(12) United States Patent (10) Patent No.: US 8,815,936 B2 Grant Et Al

USOO8815936B2

(12) United States Patent (10) Patent No.: US 8,815,936 B2 Grant et al. (45) Date of Patent: Aug. 26, 2014

(54) PHARMACEUTICAL FORMULATIONS OF 2006/0276393 A1 12/2006 Milburn et al. RESVERATROLAND METHODS OF USE 2006/0292099 A1 12/2006 Milburn et al. 2008, OO15218 A1 1/2008 Vazquez-Anon et al. THEREOF FORTREATING CELL 2009,0169585 A1* 7/2009 Sardi ...... 424/400 DSORDERS FOREIGN PATENT DOCUMENTS (75) Inventors: Ross Stewart Grant, Kellyville (AU); Nady Braidy, Riverwood (AU); Gilles JP 2001514661 A 9, 2001 WO 99.0381.6 A1 1, 1999 Guillemin, Mount Colah (AU); George WO OO12534 A2 3, 2000 Smythe, Leichhardt (AU) WO WOO1/3O336 5, 2001 WO O3072187 A2 9, 2003 (73) Assignee: Nad Life Pty Ltd, Thornleigh, New WO 2004.000302 A1 12/2003 WO 2004O16726 A2 2, 2004 South Wales (AU) WO 20060O8470 A2 1, 2006 WO WO 2006/OO.1982 1, 2006 (*) Notice: Subject to any disclaimer, the term of this WO 2006O72809 A2 T 2006 patent is extended or adjusted under 35 WO WO 2006/128009 11, 2006 U.S.C. 154(b) by 493 days. WO 2008091710 A2 T 2008 WO 2009108999 A1 9, 2009 (21) Appl. No.: 12/735,931 OTHER PUBLICATIONS (22) PCT Filed: Mar. 3, 2009 Yu et al. Constitutive Accumulation of CIS-Piceid in Transgenic (86). PCT No.: PCT/AU2O09/OOO255 Arabidopsis Overexpressing a Sorghum Stilbene Gene; Plant Cell Physiology, vol. 47, No. 7 (2006) pp. 1017-1021.* S371 (c)(1), Svoboda et al. Natural Phenolics in the Prevention of UV-Induced (2), (4) Date: Nov. 29, 2010 Skin Damage. A Review; Biomedical Papers, vol. 147, No. 2 (2003) pp. 137-145.* (87) PCT Pub. No.: WO2009/108999 Slominski et al. On the Role of Melatonin in Skin Physiology and Pathology; Endocrine, vol. 27, No. 2 (2005) pp. 137-148.* PCT Pub. Date: Sep. 11, 2009 Otto et al. Differential Behaviors Toward Ultraviolet A and B Radioation in Fibroblasts and Keratinocytes From Normal and DNA (65) Prior Publication Data Repair-Deficient Patients; Cancer Research, vol. 59(1999) pp. 1212 US 2011 FO110913 A1 May 12, 2011 1218.* Marambaued et al. Resveratrol Promotes Clearance of Alzheimer'S (30) Foreign Application Priority Data Disease Amyloid-Beta Peptides; The Journal of Biological Chemis try, vol. 280, No. 45 (2005) pp. 37377-37382.* Mar. 3, 2008 (AU) ...... 20O8901036 European Search Report for Application No. 09716592.2 dated Jan. 4, 2013. (51) Int. Cl. Athar et al., Resveratrol: A Review of Preclinical Studies for Human AOIN 43/38 (2006.01) Cancer Prevention, Toxicology and Applied Pharmacology, 2007. AOIN 43/16 (2006.01) pp. 274-283, vol. 224, ScienceDirect. AOIN 43/08 (2006.01) Ara et al., Protective Effect of Resveratrol Against Oxidative Stress in AOIN37/00 (2006.01) Cholestasis, Journal of Surgical Research, 2005, pp. 112-117, vol. AOIN3L/04 (2006.01) 127. C07C39/12 (2006.01) Anekonda, Thimmappa S. Resveratrol, A Boon for Treating Alzheimer's Disease? Brain Research Reviews, 2006, pp. 316-326, (52) U.S. Cl. ScienceDirect. USPC ...... 514/435: 514/458: 514/474: 514/578; 514/725; 568/729 (Continued) (58) Field of Classification Search USPC ...... 514/435, 458, 474, 578, 725; 568/729 Primary Examiner — Susan Hanley See application file for complete search history. Assistant Examiner — Paul Martin (74) Attorney, Agent, or Firm — TraskBritt, P.C. (56) References Cited (57) ABSTRACT U.S. PATENT DOCUMENTS Described is a composition for preventing or treating an oxi 6,008,260 A 12/1999 PeZZuto et al. dative stress related disease or condition in a subject. The 6,270,780 B1 8/2001 Carson et al. 6,414,037 B1 7/2002 Pezzuto et al. disease or condition is characterized by the presence of excess 6,790,869 B2 9, 2004 Ghai et al. oxidative compounds in the Subject, and the composition 6,878,751 B1 4/2005 Donnelly et al. includes a synergistic combination of therapeutically effec 7,026,518 B2 4/2006 Gokaraju et al. tive amounts of resveratrol to promote NAD" synthesis in the 2001/0033848 A1 10/2001 Jacobson et al. Subject; a chelating agent to reduce production of additional 2002fOO28852 A1 3, 2002 Ghai et al. 2002/0173472 A1 11/2002 Pezzuto et al. oxidative compounds in the Subject; and an antioxidant to 2006,0002914 A1 1/2006 Milbrandt et al. minimize the oxidative activity in the subject. 2006/0269616 A1 11/2006 Giampapa 2006/0270732 A1 11/2006 Giampapa 12 Claims, 21 Drawing Sheets US 8,815,936 B2 Page 2

(56) References Cited Bradford, Marion M., "A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Prin ciple of Protein-Dye Binding” Anal. Biochem (1976) 53: 452-458. OTHER PUBLICATIONS Cheung et al., “A Scintillation Proximity Assay for Poly(ADP Quincozes-Santos et al., Resveratrol Attenuates Oxidative-Induced ribose) Polymerase” Anal Biochem (2000) 282: 24-28. DNA damage in C6 Glioma Cells, NeuroToxicology, 2007, pp. 886 Farina et al.; "An Improved Synthesis of Resveratrol” Nat. Prod. Res. 891, vol. 28, ScienceDirect. (2006) 20: 247-52. Burkhardt et al., DNA Oxidatively Damaged by Chromium(III) and Goldberg et al.; “Direct Injection Gas Chromatographic Mass Spec HO, is Protected by the Antioxidants Melatonin, N-acetyl-N'- trometric Assay for trans-Resveratrol” Anal. Chem. (1994) 66:3959 formyl-5-methoxylcynuramine, Resveratrol and Uric Acid, The 63. International Journal of Biochemistry & Cell Biology, 2001, pp. Grant et al., “Murine Glial Cells Regenerate NAD. After Peroxide 775-783, vol. 33, Elsevier. Induced Depletion. Using Either Nicotinic Acid, Nicotinamide, or Quinolinic Acid as Substrates' J. Neurochem. (1998) 70(4): 1759 Database Medline Online US National Library of Medicine 1763. (NLM), Bethesda, MD, US, 2003, Morin et al., Evidence for Jeandet et al. “The Production of Resveratrol (3,5,4'- Resveratrol-Induced Preservation of Brain Mitochondria Functions trihydroxyStilbene) by Grape Berries in Different Developmental After Hypoxia-Reoxygenation, XP002688764. Database accession Stages” Am. J. Enol. Vitic. (1991) 42:41-46. No. NLMI5134379 (Abstract). Klaidman et al., "High-Performance Liquid Chromatography Analy Lagouge et al., Resveratrol Improves Mitochondrial Function and sis of Oxidized and Reduced Pyridine Dinucleotides in Specific Protects Against Metabolic Disease by Activating SIRT1 and PGC Brain Regions” Anal Biochem (1995) 225: 312-317. lo, Cell, 2006, pp. 1109-1122, vol. 127, Elsevier. Kumar, “Nano and Microparticles as Controlled Drug Delivery Devices' J Pharm Pharmaceut Sci (2000) 3 (2): 234-258. Belenky et al., NAD+ Metabolism in health and disease, Trends in Lamuela-Raventos, “Direct HPLC Analysis of cis- and trans Biochemical Sciences, 2006, pp. 12-19, vol. 32, No. 1, Resveratrol and Piceid Isomers in Spanish Red Vitis vinifera Wines' ScienceDirect. Agric. Food Chem (1995)(43): 281-283. PCT International Search Report, PCT/AU2009/000255, dated May Nisselbaum et al., “A Simple Ultramicro Method for Determination 25, 2009. of pyridine Nucleotides in Tissues' Anal Biochem (1969) 27: 212 Della-Morte et al., Resveratrol pretreatment protects rat brain from 217. cerebral ischemic damage via a sirtuin I-uncoupling protein 2 path Putt et al., “An enzymatic assay for poly(ADP-ribose) polymerase-1 way, Neuroscience, Mar. 31, 2009, pp. 993-1002, vol. 159, No. 3. (PARP-1) via the chemical quantitation of NAD+: application to the Raval et al., Resveratrol and ischemic preconditioning in the brain, high-throughput Screening of Small molecules as potential inhibi Abstract, Curr. Med. Chem., 2008, pp. 1545-1551, vol. 15, No. 15, tors’ Anal Biochem (2004) 326:78-86. abstract only. Putt et al., “Direct Quantitation of Poly(ADP-Ribose) Polymerase Howitz et al., (2003). "Small molecule activators of sirtuins extend (PARP) Activity as a Means to Distinguish Necrotic and Apoptotic Saccharomyces cerevisiae lifespan'Nature 425 (6954): pp. 191-196. Death in Cell and Tissue Samples' Chem. Bio. Chem (2005) 6: Wood et al., (2004). “Sirtuin activators mimic caloric restriction and 53-55. delay aging in metazoans' Nature 430 (7000): pp. 686-689. Sirtuin 1 obtained from http://en.wikipedia.org/wiki/Sirtuin Jang et al., (1997). “Cancer chemopreventive activity of resveratrol. 1Sirtuin 1 retrieved Dec. 17, 2013. a natural product derived from grapes' Science 275 (5297): pp. Wang et al., “An LC-MS Method for Analyzing Total Resveratrol in 218-220. GrapeJuice, Cranberry Juice and in Wine'JAgric Food Chem (2002) Sun et al., (Jun. 1, 2002). “Induced overexpression of mitochondrial 50: 431-435. Mn-Superoxide dismutase extends the life span of adult Drosophila International Preliminary Report on Patentability for PCT/AU2009/ melanogaster'. Genetics 161 (2): pp. 661-172. 000255 dated Jun. 6, 2010. Hu et al., (Mar. 2007). “Hippocampal long-term potentiation, Chapter II Demand for PCT/AU2009/000255 dated Nov. 16, 2009. memory, and longevity in mice that overexpress mitochondrial Response to Written Opinion for PCT/AU2009/000255 dated Feb. 2, Superoxide dismutase'. Neurobiol Learn Mem 87 (3): pp. 372-384. 2010. Wong GH (May 1995). "Protective roles of cytokines against radia Baxter et al., Anti-aging properties of resveratrol: Review and report tion: induction of mitochondrial MnSOD'. Biochim. Biophys. Acta of a potent new antioxidant skin care formulation, Journal of Cos 1271 (1): pp. 205-209. metic Dermatology, vol. 7 No. 1, Mar. 1, 2008, pp. 207, abstract only. Berofsky et al., “An Improved Cycling Assay for Nicotinamide Adenine Dinucleotide” Anal Biochem (1973) 53: 452-458. * cited by examiner U.S. Patent Aug. 26, 2014 Sheet 1 of 21 US 8,815,936 B2

Salvage Pathway

De Novo A. ." PBEF trampty stress Pathway frt inducted ryptophan----- NAMN FARPs recycling NuN - NR finia88.8 SirisC3 --- ARs. :

NA assessessessor N '-- RAt ce

sistasi protective

FIG. 1 (PRIOR ART) U.S. Patent Aug. 26, 2014 Sheet 2 of 21 US 8,815,936 B2

Tryptophan |Do N-Formyl kynurenine

Anthranilic acid Kynureninel kynurenic acid

3-OH Kynurenine

3-OH Anthranilicl acid

Picolinic acid Acetyl Co-A Quinolinic acid QPRT NAMNl NAD

FIG. 2 (PRIOR ART) U.S. Patent Aug. 26, 2014 Sheet 3 of 21 US 8,815,936 B2

Effect of IDO inhibition in the presence of L Tryp & Nic. A

NAD (nging protein)

Control 1-MT (100 M L-Tryp. (100 uM) Na (100 uM)

1-MT (100 uM)

