespan modulating compounds demon sis); characterize the effects of any lifespan influencing Sub strated a reduction in the gene expression (and thus indicative stance (such as the compounds and compositions discussed of shortened/damaged lifespan) of TERT by 4.6 fold and in herein) on different cell types (e.g., keratinocytes, melano PARP1 (a gene involved in DNA repair and apoptosis) a 5.1 cytes, liver, cardiac cells, brain cells, muscle cells, cells from fold reduction. Cells of the same age and from the same cell blood, and so forth), different animals or other organisms, line, when exposed to various lifespan modulating com cells/tissues/organs/organisms of different ages than charac pounds (those tested in these experiments included, polyphe terized herein, cells/tissues/organs/organisms under specific nols, coffee cherry and idebenone) demonstrated an stresses (e.g., Smoking, hypoxia, infection or other disease, UPREGULATION of those same genes in some cases 11 fold injury, environmental toxin exposure, radiation exposure, dif for PARP1 and 12 fold for TERT when compared to UVB ferent nutritional regimens, undergoing treatments with exposed control cells (for the exact numbers and to view the known or experimental drugs, and so forth); characterize the similar effect on TERT on even 50 year old cells tested, see effects of lifespan influencing Substance(s) when applied via Example 6 above) indicating an increase in the lifespan and a different route than detailed herein; and so forth. greater repair mechanisms in action for the telomere length 0665 Also contemplated are uses of the arrays to analyze and associated structures. biological samples with regard to longevity/healthy longev ity/lifespan separate from the lifespan influencing composi Example 31 tions discussed herein. For instance, the provided arrays can be used to characterize changes in (longevity or lifespan Use of Modulating Compounds in Conjunction with related) gene expression due to aging (e.g., by testing samples Other Anti Aging Skin Care Products from Subjects of different ages, or from the same Subject at different times), environmental exposure (e.g., by testing 0660 A skin care cream containing 0.5% coffee cherry samples from Subjects exposed to known or Suspected toxins extract and 0.1% each of the antioxidants vitamin E, vitamin or other environmental conditions), chemical or radiation C. Superoxide dismutase, phloretin, kinetin, alpha lipoic acid, exposure, disease (including for instance acute or chronic coenzyme Q10, green tea and grape seed extracts, along with diseases, genetic diseases, infectious diseases, and so forth), appropriate other ingredients to produce a stable formulation, dietary or wellness programs (e.g., to evaluate the effective is applied once or twice daily to areas of (prematurely) aging ness of a selected program), and myriad other uses that will skin on the face, neck, chest, hands and other body areas to now be recognized in view of the teachings provide herein. achieve two primary benefits. The first benefit is to prevent or 0666. The provided arrays are also useful in epidemiology delay aging and the second benefit is to repair or reverse studies, for instance to look at disparities of health care, existing premature aging of skin cells and related tissues so differences in geography, people groups, diet, and so forth. that the longevity and vitality of these cells is extended and Assays of the expression of lifespan related genes can be used also the appearance of the skin is maintained in a more youth to test subjects periodically to determine (like an early warn ful state. ing system) if something may be going wrong in critical Example 32 lifespan-involved system (such as telomere maintenance, mitochondrial respiration or biogenesis, and so forth). The Manufacture of a Lifespan-Influencing Array arrays could be used as diagnostic tools as well. 0667 The actual methods of assaying the array are con 0661 While representative lifespan-influencing gene ventional, and one of ordinary skill in the art will understand arrays are described herein, other arrays can be constructed how to prepare and label nucleic acid molecules to be applied using art recognized techniques (such as those described or to “probe' the arrays.