FIG. 3 U.S. Patent Aug. 26, 2014 Sheet 4 of 21 US 8,815,936 B2

PARP-1 Activation in response to different concentration of Hydrogen Peroxide

2OOOO

o 1) 2O 5 H2O2 conc. (M)

FIG. 4

Effect of H2O2 O NAD

50 100 HO Conc. ()

F.G. 5 U.S. Patent Aug. 26, 2014 Sheet 5 of 21 US 8,815,936 B2

Effect of H2O2 on LDH release

350 300 250 200 150 100 50 O 10 50 100 1 OOO HO conc. (M)

FIG. 6

Effect of Quercetin of H2O2 mediated NAD depletion 6OOO -

3OOO -

2OOO -

Control DMSO (0.2%) H2O2 (10OuM) quercetin quercetin + H2O2 (100uM)

FIG. 7 U.S. Patent Aug. 26, 2014 Sheet 6 of 21 US 8,815,936 B2

3000 -

2500 -

2000 -

15OO

1000 -

Ctrl 20 uM Mel 100 uMH2O2 2 uM Mel 20 uM Mel 200 IM Mel

100 MH20 FIG. 8

3000 -

2500 -

Ctrl 2 M Vit E 100 MH202 2 puM Vit E 20 M Vit E 200 IM Vit E 100 MH2O2

FIG. 9 U.S. Patent Aug. 26, 2014 Sheet 7 of 21 US 8,815,936 B2

350

sk 3000 asool

2000 -

150

1 OOO -

500

Ctrl C CR re 1 M C Q i O M C C. 100 M C Q

Fe + H2O FIG. 10

Effect of RNG2 on H2O2 mediated NAD depletion

6OOO -

5OOO -

4OOO -

3OOO -

2OOO

1 OOO -

O - Control H2O2 (1OOuM) RNG2 RNG2 + H2O2 (1OOuM)

FIG. 11 U.S. Patent Aug. 26, 2014 Sheet 8 of 21 US 8,815,936 B2

Effect of Kynure nine Pathway inhibition on Resveratrol induced NAD biosynthesis in Primary cells 12 OOO – 1 OOOO - 8 OOO - 6000 - 4000 - 2OOO - O 1 2 3 4. 5 6

FIG. 12

Tannic Acid - Dose Response Curve

t -- O u O s S. an o % rom C s 2. m

1 O 1 OO conc. (uM) FIG. 13 U.S. Patent Aug. 26, 2014 Sheet 9 of 21 US 8,815,936 B2

4OOO - i g - 5 3000 - O g - 5, 2000

Y a 1000 - C 2. -- Q - Cont Tan ReS Res + Tan A

Effect of RNG2 on NMNAT Activity

OO 12

0.01

OOO8 2 9 Y E 0.006 H. E s 2SF 0.004 2

OOO2

O nO RNG2 RNG2 Treatment

FIG. 15 U.S. Patent Aug. 26, 2014 Sheet 10 of 21 US 8,815,936 B2

No ReSVeratrol 115

Resveratrol 50 um 13O

Resveratrol 100 um

Resveratrol 200 pum

FIG. 16

iryptophan

-1 -"------t --- ?o1. 1. Tryptophan Colinic acid Kynurenine Nica Quinolinic acid Nica -

\ / 3 DNA dam \\\ 1902 A Fe++ ha r Drug targets \ - / | N. N.

FIG. 17 (PRIOR ART) U.S. Patent Aug. 26, 2014 Sheet 11 of 21 US 8,815,936 B2

3500 X k 3000 - g e 2500 - ? O 2000 1500 S. 1000 C 2 500 O Ctrl 100 uM Ho, 10 uMCQ 20 MWitE 10 PMCQ 20 MWitE 10 uM Fe 10 uM Fe 100 uMHO 10 uM Fe

PAM CLO 2 sooo S 25000 9 : 3 9 -- 9, 20000 S SUV-B S 15000 UV-B g No U-VB E. 10000 a No UV-B 2a 2 5000 st : Control 0.1 M 1 M 10M 50M 1C0 M Control 0,1 M 1 M 10M 50M 100 M PAM PAM PAM PAM PAM CLO CLO CLO CO CLQ FIG. 19a F.G. 19b

MEL RES

UV-B it No UV-B

Control 0.1 M 1 M 10 AM 50 IM 100 M Control 0,1 M 1 AM 10 M 50 AM 100 M ME ME MEL MEL MEL RES RES RES RES RES FIG. 19C F.G. 19C U.S. Patent Aug. 26, 2014 Sheet 12 of 21 US 8,815,936 B2

g 140 Szoo s 20 9 c.100 as 150 E 80 UWB S 10 60 " S 40 A No UV-B S at 50 - 20 ? A - 0 - 0 Control 0.1 M 1 M 10M 50M 100M : Control 0.1M 1 M 10M 50 pill 100 M PAM PAM PAM PAM PAM CLO CLO CO CLO CLO

MEL 140

- asS. is 140 9 120 : i a . . . . 9 O 100 ; : S UV-B 52 80 a UV-B is s No UV-B S 40 a No UV-B s 2O ; : S S i. Control 0.1M 1 M 10 p.M 50 IM 100 M | Control 0.1M 1 AM 10M 50M 1COM ME ME ME ME MEL | RES RES RES RES RES FIG. 19g FIG. 19h U.S. Patent Aug. 26, 2014 Sheet 13 of 21 US 8,815,936 B2

5A, e, is for 3 sea list Cir.

** ||

$º

FG ... 20a

is Series 1

cr: Control RES50/MEL50 RES 10/CLO90 RES 90/PAM10 (UV) (UV) (UV)

FIG. 20b U.S. Patent Aug. 26, 2014 Sheet 14 of 21 US 8,815,936 B2

16000 14OOO 12000 1OOOO in 8000 6OOO 4OOO | 2OOO Control (UV) RES/MEL/CLO RES/MEL/PAM (UV) (UV) FIG.21

L -::::: E o 8 ix e FIG. 22b U.S. Patent Aug. 26, 2014 Sheet 15 of 21 US 8,815,936 B2

: is: i.

:... i. U.S. Patent Aug. 26, 2014 Sheet 16 of 21 US 8,815,936 B2

|wa |dwe |ovih |)quiárepuosas

egz'914

TETTERETTET |afieuwepwnasnaíonnae

U.S. Patent Aug. 26, 2014 Sheet 17 of 21 US 8,815,936 B2

5 go g gs g : 3 s 2

FIG. 23b U.S. Patent Aug. 26, 2014 Sheet 18 of 21 US 8,815,936 B2

80 - 70 50 - 40 | 30 20

---. ------. control (UV) PAM 100 CLO 100 RES 100 MEL100 (UV) (UV) (UV) (UV)

6 O

20 1.34.5OOOOO l --- w U.S. Patent Aug. 26, 2014 Sheet 19 of 21 US 8,815,936 B2

O Control (UV) RES/MEL/CLO (UV) RES/MEL/PAM (UV)

90 80 70 60

O 50 40 30 2O 10

Control (uw) MEL (uv) RES (uv)

FIG. 27 U.S. Patent Aug. 26, 2014 Sheet 20 of 21 US 8,815,936 B2

90 80 - 70 - 50 - 40 30 20 10 |

Control (uw) RES/MEL (uv) RES/CLO (uv) RES/PAM (uw)

120 100 8 O

6 O

4.O

2 O O - Control (uw) RES/MEL/CLC (uw) RES/MEL/PAM (uw) U.S. Patent Aug. 26, 2014 Sheet 21 of 21 US 8,815,936 B2

6OOO -

5000 -

4000

3000 -

2OOO -

1000 -

O H2O2 Melatonin Clioquinol RNG2

FIG. 30 US 8,815,936 B2 1. 2 PHARMACEUTICAL FORMULATIONS OF mediated by inflammatory changes associated with oxidative RESVERATROLAND METHODS OF USE stress and accelerated DNA damage. THEREOF FORTREATING CELL DNA strand breaks caused by oxidative damage require a DISORDERS rapid repair response which uses up the cells vital NAD" resource. While NAD" serves as a substrate for a number of TECHNICAL FIELD enzymes, the most significant contributor to rapid NAD" turnover and depletion is activation of Poly(ADP-ribose) The invention relates to methods and compositions for polymerase (PARP) enzyme family members, in particular inducing DNA repair and NAD" synthesis in a subject. The PARP-1. As mentioned previously, PARP-1 is a DNA binding invention further relates to methods and pharmaceutical com 10 enzyme activated by double or single stranded breaks to the positions for the prevention and treatment of conditions and DNA and is critical to the base excision repair (BER) process. diseases associated with oxidative stress and/or DNA dam Although many proteins are involved in repairing DNA dam age. age the majority of DNA lesions are repaired by BER. 15 Accordingly, NAD" plays a central role in DNA repair and BACKGROUND ART intracellular levels are rapidly reduced during oxidative stress. Improving antioxidant capacity and DNA repair is Nicotinamide adenine dinucleotide (NAD") is the parent therefore a mechanism by which cell viability may be pro compound of the pyridine nucleotide family of coenzymes moted and retained. (NADH, NADP, NADPH) that act as essential cofactors and It is clear that maintaining NAD" levels within both the electron transporters in a number of metabolic processes cytoplasm and nucleus is vital to Sustaining nuclear integrity, including alcohol, lactate and amino acid metabolism and cell viability and growth. Accordingly, there is a general need energy (ATP) production. NAD" is an essential substrate for for treatments capable of increasing cellular levels of NAD" a number of important NAD-dependent enzymes including in circumstances where NAD" is depleted. Therapies for the Poly(ADP-ribose) polymerase (PARP), Sir2-homolog 25 treatment of oxidative stress have largely focused on the (SIRT1) and NAD glycohydrolase (CD38"). PARP is a prevention of free radical production (chelation therapy) or nuclear enzyme which mediates repair of DNA double or the reduction of molecular damage (antioxidant therapy). single strand breaks, while the NAD-dependent deacetylase Such treatments do not provide a means of alleviating DNA SIRT1 affects gene silencing and cellular longevity. NAD damage caused by ROS, and thus have limited potential in glycohydrolase catalyzes the production of cyclic ADP-ri 30 reducing cell death and deregulated cell growth (i.e. tumour bose (cADPR) from NAD" and affects intracellular calcium signalling. The role of NAD" in these and other cellular proliferation). Hence, there is a need for treatments capable of functions suggest that in addition to being a regulator of enhancing DNA repair in diseases and conditions associated metabolic activity, NAD" plays a central role in the control of with chronic or acute oxidative stress. fundamental cellular processes. 35 Maintenance of NAD" levels within both the cytoplasm DISCLOSURE OF INVENTION and nucleus is therefore vital to Sustaining nuclear integrity, cell viability and growth. Reduced levels of cellular NAD" In one aspect, the invention provides a method of inducing consistently correlate with death in a number of cell types, NAD" synthesis in a subject, said method comprising admin and are prevalent in degenerative disorders associated with 40 istering to the subject a therapeutically effective amount of oxidative stress. Oxidative stress is characterised by the pres resveratrol or a functionally equivalent analogue orderivative ence of excess oxidative compounds that may induce oxida thereof. In one embodiment of the first aspect, the induction tive stress and/or damage, for example, reactive oxygen spe NAD" synthesis increases the activity of Poly(ADP-ribose) cies (ROS), superoxide radical, hydroxyl radical, nitric oxide, polymerase (PARP) enzymes. In one embodiment of the first oZone, thiyl radicals, and carbon-centred radicals (e.g., 45 aspect the PARP enzyme is PARP-1 or PARP-2. trichloromethyl radical). ROS such as HO. O. , OH and In one embodiment of the first aspect, the induction of NO, have detrimental effects including inactivation of spe NAD" synthesis increases the activity of sirtuin enzymes. In cific enzymes via oxidation of their co-factors, oxidation of one embodiment of the first aspect, the sirtuin enzyme is polydesaturated fatty acids in lipids, oxidation of amino acids selected from the group consisting of SIRT1, SIRT2, SIRT3, within proteins and DNA damage. Oxygen free radical activ 50 SIRT4, SIRT5, SIRT6 and SIRT7. ity is responsible for several important molecular cascades In a second aspect, the invention provides a method of that underlie a number of pathologic processes including preventing or treating a disease or condition associated with neurodegeneration, ischemia-reperfusion injury, atheroscle oxidative stress, said method comprising administering to the rosis, inflammation, DNA damage in skin cells (e.g. kerati subject a therapeutically effective amount of resveratrol or a nocytes and fibroblasts) and potentially tumor generation 55 functionally equivalent analogue or derivative thereof, through deregulated cell signaling following DNA damage. wherein said administration induces NAD" synthesis in the Increased ROS production is known to result from expo Subject. Sure to U.V. or ionising radiation (X-ray, Y-rays), chemical In a third aspect, the invention provides a method of pre agents, infection, inflammation or reduced mitochondrial venting or treating a disease or condition associated with efficiency. Any one or combination of these factors may be 60 DNA damage, said method comprising administering to the prevalent in chronic degenerative states such as normal cel subject a therapeutically effective amount of resveratrol or a lular aging, accelerated aging of the skin, Alzheimer's disease functionally equivalent analogue or derivative thereof. and Parkinson's disease, as well as chronic or acute UV In one embodiment of the second and third aspects, the induced damage in skin cells. Alzheimer's and Parkinson's disease or condition is a neurodegenerative disorder. In one diseases are examples of neurodegenerative disorders char 65 embodiment of the second and third aspects, the neurodegen acterised by progressive loss of neuronal cells. The primary erative disorder is Alzheimer's disease or Parkinson's dis cause of brain cell death is not known but appears to be CaSC. US 8,815,936 B2 3 4 In one embodiment of the second and third aspects, the carotene, retinol, catechins, epicatechins, epigallocatechin-3- disease or condition is associated with accelerated aging of gallate, flavenoids, L-ergothioneine, idebenone and sele the skin. nium. In one embodiment of the second and third aspects, the In one embodiment of the seventh aspect, the chelating disease or condition is UV induced DNA damage in skin cells agent is selected from the group consisting of ethylenedi and the skin cells may include keratinocytes and fibroblasts. aminetetraacetic acid (EDTA), Ethylenediamine tetraacetic In one embodiment of the second and third aspect, the acid (calcium disodium versante) (CaNa-EDTA), Ethylene resveratrol or a functionally equivalent analogue orderivative glycol tetraacetic acid (EGTA), dimercaptosuccinic acid thereof is administered as a triple therapy in a therapeutically (DMSA), Alpha lipoic acid (ALA), 2,3-dimercapto-1-pro effective amount. 10 panesulfonic acid (DMPS), Dimercaprol (BAL), Deferoxam In one embodiment of the second and third aspects the ine, D-penicillamine, dimercaprol, Aminophenoxyethane disease or condition associated with DNA damage is cancer. In one embodiment of the second and third aspects, the tetraacetic acid (BAPTA) Defarasirox, Diethylene triamine disease or condition results from exposure to ultra-violet pentaacetic acid (DTPA), 2-pyridinecarboxylic acid (pi light, ionising radiation, exposure to chemical agents, infec 15 colinic acid), 2.3-pyridinedicarboxylic acid (quinolinic acid), tion, inflammation, reduced mitochondrial efficiency or com 2-aminobenzoic acid (anthranilic acid), kynurenic acid, Xan binations thereof. thurenic acid and 8-hydroxyquinoline (and functional deriva In one embodiment of the first, second and third aspects, tives thereof). the resveratrol or functionally equivalent analogue or deriva In an eighth aspect, the invention provides a method for the tive is a trans-isomer. treatment of a disease or condition associated with oxidative In one embodiment of the first, second and third aspects stress said method comprising administering to the Subject a method according to any one the functionally equivalentana therapeutically effective amount of a pharmaceutical compo logue of resveratrol is selected from the group consisting of sition comprising a synergistic combination of at least one hydroxylated resveratrol analogues, methoxylated resvera agent capable of inducing NAD" synthesis, and an effective trol analogues, cis-resveratrol glucoside (cis-piceid) and 25 amount of one or both of trans-resveratrol-3-O-B-glucoside (trans-piceid). (a) at least one antioxidant In a fourth aspect, the invention provides a pharmaceutical (b) at least one chelating agent composition when used for increasing NAD" synthesis com In a ninth aspect, the invention provides a method for the prising a therapeutically effective amount of resveratrol or a treatment of a disease or condition associated with DNA functionally equivalent analogue or derivative thereof. 30 damage said method comprising administering to the Subject In a fifth aspect, the invention provides pharmaceutical a therapeutically effective amount of a pharmaceutical com composition when used for the prevention or treatment of a position comprising a synergistic combination of at least one disease or condition associated with oxidative stress compris agent capable of inducing NAD" synthesis, and an effective ing a therapeutically effective amount of resveratrol or a amount of one or both of functionally equivalent analogue or derivative thereof, 35 wherein said composition induces NAD" synthesis in the (a) at least one antioxidant Subject. (b) at least one chelating agent In a sixth aspect, the invention provides pharmaceutical In one embodiment of the eighth aspect, the disease or composition when used for the prevention or treatment of a condition is UV induced DNA damage in skin cells and the disease or condition associated with DNA damage compris 40 skin cells may include keratinocytes and fibroblasts. ing a therapeutically effective amount of resveratrol or a In one embodiment of the eighth aspect, the resveratrol or functionally equivalent analogue or derivative thereof, a functionally equivalent analogue or derivative thereof is wherein said composition induces NAD" synthesis in the administered as a triple therapy in a therapeutically effective Subject. amount. In one embodiment of the sixth aspect, the disease or 45 In one embodiment of the eighth and ninth aspects, the condition is UV induced DNA damage in skin cells and the least one agent capable of inducing NAD" synthesis is res skin cells may include keratinocytes and fibroblasts. Veratrol or a functionally equivalent analogue or derivative In one embodiment of the sixth aspect, the resveratrol or a thereof. functionally equivalent analogue or derivative thereof is In one embodiment of the eighth and ninth aspects, the administered as a triple therapy in a therapeutically effective 50 antioxidant is selected from the group consisting of melato amount. nin, Vitamin E. Vitamin C, methionine, taurine, Superoxide In a seventh aspect, the invention provides pharmaceutical dismutase (SOD), catalase (CAT), and glutathione peroxi composition when used for the prevention or treatment of a dase (GPX), L-ergothioneine N-Acetyl Cysteine (NAC), disease or condition associated with oxidative stress, said Vitamin A, beta-carotene, retinol, catechins, epicatechins, composition comprising a synergistic combination of at least 55 epigallocatechin-3-gallate, flavenoids, L-ergothioneine, ide one agent capable of inducing NAD" synthesis, and an effec benone and selenium. tive amount of one or both of In one embodiment of the eighth and ninth aspects, the (a) at least one antioxidant chelating agent is selected from the group consisting of eth (b) at least one chelating agent ylenediaminetetraacetic acid (EDTA), Ethylenediamine tet In one embodiment of the seventh aspect, at least one agent 60 raacetic acid (calcium disodium versante) (CaNa-EDTA), capable of inducing NAD" synthesis is resveratrol or a func Ethylene glycol tetraacetic acid (EGTA), dimercaptosuccinic tionally equivalent analogue or derivative thereof. acid (DMSA), Alpha lipoic acid (ALA), 2,3-dimercapto-1- In one embodiment of the seventh aspect, the antioxidantis propanesulfonic acid (DMPS), Dimercaprol (BAL), Defer selected from the group consisting of melatonin, vitamin E, oxamine, D-penicillamine, dimercaprol, Aminophenoxy Vitamin C, methionine, taurine, Superoxide dismutase 65 ethane-tetraacetic acid (BAPTA) Defarasirox, Diethylene (SOD), catalase (CAT), and glutathione peroxidase (GPX), triamine pentaacetic acid (DTPA) 2-pyridinecarboxylic acid L-ergothioneine N-Acetyl Cysteine (NAC), vitamin A, beta (picolinic acid), 2.3-pyridinedicarboxylic acid (quinolinic US 8,815,936 B2 5 6 acid), 2-aminobenzoic acid (anthranilic acid), kynurenic As used herein, the term “oxidative stress' is used in gen acid, Xanthurenic acid and 8-hydroxyquinoline (and func eral context and refers to enhanced generation of free radicals tional derivatives thereof). or reactive oxygen species (ROS) (such as C-hydroxy ethyl In a tenth aspect, the invention provides a kit for increasing radical, hydrogen peroxide, peroxy radical, hydroxy radical, NAD" synthesis in a subject comprising resveratrol or a func and Superoxide radical) and/or a depletion in antioxidant tionally equivalent analogue or derivative thereof. defense system causing an imbalance between prooxidants In an eleventh aspect, the invention provides a kit for treat and antioxidants. In general, oxidative stress involves the ing a disease or condition associated with oxidative stress in accumulation of free-radicals within the cell or the cell envi a subject comprising resveratrol or a functionally equivalent ronment which may result in oxidative damage. Oxidative analogue or derivative thereof. 10 stress may arise from biotic (living) and abiotic (non-living) In a twelfth aspect, the invention provides a kit for treating Sources, for example, exposure to U.V. or ionising radiation a disease or condition associated with DNA damage in a or chemical agents, infection by different infectious agents, Subject comprising resveratrol or a functionally equivalent inflammation or reduced mitochondrial efficiency analogue or derivative thereof. 15 BRIEF DESCRIPTION OF THE DRAWINGS In one embodiment of the twelfth aspect, the disease or condition is UV induced DNA damage in skin cells and the A preferred embodiment of the present invention will now skin cells may include keratinocytes and fibroblasts. be described, by way of an example only, with reference to the In one embodiment of the twelfth aspect, the resveratrol or accompanying drawings wherein: a functionally equivalent analogue or derivative thereof is FIG. 1 is a diagram showing the synthesis of NAD" via the administered as a triple therapy in a therapeutically effective salvage pathway from nicotinamide (NAM) or Nicotinic acid amount. (NA): In one embodiment of the tenth, eleventh and twelfth FIG. 2 is a diagram showing the synthesis of NAD" via the aspects, the resveratrol or functionally equivalent analogue or de novo pathway from tryptophan; derivative is a trans-isomer. 25 FIG. 3 is a graph showing the effect of the indoleamine In one embodiment of the tenth, eleventh and twelfth 2,3-dioxygenase (IDO) inhibitor 1-MT on intracellular aspects, the functionally equivalent analogue is selected from NAD" levels in cultured human foetal astrocytes. Human the group consisting of hydroxylated resveratrol analogues, foetal astrocytes were treated with 1-MT alone, or combina methoxylated resveratrol analogues, cis-resveratrol gluco tions of 1-MT/L-tryptophan (1-Tryp) and 1-MT/nicotinic side (cis-piceid) and trans-resveratrol-3-O-B-glucoside 30 acid (Nica). *p-0.05 compared to control, **p<0.05 com (trans-piceid). pared to 1-MT treatment alone, ****p<0.05 compared to Definitions L-tryp+1-MT treated cells; As used in this application, the singular form 'a', 'an' and FIG. 4 is a graph showing the effect of different concen “the include plural references unless the context clearly trations of hydrogen peroxide (HO) on PARP activity in dictates otherwise. For example, the term “a stem cell also 35 cultured human foetal astrocytes. p-0.05 compared to no includes a plurality of stem cells. H.O.;p-0.05 compared to 10 uMH.O.; *p<0.05 compared As used herein, the term "comprising means “including.” to 20 uM H.O.;p-0.05 compared to 50 MHO: Variations of the word “comprising, such as “comprise' and FIG. 5 is a graph showing the effect of different concen “comprises have correspondingly varied meanings. Thus, trations of hydrogen peroxide (H2O) on intracellular NAD" for example, a polynucleotide “comprising a sequence 40 in cultured human foetal astrocytes. *p-0.05 compared to 0 encoding a protein may consist exclusively of that sequence uM HO control, **p<0.05 compared to 50 uM H.O., or may include one or more additional sequences. ***p<0.05 compared to 100 MHO: As used herein, the term “resveratrol encompasses either FIG. 6 is a graph showing lactate dehydrogenase (LDH) the cis-isomer of resveratrol, the trans-isomer of resveratrol, release in cultured human foetal astrocytes following expo or a mixture of the two isomers. The term encompasses both 45 Sure to different concentrations of hydrogen peroxide (H2O). the naturally occurring and chemically synthesized active *p-0.05 compared to 0 uM HO control, **p<0.05 com agent and the compound as it may be in the laboratory. Fur pared to 10 uMHO, ***p<0.05 compared to 50 uMHO, ther, when the term “resveratrol' is used herein, it is intended up <0.05 compared to 100DuM HO: to encompass pharmacologically acceptable salts, esters, FIG. 7 is a graph showing the effect of quercetin on HO amides, prodrugs and derivatives and analogues of resvera 50 mediated NAD" depletion in cultured human foetal astro trol. cytes. *p-0.05, **p<0.005 compared to HO, alone; As used herein, the term "synergistic’ refers to a greater FIG. 8 is a graph showing the effect of H2O, on intracel than additive effect that is produced by a combination of the lular NAD" levels in cultured human neuroblastoma cells agents, which exceeds the effect that would otherwise result following treatment with melatonin. p-0.05 vs Ctrl, from use of the agents alone. 55 **p<0.05 100 uMHO, vs 20 uM Mel-HO, ***p<0.052 A “therapeutically effective amount', as used herein, uM Mel+HO, vs 200 uM Mel+H.O. includes within its meaning a non-toxic but Sufficient amount FIG. 9 is a graph showing the effect of H2O, on intracel of the particular therapeutic compound to which it is referring lular NAD" levels in cultured human neuroblastoma cells to provide the desired therapeutic effect. The exact amount following treatment with vitamin E. p <0.05 vs Ctrl; required will vary from Subject to Subject depending on fac 60 FIG. 10 is a graph showing the dose response effect of the tors such as the patient’s general health, the patient’s age and lipophilic chelator clioquinol (CQ) on intracellular NAD con the stage and severity of the condition. centrations in human neuroblastoma cells following treat As used herein, the term “neurodegenerative disorder ment with HO in the presence of ascorbic acid and ferrous refers to a disease or condition in an animal wherein there is iron. *p-0.05 vs Ctrl, **p-0.05 vs H.O+Fe, ***p-0.05 vs a degeneration or inactivation of nerve cells in any location of 65 10 uMCQ+H2O+Fe: the body including the brain, central nervous system and FIG. 11 is a graph showing the effect of resveratrol on periphery. HO-mediated NAD depletion. Resveratrol significantly US 8,815,936 B2 7 8 increased intracellular NAD+ above control (p<0.05), RES+MEL RES+CLQ, RES+PAM for 24 hr after UVB **p<0.05 compared to HO, alone in human primary astro exposure, inhuman primary Fibroblasts. Concentrations used cytes; were equivalent to ECso/ECso, EC/ECo EC/EC FIG. 12 is a graph showing the effect of the kynurenine respectively; pathway inhibitor 1-MT on resveratrol-induced NAD" syn FIG. 26 is a graph showing the effect of triple therapy thesis via the de novo pathway; supplementation on DNA repair following treatment with FIG. 13 is a graph of a dose response curve showing the RES+MEL+CLQ or RES+MEL+PAM for 24 hr after UVB effect of the nicotinamide mononucleotide adenylyl trans exposure, in human primary Fibroblasts; ferase (NMNAT) inhibitor tannic acid on NAD" synthesis. FIG. 27 is a graph showing the effect of mono therapy IC-inhibitory concentration: 10 supplementation on DNA repair following treatment with FIG. 14 is a graph showing the effect of the nicotinamide RES (100 uM) or MEL (100 uM) or CLO (0.1 uM) or PAM mononucleotide adenylyl transferase (NMNAT) inhibitor (100 uM) for 24 hr after UVB exposure, in human primary tannic acid on resveratrol-mediated NAD" synthesis. Keratinocytes; *p-0.05 compared to resveratrol treatment alone. **p-0.05 FIG. 28 is a graph showing the effect of dual therapy compared to control; 15 supplementation on DNA repair following treatment with FIG. 15 is a graph showing the effect of resveratrol on RES+MEL RES+CLQ, RES+PAM for 24 hr after UVB nicotinamide mononucleotide adenylyl transferase (NM exposure, in human primary Keratinocytes. Concentrations NAT) activity in human brain astrocytes; used were equivalent to ECso/ECso, EC/ECo EC/EC FIG. 16 is a table showing the effect of various concentra respectively; tions of resveratrol on the activity of nicotinamide mono FIG. 29 is a graph showing the effect of triple therapy nucleotide adenylyl transferase (NMNAT) activity in human supplementation on DNA repair following treatment with brain astrocytes; RES+MEL+CLQ or RES+MEL+PAM for 24 hr after UVB FIG. 17 is a diagram showing a mechanism common by exposure, in human primary Keratinocytes; and which oxidative stress induces DNA damage: FIG.30 is a graph showing the effect of 24hr pre-treatment FIG. 18 is a graph showing NAD" production in neuroblas 25 with an antioxidant (melatonin) and/or clioquinol), a chelator toma cells treated with chelators (clioquinol—CQ) and an and/or resveratrol on intracellular NAD levels in human foe antioxidant (vitamin E) after exposure to the pro oxidant tal astrocytes following 15 min of H-O-induced oxidative (HO) and iron (Fe); stress. *p-0.05, **p<0.05 compared to HO, alone, FIGS. 19a-d show graphical representations of intracellu ***p-0.05 compared to each dual therapy treatment. lar NAD concentrations +/-UV exposure, +/-Supplementa 30 tion; MODE(S) FORCARRYING OUT THE FIGS. 19e-h show graphical representations of supernatant INVENTION LDH activity+/-UV exposure, +/-supplementation; FIG.20a is a graph showing the effect of dual supplemen The invention relates to the finding that the intracellular tation on NAD concentrations following treatment with 35 synthesis of NAD" are increased by resveratrol (3,5,4'-trihy RES+/-PAM+/-CLQ+/-MEL on NAD recovery 24 hr after droxystilbene). While not being bound or limited to a particu UVB exposure, in human primary Keratinocytes; lar mechanism, the inventors have demonstrated that resvera FIG. 20b is a graph showing the effect of dual supplemen trol induces NAD" synthesis via the salvage pathway (shown tation on LDH release (cell death) following treatment with in FIG. 1), specifically, by upregulation of nicotinamide RES+MEL or RES+CLQ or RES+PAM for 24 hr after UVB 40 mononucleotide adenylyl transferase (NMNAT) activity. exposure, in human primary Keratinocytes; Accordingly, the invention provides a means by which the FIG. 21 is a graph showing the effect of triple supplemen intracellular synthesis of NAD" may be induced in a subject. tation (selected combinations) on NAD concentrations fol The inventors have also demonstrated that the induction of lowing treatment with RES+MEL+CLQ or RES+MEL+ NAD" synthesis by administration of resveratrol provides a PAM on NAD recovery 24 hr after UVB exposure, in human 45 means of inducing DNA repair and promoting cellular viabil primary Keratinocytes; ity and longevity. Again without being bound by a particular FIG. 22a is an immunocytochemistry visualisation of mechanism, it is believed that the upregulation of NMNAT Keratinocytes depicting intense staining for DNA damage activity by resveratrol has the effect of increasing the metabo (middle column) for UV treated cells without supplementa lism of nicotinamide (NAM), a precursor of NAD" and a tion; 50 by-product of Poly(ADP-ribose) polymerase (PARP) FIG. 22b is a graph showing the semiquantitation of fluo enzyme family members. NAM is an inhibitor of PARP rescent intensities for each of the ICC stained sections of enzymes (e.g. PARP-1) and mammalian sirtuin enzyme fam Keratinocytes; ily members (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6 FIG. 23a is an immunocytochemistry visualisation of and SIRT7). Metabolism of NAM induced by administration Fibroblasts depicting intense staining for DNA damage 55 of resveratrol thus serves to increase available NAD" levels, (middle column) for UV treated cells without supplementa while reducing inhibition of both PARP activity (thereby tion; promoting DNA repair) and the activity of sirtuin enzymes FIG. 23b is a graph showing the semiquantitation of fluo (thereby promoting cell viability and longevity). rescent intensities for each of the ICC stained sections of The invention further relates to the use of a promoter of Fibroblasts; 60 NAD" synthesis, for example resveratrol, in combination FIG. 24 is a graph showing the effect of mono therapy with chelating agents and/or antioxidants to produce a syner supplementation on DNA repair following treatment with gistic effect for the treatment of conditions associated with RES (100 uM) or MEL (100 uM) or CLO (0.1 uM) or PAM oxidative stress. The inventors have demonstrated that the (100 uM) for 24 hr after UVB exposure, in human primary benefits derived from combination drug therapies consisting Fibroblasts; 65 of an agent for chelating redox-active metals and an antioxi FIG. 25 is a graph showing the effect of dual therapy dant to reduce damage caused by residual oxygen free radi supplementation on DNA repair following treatment with cals may be enhanced in a synergistic manner by the inclusion US 8,815,936 B2 10 of a promoter of NAD" synthesis. It is believed that this 42:41-46, Goldberg et al. Anal. Chem. (1994) 66: 3959-63, synergistic effect arises from improved DNA repair (through and Farina et al. Nat. Prod. Res. (2006). 20: 247-52, the at least increased PARP enzyme activity), and improved cell contents of which are incorporated herein by cross-reference. viability and longevity (through at least increased sirtuin Additionally or alternatively, resveratrol for use in the inven activity), via increased production of their essential Substrate 5 tion may be obtained from commercial sources (e.g. from NAD. Sigma, St. Louis, Mo.). Resveratrol and NAD" Synthesis Resveratrol for use in accordance with the invention may Nicotinamide adenine dinucleotide (NAD") can be synthe be in the form of a cis isomer, a trans isomer, or a mixture sised from several different starting compounds. In the “de thereof. Cis-resveratrol may be derived from trans-resvera novo’ pathway (shown in FIG. 2) NAD" is produced from the 10 trol using methods known in the art, for example, by heating essential amino acid tryptophan. This pathway involves the or exposing trans-resveratrol to ultraviolet irradiation fol generation of quinolinic acid from tryptophan, which is con lowed by separation by HPLC and identification by mass verted to nicotinic acid mononucleotide (NaMN) via the spectrometry (MS). The invention also contemplates the use transfer of a phosphoribose group. An adenylate group is then of functionally equivalent resveratrol derivatives and ana transferred to form nicotinic acid adenine dinucleotide 15 logues. A resveratrol analogue is a compound that is based on (NaAD). The nicotinic acid group of NaAD is then amidated resveratrol with substituted groups attached to the parent to a nicotinamide group, forming NAD. Alternatively, nico compound to produce a chemically-modified resveratrol tinamide or pre-formed compounds containing nicotinamide compound. Examples of Substituent groups include, but are such as nicotinamide riboside are used to generate NAD" via not limited to C-C alkyl (such as methyl, ethyl, propyl), the “salvage' pathway (see FIG. 1). Inhibition of enzymes in CH2OH, halogen (e.g., fluoro, chloro, bromo, iodo). Any one either the de novo or salvage pathway results in significant or more of the hydroxyl groups may be functionalised, e.g., NAD" depletion highlighting the importance of both these with a protecting group. Suitable protecting groups are pathways to the maintenance of cellular NAD" levels. known to those skilled in the art and reference may be had to The invention provides a method of increasing the synthe “Protective Groups in Organic Synthesis” by Theodora sis/level of intracellular NAD" in a subject. The method com 25 Greene and Peter Wuts (third edition, 1999, John Wiley and prises administering to the Subject a therapeutically effective Sons). Alternatively, any one or more of the hydroxyl groups amount of resveratrol or a functionally equivalent analogue or may be functionalised with, for example, C-C alkyl (to form an ether), or a carboxylic acid group (to form an ester). derivative thereof. Examples of Such compounds include hydroxylated or 30 methoxylated resveratrol analogues. The functionally OH equivalent resveratrol analogue may be cis-resveratrol gluco side (cis-piceid) or trans-resveratrol-3-O-P-glucoside (trans piceid). Derivatives of resveratrol include those in which one or more hydroxyl groups, typically the 3-hydroxyl group, is / ( ) 35 conjugated to a mono-saccharide or di-saccharide. In general, the 3-hydroxyl group may be conjugated to the 1-position of OH a monosaccharide. Examples of saccharides which may be conjugated to resveratrol include, but are not limited to, glu cose, maltose, lactose, Sucrose and galactose. Other deriva Chemical Structure of Resveratrol 40 tives and analogues of resveratrol suitable for use in the (3,5,4'-trihydroxy stilbene) methods of the invention are described in, for example, U.S. Pat. No. 7,026,518, U.S. Pat. No. 6,790,869, PCT publication In accordance with the methods of the invention, resvera No. WO/2003/055444, PCT publication No. WO/2004/ trol (also known as 3.5,4'-trihydroxy stilbene, trans-3,5,4'-tri 000302, PCT publication No. WO/1999/003816, the contents hydroxy stilbene: 3,4,5-Stilbenetriol; trans-resveratrol; (E)- 45 of which are incorporated herein by cross reference. The 5-(p-Hydroxystyryl) resorcinol and resorcinol) may be skilled addressee will recognise that the derivatives and ana administered in natural form, for example, as isolated from logues of resveratrol described herein are non-limiting grape skins, wine or other plant-derived compositions using examples and the invention encompasses the use of other Such methods known in the art (see, for example, methods derivatives and analogues of resveratrol capable of inducing a described in Lamuela-Raventos, J. Agric. Food Chem. (1995) 50 functionally equivalent effect. (43): 281-283, and Wang et al. J Agric Food Chem. (2002) The invention also contemplates pharmaceutically accept 50:431-435, the contents of which are incorporated herein by able salts of resveratrol and analogues thereof. S. M. Berge et cross-reference). For example, ground plant material may be al. describe pharmaceutically acceptable salts in detail in J. extracted using a suitable solvent such as methanol followed Pharmaceutical Sciences, 1977, 66:1-19. The salts can be by concentration and dilution with water. After washing with 55 prepared in situ during the final isolation and purification of an appropriate nonpolar organic solvent Such as hexane the the compounds of the invention, or separately by reacting the aqueous layer may be partitioned using, for example, ethyl free base function with a suitable organic acid. Representative acetate. The ethyl acetate extract may then be separated into acid addition salts include acetate, adipate, alginate, ascor fractions over a silica gel chromatographic column using, for bate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, example, chloroform-methanol as eluent. Fractions contain 60 butyrate, camphorate, camphorsulfonate, citrate, diglucon ing increased resveratrol concentration may be pooled ate, cyclopentanepropionate, dodecylsulfate, ethane together and Subjected to further column chromatography. Sulfonate, fumarate, glucoheptonate, glycerophosphate, Additionally or alternatively, resveratrol may be chemi hemisulfate, heptonate, hexanoate, hydrobromide, hydro cally synthesized, for example, using a Wittig reaction chloride, hydroiodide, 2-hydroxy-ethanesulfonate, lacto wherein two substituted phenols are linked through a styrene 65 bionate, lactate, laurate, lauryl Sulfate, malate, maleate, mal double bond, as described by Moreno-Manas et al. Anal. onate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, Ouim (1985)81:157-61, Jeandet al. Am. J. Enol. Vitic. (1991) nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persul US 8,815,936 B2 11 12 fate, 3-phenylpropionate, phosphate, picrate, pivalate, propi PARP enzymes consume NAD" as an adenosine diphos onate, Stearate. Succinate, Sulfate, tartrate, thiocyanate, tolu phate ribose (ADPR) donor and synthesize poly(ADP-ri enesulfonate, undecanoate, Valerate salts, and the like. bose) onto nuclear proteins such as histones and PARP itself. Representative alkali or alkaline earth metal salts include Overactivation of PARP, particularly during oxidative stress, Sodium, lithium potassium, calcium, magnesium, and the can cause significant depletion of cellular NAD" leading to like, as well as non-toxic ammonium, quaternary ammonium, cellular necrosis. Administration of resveratrol in accordance and amine cations, including, but not limited to ammonium, with the methods of the invention overcomes this problem tetramethylammonium, tetraethylammonium, methylamine, firstly by increasing NAD" synthesis thus providing increas dimethylamine, trimethylamine, triethylamine, ethylamine, ing the availability of substrate for PARP-mediated DNA triethanolamine and the like. 10 In accordance with the methods of the invention, the repair, and secondly through the increased metabolism of administration of therapeutically effective amounts of res nicotinamide (NAM) has the effect of removing an active Veratrol to a subject may be used to induce the synthesis of inhibitor of PARP activity. PARP-1 is activated by DNA intracellular NAD". Intracellular NAD" can be measured by strand breaks and has been implicated in multiple DNA repair methods known in the art and are described in, for example, 15 pathways, including the base excision repair (BER), single Grant and Kapoor, J. Neurochem. (1998) 70(4): 1759-1763. strand break (SSB) and double-strand break (DSB) pathways. Intracellular levels of NAD" can be measured, for example, Binding of PARP-1 to damaged DNA via a double zinc finger using a high-performance liquid chromatography (HPLC) DNA-binding domain potently activates PARP-1 activity technique such as described by Klaidman et al., Anal Biochem thus allowing PARP-1 to also function as a DNA damage (1995) 228:312-317, the contents of which are incorporated sensor. PARP-2 is also stimulated by damaged DNA and been herein by reference. Additionally or alternatively, a spectro implicated in base excision repair (BER) pathway via inter photometric, microcycling assay may be used to measure actions with PARP-1 and X-ray repair cross-complementing intracellular levels of NAD", such as that described by Nis group 1 (XRCC-1). selbaum and Green, Anal Biochem (1969) 27: 212-217 or Nicotinamide (NAM) also inhibits the activity of mamma Berofsky and Swan, Anal Biochem (1973) 53: 452-458, the 25 lian sirtuin family enzymes such as SIRT1 on the Internet at contents of which are incorporated herein by reference. For en.wikipedia.org/wiki/Sir2-note-1. Administration of res example, tissue/cell homogenate may be incubated in a pH Veratrol in accordance with the methods of the invention controlled (buffered) solution, (reaction mixture), containing increases NAD" synthesis thus increasing the availability of essential ingredients to facilitate the reaction of NAD" to a Substrate necessary for sirtuin activity, while also reducing measurable compound. This reaction mixture may contain, 30 inhibition of sirtuin activity by inducing the increased for example ethanol, alcohol dehydrogenase, phenazine metabolism of NAM. Accordingly, the invention provides a methosulfate and 3-4,5-Dimethylthiazol-2-yl)-2,5-diphe method of inducing sirtuin activity by the administration of nyltetrazolium bromide, (MTT) (in bicine buffered solution). resveratrol. After a suitable incubation time, the reaction is then stopped Pharmaceutical Compositions and Routes of Administration with a Suitable stopping reagent, for example, iodoacetic acid, 35 The invention provides pharmaceutical compositions com and the level of NAD" in the sample determined by measure prising a therapeutically effective amount of resveratrol or a ment against a reagent blank in a microplate reader. functionally equivalent analogue or derivative thereof. The NAD"-dependent enzymes play an important role in DNA pharmaceutical compositions of the invention may be used repair, particularly under conditions of oxidative stress. for increasing NAD" synthesis in a subject in need thereof. Accordingly, the invention provides a method of inducing 40 Additionally or alternatively, the pharmaceutical composi DNA repair in a Subject, the method comprising administer tions of the invention may be used for the prevention or ing to the Subject a therapeutically effective amount of res treatment of a disease or condition associated with oxidative Veratrol or a functionally equivalent analogue or derivative stress and/or DNA damage. thereof. The induction of DNA repair in a subject may arise Resveratrol in the pharmaceutical compositions of the from the increased activity of one or more Poly(ADP-ribose) 45 invention may be in the form of a cis isomer, a trans isomer, or polymerase (PARP) enzyme family members. The activity of a mixture thereof. Examples of resveratrol analogues Suitable PARP enzymes can be measured using methods known in the for the pharmaceutical compositions of the invention include, art. For example PARP enzyme activity may be assayed by but are not limited to hydroxylated or methoxylated resvera scintillation proximity assay (SPA) using biotinylated NAD trol analogues. The functionally equivalent resveratrol ana as described by Cheung and Zhang Anal Biochem (2000) 282: 50 logue may be cis-resveratrol glucoside (cis-piceid) or trans 24-28, the contents of which are incorporated herein by ref resveratrol-3-O-B-glucoside (trans-piceid). Derivatives of erence. Additionally or alternatively, the activity of PARP resveratrol suitable for the pharmaceutical compositions of enzymes may be measured using an enzymatic assay via the the invention include those in which one or more hydroxyl chemical quantitation of NAD as described by Karson et al., groups, typically the 3-hydroxyl group, is conjugated to a Anal Biochem (2004) 326: 78-86 or Putt et al., Chem. Bio. 55 mono-saccharide or di-saccharide. In general, the 3-hydroxyl Chem. (2005) 6:53-55, the contents of which are also incor group may be conjugated the 1-position of a monosaccharide. porated herein by reference. For example, plated cells or Examples of saccharides which may be conjugated to res tissue are washed and then lysed using a suitable buffer solu Veratrol include, but are not limited to, glucose, maltose, tion containing excess NAD". A suitable buffer solution may lactose, Sucrose and galactose. Other suitable resveratrol comprise, for example, MgCl2 (10 mM), Triton X-100 (1%), 60 derivatives and analogues are described in, for example, U.S. and NAD" (20 uM) in Tris buffer (50 mM, pH 8.1). As NAD" Pat. No. 7,026,518, U.S. Pat. No. 6,790,869, PCT publication is the exclusive substrate for the PARP enzyme and is No. WO/2003/055444, PCT publication No. WO/2004/ degraded during this reaction, total enzyme activity is deter 000302, PCT publication No. WO/1999/003816, the contents mined by the reduction in NAD" concentration in the buffer of which are incorporated herein by cross-reference. The solution over time. Following incubation for a suitable time 65 skilled addressee will recognise that the derivatives and ana period, the amount of NAD" consumed is measured by the logues of resveratrol Suitable for the pharmaceutical compo NAD(H) microcycling assay as described above. sitions of the invention are non-limiting examples and the US 8,815,936 B2 13 14 invention encompasses the use of other Such derivatives and cation, the compositions may be administered prophylacti analogues of resveratrol capable of inducing a functionally cally in conditions expected to increase oxidative stress and/ equivalent effect. or DNA damage or to a patient already Suffering from a Combination drug therapies consisting of an agent for disease or condition associated with oxidative stress and/or chelating redox-active metals and/or an antioxidant to reduce 5 DNA damage, or at least partially arrest the disease or con damage caused by residual oxygen free radicals can be dition associated with oxidative stress and its complications. enhanced in a synergistic manner by the inclusion of a pro Single or multiple administrations of the pharmaceutical moter of NAD" synthesis. Accordingly, the invention pro compositions can be carried out with dose levels and pattern vides pharmaceutical compositions for the treatment of con being selected by the treating physician. ditions and diseases associated with oxidative stress 10 The therapeutically effective dose level for any particular comprising a synergistic combination of at least one agent to patient will depend upon a variety of factors including: the promote NAD" synthesis, at least one antioxidant and/or at severity of the disease or condition associated with oxidative least one chelating agent. stress and/or DNA damage, including UV induced DNA The inclusion of an agent to promote NAD" synthesis in damage in skin cells, the composition employed, the age, compositions containing an antioxidant and/or a chelating 15 body weight, general health and diet of the patient, the time of agent provides a synergistic effect for the prevention, treat administration, the route of administration, the duration of the ment or alleviation of oxidative stress. In a preferred embodi treatment, drugs used in combination or coincidental with the ment, the agent to promote of NAD" synthesis is resveratrol synergistic composition, together with other related factors or a functional analogue or equivalent thereof. Without being well known in medicine. bound to a particular mechanism, the provision of increased One skilled in the art would, by routine experimentation, be NAD" during conditions of oxidative stress may be beneficial able to determine an effective, non-toxic amount of this treat to the function of other NAD-dependent enzymes, for ment regime which would be required to treat a disease or example, NAD glycohydrolase, providing further beneficial condition associated with oxidative stress and/or DNA dam effects for cells under oxidative stress. Additionally or alter age, including UV induced DNA damage in skin cells, with natively, NAD" synthesis-promoting agents may have the 25 the pharmaceutical compositions of the present invention. capacity to increase activity of the NAD" dependant deacety In the methods of the invention, the pharmaceutical com lases Such as sirtuin family enzymes (e.g. SIRT1) and pro positions administered may be in the range of about 0.0001 mote improved DNA repair by enhancing PARP enzyme mg to about 1000 mg per kg body weight; for example, about activity through increased production of their essential Sub 0.001 mg to about 750 mg per kg body weight; about 0.01 mg strate NAD". Additionally or alternatively, NAD" synthesis 30 to about 500 mg per kg body weight; about 0.1 mg to about promoting agents may also be capable of increasing NAM 500 mg per kg body weight; about 0.1 mg to about 250 mg per metabolism via induction of NMNAT activity, thereby kg body weight; or about 1.0 mg to about 250 mg per kg body removing an inhibitor of PARP/sirtuins and further inducing weight. More Suitably, an effective dosage per 24 hours may the activity of those enzymes. be in the range of about 1.0 mg to about 200 mg per kg body Chelating agents suitable for the synergistic compositions 35 weight; about 1.0 mg to about 100 mg per kg body weight; of the invention include, but are not limited to, ethylenedi about 1.0 mg to about 50 mg per kg body weight; about 1.0 aminetetraacetic acid (EDTA), Ethylenediamine tetraacetic mg to about 25 mg per kg body weight; about 5.0 mg to about acid (calcium disodium versante) (CaNa-EDTA), Ethylene 50 mg per kg body weight; about 5.0 mg to about 20 mg per glycol tetraacetic acid (EGTA), dimercaptosuccinic acid kg body weight; or about 5.0 mg to about 15 mg per kg body (DMSA), Alpha lipoic acid (ALA), 2,3-dimercapto-1-pro 40 weight. panesulfonic acid (DMPS), Dimercaprol (BAL), Deferoxam Further, it will be apparent to one of ordinary skill in the art ine, D-penicillamine, dimercaprol, Aminophenoxyethane that the optimal quantity and spacing of individual dosages of tetraacetic acid (BAPTA) Defarasirox, Diethylene triamine the compositions of the present invention will be determined pentaacetic acid (DTPA) 2-pyridinecarboxylic acid (picolinic by the nature and extent of the condition the form, route and acid), 2.3-pyridinedicarboxylic acid (quinolinic acid), 2-ami 45 site of administration, and the nature of the particular patient nobenzoic acid (anthranilic acid), kynurenic acid, Xan being treated. Also, Such optimum conditions can be deter thurenic acid and 8-hydroxyquinoline (and functional deriva mined by conventional techniques. tives thereof). In general, the chelating agent will be capable It will also be apparent to one of ordinary skill in the art that of forming complexes with redox-active metals in which the the optimal course of treatment, such as the number of doses metal ion is generally bound to two or more atoms of the 50 of the composition of the present invention given per day for chelating agent thereby reducing hydroxyl radical produc a defined number of days, can be ascertained by those skilled tion. The bonds may be any combination of coordination or in the art using conventional course of treatment determina ionic bonds. Examples of redox-active metals that may be tion tests. bound and complexed by chelating agents include, but are not In general, pharmaceutical compositions of the present limited to Fe", Cut, Cr", Mn", Co", Ni" Ag". 55 invention may be prepared according to methods which are Antioxidants suitable for the synergistic compositions of known to those of ordinary skill in the art, and accordingly the invention include, for but are not limited to, melatonin, may include a pharmaceutically acceptable carrier, diluent Vitamin E. Vitamin C, methionine, taurine, Superoxide dismu and/or adjuvant. tase (SOD), catalase (CAT), and glutathione peroxidase According to the methods of present invention, the phar (GPX) and L-ergothioneine N-Acetyl Cysteine (NAC), vita 60 maceutical compositions may be administered by any Suit minA, beta-carotene, retinol, catechins, epicatechins, epigal able route, either systemically, regionally or locally. The par locatechin-3-gallate, flavenoids, L-ergothioneine, idebenone ticular route of administration to be used in any given and selenium. Other suitable antioxidants are described in US circumstance will depend on a number of factors, including Patent No. 2008015218, the contents of which are incorpo the nature of the disease or condition to be treated, the severity rated herein by cross-reference. 65 and extent of the disease or condition, the required dosage of The pharmaceutical compositions of the present invention the particular compounds to be delivered and the potential may be administered therapeutically. In a therapeutic appli side-effects of the compounds. US 8,815,936 B2 15 16 For example, in circumstances where it is required that coated with compounds such as glyceryl monostearate or appropriate concentrations of the desired compounds are glyceryl distearate which delay disintegration. delivered directly to the site in the body to be treated, admin Adjuvants typically include emollients, emulsifiers, thick istration may be regional rather than systemic. Regional ening agents, preservatives, bactericides and buffering administration provides the capability of delivering very high 5 agents. local concentrations of the desired compounds to the required Solid forms for oral administration may contain binders site and thus is suitable for achieving the desired therapeutic acceptable in human and Veterinary pharmaceutical practice, or preventative effect whilst avoiding exposure of other Sweeteners, disintegrating agents, diluents, flavourings, coat organs of the body to the compounds and thereby potentially ing agents, preservatives, lubricants and/or time delay agents. reducing side effects. 10 Suitable binders include gum acacia, gelatine, corn starch, By way of example, administration according to embodi gum tragacanth, Sodium alginate, carboxymethylcellulose or ments of the invention may be achieved by any standard polyethylene glycol. Suitable Sweeteners include Sucrose, routes, including intracavitary, intravesical, intramuscular, lactose, glucose, aspartame or saccharine. Suitable disinte intraarterial, intravenous, Subcutaneous, topical or oral. Int grating agents include corn starch, methylcellulose, polyvi racavitary administration may be intraperitoneal or intrapleu 15 nylpyrrolidone, guar gum, Xanthan gum, bentonite, alginic ral. In particular embodiments, administration may be via acid or agar. Suitable diluents include lactose, Sorbitol, man intravenous infusion or intraperitoneal administration. nitol, dextrose, kaolin, cellulose, calcium carbonate, calcium If desired, devices or compositions containing the pharma silicate or dicalcium phosphate. Suitable flavouring agents ceutical compositions Suitable for Sustained or intermittent include peppermint oil, oil of wintergreen, cherry, orange or release could be, in effect, implanted in the body or topically raspberry flavouring. Suitable coating agents include poly applied thereto for the relatively slow release of such materi mers or copolymers of acrylic acid and/or methacrylic acid als into the body. and/or their esters, waxes, fatty alcohols, Zein, shellac or The carriers, diluents and adjuvants must be “acceptable' gluten. Suitable preservatives include Sodium benzoate, Vita in terms of being compatible with the other components of the min E, alpha-tocopherol, ascorbic acid, methyl paraben, pro composition, and not deleterious to the recipient thereof. 25 pyl paraben or sodium bisulphite. Suitable lubricants include Examples of pharmaceutically acceptable carriers or diluents magnesium Stearate, Stearic acid, sodium oleate, sodium are demineralised or distilled water, saline solution, vegetable chloride or talc. Suitable time delay agents include glyceryl based oils such as peanut oil, safflower oil, olive oil, cotton monostearate or glyceryl distearate. seed oil, maize oil, Sesame oils such as peanut oil, safflower Liquid forms for oral administration may contain, in addi oil, olive oil, cottonseed oil, maize oil, Sesame oil, arachis oil 30 tion to the above agents, a liquid carrier. Suitable liquid car or coconut oil; Silicone oils, including polysiloxanes, such as riers include water, oils such as olive oil, peanut oil, Sesame methyl polysiloxane, phenyl polysiloxane and methylphenyl oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid polySolpoxane; Volatile silicones, mineral oils such as liquid paraffin, ethylene glycol, propylene glycol, polyethylene gly paraffin, soft paraffin or squalane; cellulose derivatives Such col, ethanol, propanol, isopropanol, glycerol, fatty alcohols, as methyl cellulose, ethyl cellulose, carboxymethylcellulose, 35 triglycerides or mixtures thereof. sodium carboxymethylcellulose or hydroxypropylmethylcel Suspensions for oral administration may further comprise lulose, lower alkanols, for example ethanol or iso-propanol: dispersing agents and/or Suspending agents. Suitable Sus lower aralkanols, lower polyalkylene glycols or lower alky pending agents include Sodium carboxymethylcellulose, lene glycols, for example polyethylene glycol, polypropylene methylcellulose, hydroxypropylmethyl-cellulose, poly-vi glycol, ethylene glycol, propylene glycol. 1,3-butylene gly 40 nyl-pyrrolidone, Sodium alginate or acetyl alcohol. Suitable color glycerine, fatty acid esters such as isopropyl palmitate, dispersing agents include lecithin, polyoxyethylene esters of isopropyl myristate or ethyl oleate, polyvinylpyrridone, agar, fatty acids such as Stearic acid, polyoxyethylene Sorbitol carrageenan, gum tragacanth or gum acacia, and petroleum mono- or di-oleate, -Stearate or -laurate, polyoxyethylene jelly. The carrier or carriers may form from between 10% to Sorbitan mono- or di-oleate, -Stearate or -laurate and the like. 99.9% by weight of the compositions. 45 Pharmaceutical compositions of the invention may be pre The pharmaceutical compositions of the invention may be pared by blending, grinding, homogenising, Suspending, dis in the form of a composition in a form Suitable for adminis Solving, emulsifying, dispersing and/or mixing the selected tration by oral ingestion (such as capsules, tablets, caplets and therapeutic compound with the selected excipient(s), elixirs, for example), in the form of an ointment, cream or carrier(s), adjuvant(s) and/or diluent(s). lotion suitable for topical administration, which would be 50 One type of pharmaceutical composition of the invention particularly preferred for the treatment and repair of UV in the form of a tablet or capsule may be prepared by (a) induced DNA damage in skin cells, in a form suitable for preparing a first tablet or a capsule comprising a first thera delivery as an eye drop, in anaerosol form Suitable for admin peutic compound, together with any desired excipient(s), car istration by inhalation, such as by intranasal inhalation or oral rier(s), adjuvant(s) and/or diluent(s), and (b) preparing a sec inhalation, in a form Suitable for parenteral administration, 55 ond tablet or a capsule, wherein the second tablet or the that is Subcutaneous, intramuscular or intravenous injection. capsule includes a second therapeutic compound and the first For administration as an injectable solution or Suspension, tablet or capsule. non-toxic parenterally acceptable diluents or carriers can Another type of pharmaceutical composition of the inven include Ringer's solution, isotonic saline, phosphate buffered tion in the form of a capsule may be prepared by (a) preparing saline, ethanol and 1.2-propylene glycol. 60 a first capsule comprising a first therapeutic compound Some examples of Suitable carriers, diluents, excipients together with any desired excipient(s), carrier(s), adjuvant(s) and adjuvants for oral use include peanut oil, liquid paraffin, and/or diluent(s), and (b) preparing a second capsule, wherein Sodium carboxymethylcellulose, methylcellulose, sodium the second capsule includes a second therapeutic compound alginate, gum acacia, gum tragacanth, dextrose, Sucrose, Sor and the first capsule. bitol, mannitol, gelatine and lecithin. In addition, these oral 65 A further type of pharmaceutical composition of the inven formulations may contain Suitable flavouring and colourings tion in the form of a tablet may be prepared by (a) preparing agents. When used in capsule form the capsules may be a capsule comprising antherapeutic compound together with US 8,815,936 B2 17 18 any desired excipient(s), carrier(s), adjuvant(s) and/or formed by mono- or multi-lamellar hydrated liquid crystals diluent(s), and (b) preparing a tablet, wherein the tablet that are dispersed in an aqueous medium. Specific examples includes the second therapeutic compound and the capsule. of liposomes used in administering or delivering a composi The emulsions for oral administration may further com tion to target cells are synthetic cholesterol (Sigma), the phos prise one or more emulsifying agents. Suitable emulsifying pholipid 1,2-distearoyl-sn-glycero-3-phosphocholine agents include dispersing agents as exemplified above or (DSPC: Avanti Polar Lipids), the PEG lipid 3-N-(-methoxy natural gums such as guar gum, gum acacia or gum traga poly(ethylene glycol)2000)carbamoyl-1,2-dimyrestyloxy canth. propylamine (PEG-cDMA), and the cationic lipid 1,2-di-o- Methods for preparing parenterally administrable compo octadecenyl-3-(N,N-dimethyl)aminopropane (DODMA) or sitions are apparent to those skilled in the art, and are 10 1,2-dilinoleyloxy-3-(N,N-dimethyl)aminopropane described in more detail in, for example, Remington's Phar maceutical Science, 15th ed., Mack Publishing Company, (DLindMA) in the molar ratios 55:20:10:15 or 48:20:2:30, Easton, Pa., hereby incorporated by cross-reference. respectively, PEG-cDMA, DODMA and DLinDMA. Any The topical compositions of the invention, comprise an non-toxic, physiologically acceptable and metabolisable therapeutic compound(s) together with one or more accept 15 lipid capable of forming liposomes can be used. The compo able carriers, and optionally any other therapeutic ingredi sitions in liposome form may contain stabilisers, preserva ents. Compositions Suitable for topical administration tives, excipients and the like. The preferred lipids are the include liquid or semi-liquid preparations Suitable for pen phospholipids and the phosphatidylcholines (lecithins), both etration through the skin to the site of where treatment is natural and synthetic. Methods to form liposomes are known required. Such as liniments, lotions, creams, ointments or in the art, and in relation to this specific reference is made to: pastes, and drops Suitable for administration to the eye, ear or Prescott, Ed., Methods in Cell Biology, Volume XIV. Aca OS. demic Press, New York, N.Y. (1976), p. 33 et seq., the con Drops according to the present invention may comprise tents of which is incorporated herein by cross-reference. sterile aqueous or oily Solutions or Suspensions. These may be The compositions may also be administered in the form of prepared by dissolving the therapeutic compound(s) in an 25 microparticles. Biodegradable microparticles formed from aqueous Solution of a bactericidal and/or fungicidal agent polylactide (PLA), polylactide-co-glycolide (PLGA), and and/or any other Suitable preservative, and optionally includ epsilon-caprolactone (e-caprolactone) have been extensively ing a surface active agent. The resulting solution may then be used as drug carriers to increase plasma half life and thereby clarified by filtration, transferred to a suitable container and prolong efficacy (R. Kumar, M., 2000, J Pharm Pharmaceut sterilised. Sterilisation may be achieved by: autoclaving or 30 Sci. 3(2) 234-258). Microparticles have been formulated for maintaining at 90° C.-100° C. for half an hour, or by filtration, the delivery of a range of drug candidates including vaccines, followed by transfer to a container by an aseptic technique. antibiotics, and DNA. Moreover, these formulations have Examples of bactericidal and fungicidal agents suitable for been developed for various delivery routes including inclusion in the drops are phenylmercuric nitrate or acetate parenteral Subcutaneous injection, intravenous injection and (0.002%), benzalkonium chloride (0.01%) and chlorhexidine 35 inhalation. acetate (0.01%). Suitable solvents for the preparation of an The compositions may incorporate a controlled release oily solution include glycerol, diluted alcohol and propylene matrix that is composed of Sucrose acetate isobutyrate glycol. (SAIB) and organic solvent or organic Solvents mixture. Lotions according to the invention include those Suitable Polymer additives may be added to the vehicle as a release for application to the skin or eye. An eye lotion may comprise 40 modifier to further increase the viscosity and slow down the a sterile aqueous solution optionally containing a bactericide release rate. SAIB is a well known food additive. It is a very and may be prepared by methods similar to those described hydrophobic, fully esterified sucrose derivative, at a nominal above in relation to the preparation of drops. Lotions or lini ratio of six isobutyrate to two acetate groups. As a mixed ester, ments for application to the skin may also include an agent to SAIB does not crystallize but exists as a clear viscous liquid. hasten drying and to cool the skin, such as an alcohol or 45 Mixing SAIB with a pharmaceutically accepted organic Sol acetone, and/or a moisturiser Such as glycerol, or oil such as vent Such as ethanol or benzyl alcohol decreases the Viscosity castor oil or arachis oil. of the mixture sufficiently to allow for injection. Atherapeutic Creams, ointments or pastes according to the invention are compound(s) may be added to the SAIB delivery vehicle to semi-solid formulations of the compound(s) for external form SAIB solution or suspension formulations. When the application. They may be made by mixing the active ingredi 50 formulation is injected subcutaneously, the solvent diffuses ent in finely-divided or powdered form, alone or in solution or from the matrix allowing the SAIB-drug or SAIB-drug-poly Suspension in an aqueous or non-aqueous fluid, with a greasy mer mixtures to set up as an in situ forming depot. or non-greasy basis. The basis may comprise hydrocarbons Those skilled in the art will appreciate that in accordance Such as hard, soft or liquid paraffin, glycerol, beeswax, a with the methods of the invention the pharmaceutical com metallic soap; a mucilage; an oil of natural origin Such as 55 positions may be administered alone or in conjunction with almond, corn, arachis, castor or olive oil; wool fat or its one or more additional agents as a combination therapy. For derivatives, or a fatty acid such as Stearic or oleic acid together example, a pharmaceutical composition of the invention may with an alcohol Such as propylene glycol or macrogols. be administered together with one or more additional agents The compositions may incorporate any suitable Surfactant capable of treating or preventing conditions associated with Such as an anionic, cationic or non-ionic Surfactant such as 60 oxidative stress and/or DNA damage, including UV induced sorbitan esters or polyoxyethylene derivatives thereof. Sus DNA damage in skin cells. pending agents such as natural gums, cellulose derivatives or For Such combination therapies, each component of the inorganic materials such as silicaceous silicas, and other combination therapy may be administered at the same time, ingredients such as lanolin, may also be included. or sequentially in any order, or at different times, so as to The compositions may also be administered or delivered to 65 provide the desired effect. Alternatively, the components may target cells in the form of liposomes. Liposomes are generally be formulated together in a single dosage unit as a combina derived from phospholipids or other lipid substances, and are tion product. When administered separately, it may be pre US 8,815,936 B2 19 20 ferred for the components to be administered by the same reduction in NAD" levels and proportionate increased levels route of administration, although it is not necessary for this to of cell death oxidative stress-induced PARP activation. be so. Diseases and conditions associated with oxidative stress Therapeutic advantages may be realised through combina and/or DNA damage, including UV induced DNA damage in tion regimens. In combination therapy the respective compo skin cells suitable for treatment by the methods of the inven sitions and any other agents may be administered simulta tion include, but are not limited to those in which increased neously or sequentially in any order. Accordingly, methods of levels of ROS result from exposure to U.V. or ionising radia treatment according to the invention may be applied in con tion (e.g. X-ray, Y rays), chemical agents, infection, inflam junction with conventional therapy, such as radiotherapy, mation or reduced mitochondrial efficiency. The methods of chemotherapy, Surgery, or other forms of medical interven 10 the invention are suitable for the treatment of normal cellular tion. Examples of chemotherapeutic agents include adriamy ageing or accelerated ageing of the skin, treatment and repair of UV induced DNA damage in skin cells (i.e. keratinocytes cin, taxol, fluorouricil, melphalan, cisplatin, oxaliplatin, and fibroblasts) and neurodegenerative diseases and disorders alpha interferon, Vincristine, vinblastine, angioinhibins, including, for example, Alzheimer's disease and Parkinson TNP-470, pentosan polysulfate, platelet factor 4, angiostatin, 15 Disease. Additionally or alternatively, the methods of the LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG invention suitable for the treatment of diseases and conditions and the like. Other chemotherapeutic agents include alkylat associated with DNA damage including, for example, cancer. ing agents such as nitrogen mustards including mechloet The skilled addressee will recognise that the diseases referred hamine, melphan, chlorambucil, cyclophosphamide and ifos to above are non-limiting examples and the compositions of famide, nitrosoureas including carmustine, lomustine, the invention may be used for the treatment of other condi Semustine and streptozocin; alkyl Sulfonates including busul tions and diseases associated with oxidative stress and/or fan; triazines including dicarbazine; ethyenimines including DNA damage. thiotepa and hexamethylmelamine; folic acid analogues Kits including methotrexate; pyrimidine analogues including The invention provides kits for increasing NAD" synthesis, 5-fluorouracil, cytosine arabinoside; purine analogues 25 the kit comprising resveratrol or a functionally equivalent including 6-mercaptopurine and 6-thioguanine; antitumour analogue or derivative thereof. antibiotics including actinomycin D; the anthracyclines Also provided are kits for treating a disease or condition including doxorubicin, bleomycin, mitomycin C and methra associated with oxidative stress in a Subject comprising res mycin; hormones and hormone antagonists including tamox Veratrol or a functionally equivalent analogue or derivative ifen and cortiosteroids and miscellaneous agents including 30 thereof. cisplatin and brequinar, and regimens such as COMP (cyclo Further provided are kits for treating a disease or condition phosphamide, Vincristine, methotrexate and prednisone), eto associated with DNA damage, including UV induced DNA poside, mBACOD (methotrexate, bleomycin, doxorubicin, damage in skin cells in a Subject comprising resveratrol or a cyclophosphamide, Vincristine and dexamethasone), and functionally equivalent analogue or derivative thereof. PROMACE/MOPP (prednisone, methotrexate (w/leucovin 35 The resveratrol or functionally equivalent analogue or rescue), doxorubicin, cyclophosphamide, taxol, etoposide/ derivative may be a cis-isomer, a trans-isomer or a mixture mechlorethamine, Vincristine, prednisone and procarbazine). thereof. Resveratrol analogues that may be included in the kit Those skilled in the art will appreciate that the invention include, but are not limited to methoxylated resveratrol ana described herein is susceptible to variations and modifica logues, cis-resveratrol glucoside (cis-piceid) and trans-res tions other than those specifically described. It is to be under 40 Veratrol-3-O-3-glucoside (trans-piceid). stood that the invention includes all Such variations and modi The kit may comprise any number of additional compo fications. nents. By way of non-limiting examples the additional com Methods of Treatment ponents may include reference samples, buffers, labels, and The invention contemplates methods for treating diseases written instructions for performing the assay. and conditions associated with oxidative stress and/or DNA 45 The invention will now be described with reference to damage, including UV induced DNA damage in skin cells. In specific examples, which should not be construed as in any one embodiment, the method comprises the administration to way limiting the scope of the invention. a subject of a therapeutically effective amount of resveratrol or a functionally equivalent analogue orderivative thereof. In EXAMPLES another embodiment, the method comprises administering to 50 the Subject a therapeutically effective amount of a pharma Example 1 ceutical composition comprising a synergistic combination of at least one agent that induces NAD synthesis, and an Inhibition of the DeNovo Pathway of NAD" effective amount of one or both of at least one antioxidant and Synthesis by the IDO Inhibitor 1-MT in Human at least one chelating agent. 55 Foetal Astrocytes The skilled addressee will recognise that administration of the compositions of the invention can be used to directly Primary human foetal astrocytes were seeded into 24 well target the stimulation of NAD" synthesis thus providing ben culture plates (~10 cells/well). Each well contained 1 ml of eficial effects for treatment of oxidative stress and/or DNA RPMI 1640 cell culture medium supplemented with 10% damage, including UV induced DNA damage in skin cells, 60 foetal bovine serum (FBS) and 0.5% Glutamax. Cultures involving increased levels of reactive oxygen species such as were left to equilibrate for 24 hours before treatment with the H2O, O...— .OH and NO. Increased synthesis of NAD" IDO inhibitor 1-methyltryptophan (1-MT) (100LLM) alone or provides additional substrate for PARP enzymes involved in in combination with either L-tryptophan (100 uM) or Nico DNA repair and sirtuin enzymes, while also increasing nico tinic acid (100 uM). Following the addition of drug, cultures tinamide metabolism thus removing a potent inhibitor of 65 were incubated for 24 hours at 37° C. in 5% CO, before PARP and sirtuin enzyme activity. Accordingly, the methods analysis of cellular NAD" concentration. Cells were washed of the invention provide a means of alleviating the critical with TRIS buffer (pH7.4) and sonicated in a homogenate US 8,815,936 B2 21 22 solution containing nicotinamide (10 mM) as a PARP inhibi being washed twice and resuspended in 300 u, PBS and tor. A sample of cell homogenate was then added to a reaction placed on ice before Sonication and immediate analysis of mixture containing bicine buffer (120 mM), ethanol (0.6M), cellular NAD levels as described in Example 1 above. Pheny Zine methylsulfate (PMS) (2 mM), MIT (0.5 mM) and Intracellular NAD" was significantly decreased in cultured alcohol dehydrogenase (1 mg/ml). NAD"+NADH levels 5 human foetal astrocytes following exposure to 10-100 uM were quantified spectrophotometrically against a reagent HO, for 20 min (FIG.5) (p<0.05 compared to 0 uMHO, blank using a 570 nm filter in a BioRad X680 microplate control, **p-0.05 compared to 50 uM HO, ***p<0.05 reader (BioRad, Hercules Calif.). compared to 100 uM HO). The reduction in NAD" levels NAD"(H) concentrations per cell were adjusted for vary was inversely correlated to measured PARP-1 activity (shown ing levels of protein by referencing against the total amount of 10 in FIG. 4). protein in the cell homogenate. Total protein was determined using the Bradford Protein Example 4 assay. Briefly, 10 ul of sample cell homogenate (used in NAD'assay) was added into each well with 230 ul of MILLI HO, Induces LDH Release in Human Foetal QR) water and 60 ul of Bradford reagent and left to equilibrate 15 Astrocytes for 10-15 min. The plate was then read using a 595 nm filter in a BioRad X680 microplate reader (BioRad, Hercules Primary human foetal astrocytes were seeded into 24 well Calif.). culture plates (~10 cells/well). Each well contained 1 ml of Complete inhibition of de novo synthesis of NAD" from RPMI 1640 cell culture medium supplemented with 10% tryptophan was induced by the competitive IDO inhibitor foetal bovine serum (FBS) and 0.5% Glutamix. Cultures were 1-MT (1 mM, 24 h) resulting in a significant decrease in left to equilibrate for 24 hours before treatment with the intracellular NAD" levels (FIG. 3). Supplementation with pro-oxidant H2O at increasing concentration between 0 and either excess L-tryptophan or the Salvage pathway Substrate 1000 uM. Following the addition of the HO cultures were nicotinic acid partially reversed the NAD" depletion com incubated for 20 minutes at 37°C. in 5% CO, before super pared to 1-MT treatment alone (FIG. 3). 25 natants were withdrawn and analysed for LDH activity. As LDH is a cytoplasmic enzyme, leakage of LDH into the cell Example 2 culture Supernatant is widely used as a measure of overall cell viability. Briefly, 50 ul of pyruvate (11.5 mM) is added to 50 H2O. Increases PARP Activity in Human Foetal ul of cell culture supernatant. A further 100 ul of NADH Astrocytes 30 solution (700 LM) is added to the wells. LDH activity is measured spectrophotometrically by monitoring the change Primary human foetal astrocytes were seeded into 24 well in absorbance at 340 nm over 5 min.., using a BioRad X680 culture plates (~10 cells/well). Each well contained 1 ml of microplate reader (BioRad, Hercules Calif.) and calculating RPMI 1640 cell culture medium supplemented with 10% the maximal rate of change in absorbance over the linear foetal bovine serum (FBS) and 0.5% Glutamix. Cultures were 35 portion of the curve. left to equilibrate for 24 hours before treatment with the LDH activity (a measure of cell death) was significantly pro-oxidant H2O at increasing concentration between OuM increased in cultured human foetal astrocytes following expo and 1000 uM. Following the addition of the H.O., cultures sure to 10-100 uMHO, for 20 min (FIG. 6) (p<0.05 com were incubated for 15 minutes at 37° C. in 5% CO, before pared to 0 uMHO, control, **p<0.05 compared to 10 uM being washed twice and homogenised/lysed in 200 uL of 40 HO, ***p<0.05 compared to 50 MHO, p<0.05 com PARPassay buffer containing MgCl, (10 mM), Triton X-100 pared to 100DuM H2O). The increase in LDH release (1%), and NAD" (20 uM) in Tris buffer (50 mM, pH 8.1) and directly correlated to the measured decrease in intracellular left to incubate for 60 minutes at 37° C. As NAD is the NAD levels (shown in FIG. 5). exclusive substrate for the PARP enzyme and is degraded during this reaction, total enzyme activity is determined by 45 Example 5 quantitating the reduction in NAD" concentration in the assay buffer solution. The amount of NAD" consumed is measured Antioxidants Inhibit Intracellular NAD" Depletion by the NAD"(H) microcycling assay as previously above. Induced by HO in Human Foetal Astrocytes and PARP-1 activity was significantly increased in cultured Human Neuroblastoma Cells human foetal astrocytes following exposure to 10-100 uM 50 HO for 20 min (FIG. 4) (p<0.05 compared to no H Oxygen radical induced stress can be ameliorated via O; p<0.05 compared to 10 uMHO:Xp<0.05 compared to effective antioxidant therapy. Quercetin is a plant derived 20 uM H.O., p<0.05 compared to 50 uM H.O.). polyphenolic compound with well demonstrated antioxidant activity. The ability of quercetin to modulate HO, induced Example 3 55 NAD depletion in human foetal astrocytes was tested. Primary human foetal astrocytes were seeded into 24 well HO, Decreases Intracellular NAD" in Human culture plates (~10 cells/well). Each well contained 1 ml of Foetal Astrocytes RPMI 1640 cell culture medium supplemented with 10% foetal bovine serum (FBS) and 0.5% Glutamix. Cultures were Primary human foetal astrocytes were seeded into 24 well 60 left to equilibrate for 24 hours before treatment with either culture plates (~10 cells/well). Each well contained 1 ml of DMSO (0.2%) alone or 0.2% DMSO+Quercetin (50 uM). RPMI 1640 cell culture medium supplemented with 10% After a further 24 hour incubation HO was added to selected foetal bovine serum (FBS) and 0.5% Glutamix. Cultures were cultures (see FIG.7) and incubated for 20 minutes at 37°C. in left to equilibrate for 24 hours before treatment with the 5% CO, before being washed twice in PBS, followed by prooxidant H2O at increasing concentration between 0 uM 65 sonication in ~300 uL PBS. The resulting homegenates were and 1000 uM. Following the addition of the HO cultures placed on ice before immediate analysis of cellular NAD" were incubated for 15 minutes at 37° C. in 5% CO, before levels as described in Example 1 above. US 8,815,936 B2 23 24 NAD" levels were significantly preserved in cultured The increased oxidative modification of macromolecules human foetal astrocytes when cultured in the presence of in neurodegenerative disorders such as Alzheimer's disease quercetin overnight followed by exposure to 10-100 uM appear to originate to a significant degree from increased HO, for 20 min (FIG. 7) (p<0.05, *p-0.005 compared to intracellular production of the membrane permeable oxidant, H2O, alone). hydrogen peroxide (H2O) by inefficient mitochondria activ The effect of melatonin on NAD" levels in human neuro ity, increased extra-neuronal production of H2O, activated blastoma cells was also investigated. Human neuroblastoma microglia, and by AB deposits. This highly reactive free radi cells (SK-N-SH) were maintained in RPMI1640 cell culture cal has the potential to induce significant DNA damage lead medium supplemented with 10% foetal bovine serum, 2 mM ing to PARP activation and NAD" depletion. I-glutamine, 1% penicillin/streptomycin, at 37° C. in a 10 humidified atmosphere containing 95% air/5% CO. Before The hypothesis that effective chelation of redox active experimentation cells were seeded into 24 well culture plates metals such as Fe" and Cu" would reduce OH mediated to a density of approximately 5x10 cells and incubated over PARP activation and subsequent NAD depletion was tested. night. On the day of the experiment, the culture medium was Human neuroblastoma cells (SK-N-SH) were maintained in aspirated and discarded. Cells were washed twice with 500 15 RPMI 1640 cell culture medium supplemented with 10% uL PBS before addition of 1 ml PBS/well containing 10 uM foetal bovine serum, 2 mMI-glutamine, 1% penicillin/strep iron, 10 Mascorbic acid (to maintain iron in its Fe" oxida tomycin, at 37° C. in a humidified atmosphere containing tion state), and 100 uM HO. The iron was incubated with 95% air/5% CO. Before experimentation cells were seeded the antioxidant Melatonin (2 uM-200 uM) for 5 mins before into 24 well culture plates to a density of approximately the addition of H2O. All treatments were incubated for a 5x10 cells and incubated overnight. On the day of the experi further 30 mins at 37° C. in 5% CO. Cultures were then ment, the culture medium was aspirated and discarded. Cells washed twice in PBS followed by sonicationin -300 uL PBS. were washed twice with 500 uL PBS before addition of 1 ml The resulting homegenates were placed on ice before imme PBS/well containing 10 uM iron, 10 uMascorbic acid (to diate analysis of cellular NAD" levels as described in maintain iron in its Fe" oxidation state), and 100 uM H.O. Example 1 above. 25 The iron was incubated with the chelator Clioquinol (1 FIG. 8 shows that the depletion of intracellular NAD" uM-100 uM) for 5 minutes before the addition of H.O.. All levels incultured human neuroblastomacells treated with 100 treatments were incubated for a further 30 mins at 37° C. in uMHO, is inhibited by pre-treatment with 2, 20 and 200 uM 5% CO. Cultures were then washed twice in PBS followed of melatonin (p<0.05 vs Ctrl, **p<0.05 100 uMHO, vs 20 by sonication in -300 uL PBS. The resulting homegenates uM Mel+HO, ***p<0.052 M Mel+HO, vs 200uMMel+ 30 were placed on ice before immediate analysis of cellular H2O). NAD" levels as described in Example 1 above. The effect of vitamin E on NAD levels in human neuro The dose response effect of the lipophilic chelator clio blastoma cells was also investigated. Human neuroblastoma quinol (CQ) on intracellular NAD" concentrations in human cells (SK-N-SH) were maintained in RPMI 1640 cell culture neuroblastoma cells treated with HO is shown in FIG. 10 medium supplemented with 10% foetal bovine serum, 2 mM 35 (p<0.05 vs Ctrl, **p<0.05 vs H.O.--Fe, ***p<0.05 vs 10 I-glutamine, 1% penicillin/streptomycin, at 37° C. in a uMCQ+HO+Fe). These results demonstrate that OH-in humidified atmosphere containing 95% air/5% CO. Before duced NAD" depletion is effectively reduced by treatment experimentation cells were seeded into 24 well culture plates with cell permeable chelating agents alone in a dose depen to a density of approximately 5x10 cells and incubated over dant fashion. night. On the day of the experiment, the culture medium was 40 aspirated and discarded. Cells were washed twice with 500 Example 7 uL PBS before addition of 1 ml PBS/well containing 10 uM iron, 10 Mascorbic acid (to maintain iron in its Fe" oxida Resveratrol Increases Intracellular NAD" tion state), and 100 uM HO. The iron was incubated with the antioxidant Vitamin E (2-200 uM) for 5 mins before the 45 Primary human foetal astrocytes were seeded into 24 well addition of H2O. All treatments were incubated for a further culture plates (~10 cells/well). Each well contained 1 ml of 30 mins at 37° C. in 5% CO. Cultures were then washed RPMI 1640 cell culture medium supplemented with 10% twice in PBS followed by sonication in -300 uL PBS. The foetal bovine serum (FBS) and 0.5% Glutamix. Cultures were resulting homegenates were placed on ice before immediate left to equilibrate for 24 hours before treatment with resvera analysis of cellular NAD" levels as described in Example 1 50 trol (100 uM). After a further 24 hour incubation, H2O, was above. FIG.9 shows that the depletion of intracellular NAD" added to selected cultures and incubated for 20 minutes at 37° levels incultured human neuroblastomacells treated with 100 C. in 5% CO, before being washed twice in PBS. Followed by uMHO is inhibited by pre-treatment with 2, 20 and 200LM sonication in ~300 uL PBS. The resulting homogenates were vitamin E (p<0.05 vs Ctrl). placed on ice before immediate analysis of cellular NAD" These results demonstrate that oxidative stress induced 55 levels as described in Example 1 above. NAD" depletion can be ameliorated by treatment with anti Resveratrol significantly increased intracellular NAD" oxidants alone at effective doses. above control levels (p<0.05), **p<0.05 compared to H.O. alone in human primary astrocytes (FIG. 11). While resvera Example 6 trol showed only modest apparent antioxidant activity (Small 60 reduction in NAD" depletion) the effect of resveratrol on Clioquinol Reduces OH-Induced NAD" Depletion intracellular NAD" in cells not subject to oxidative stress was in Human Primary Astrocytes marked, showing an approximately 75% increase in intracel lular NAD" concentration. While modest increases in NAD" Reaction of HO, with available redox active metalions via levels above untreated controls have been observed with other Fenton chemistry leads to the generation of the hydroxyl 65 antioxidants due to reduced constitutive oxidative activity, radical (OH.). the effect of resveratrol was 100% greater than the best of these other antioxidants. Example 8: Resveratrol increases US 8,815,936 B2 25 26 intracellular NAD" in the presence the IDO inhibitor 1-MT or Human brain astrocytes were trypsonised, washed twice, and the QPRT inhibitor PA in primary cells then resuspended in HEPES buffer before being homoge Primary human foetal astrocytes were seeded into 24 well nised by shorts bursts of sonication. Cellular particulates culture plates (~10 cells/well). Each well contained 1 ml of were then removed by centrifugation and the Supernatant was RPMI 1640 cell culture medium supplemented with 10% 5 used for NMNAT activity analysis. 200 uM resveratrol was foetal bovine serum (FBS) and 0.5% Glutamix. Cultures were added to a small volume of supernatant. NMNAT activity was left to equilibrate for 24 hours before treatment with resvera trol (100 uM), 1-MT (1 mM), PA (1 mM), resveratrol (100 then determined in this sample and a sample without resvera uM)+1-MT (1 mM), and resveratrol (100 uM)+PA (1 mM) trol (control) using a protocol modified from Balducci et al. After a further 24 hour incubation cells were washed twice in Anal. Biochem. (1995). 228: 64-68. The standard assay was PBS, followed by sonication in -300 uL PBS. The resulting 10 performed at 37°C. in a 1 ml, 1 cm path curvette in a final homogenates were placed on ice before immediate analysis volume of 850 ul. The reaction mixture contained 240 ul of cellular NAD" levels as described in Example 1 above. HEPES (10 mM, pH 7.4) containing 390 ul ethanol reagent, The effect of resveratrol on intracellular NAD levels MgCl, (40 mM), 100 ul ATP (12.5 mM), 50 ul ADH (0.5 either alone or in the presence of 1-MT, 1 mM (an inhibitor of mg/ml), and the appropriate amount of hr-NMNAT1. The IDO, the first enzyme in de novo NAD synthesis) and PA 1 15 reaction was started by adding nicotinamide mononucleotide mM, (an inhibitor of QPRT, a downstream enzyme in denovo (NMN) to a final concentration of 0.625 mM. The increase in NAD synthesis) (FIG.12) (p<0.05 compared to 1-MT treat absorbance for 10 minutes was recorded continuously at 340 mentalone, **p-0.05 compared to PA treatment alone). Both nm using the Cary 50BIO UV spectrophotometer (Varian, inhibitors were used at concentrations considered to com Sydney). Change in absorbance per minute was calculated pletely block the activity of their respective enzymes. Neither from the slope of the linear progress curve and the amount of inhibitor was able to abrogate the resveratrol effect suggest NADH produced perminute (nM/min) was determined using ing that resveratrol may be able to induce NAD" synthesis via an NADH standard curve. the salvage pathway. Treatment with 200 uM resveratrol increased NMNAT activity in whole cell homogenates of human brain astrocytes Example 9 25 by 67% compared to control (no resveratrol treatment). The effect of resveratrol on the activity of NMNAT was confirmed The NMNAT Inhibitor Tannic Acid Abrogates directly using human recombinant NMNAT (hrNMNAT: Resveratrol-Mediated Increases in Intracellular Alexis Biochemicals) (FIG. 16). The presence of resveratrol NAD significantly increased NMNAT activity in the whole cell 30 extract by approximately 70% (FIG. 16). Resveratrol dose Around 5 enzymes are used by the salvage pathway (see dependently increased Vmax by >500% and decreased the FIG. 1) to generate NAD" from various sources, including Kim by approximately 3 times. The standard assay was per nicotinamide mononucleotide adenylyl transferase (NM formed at 37°C. in a 1 ml, 1 cm path cuvette in a final volume NAT). NMNAT was inhibited and its effect was evaluated in of 850 ul. The reaction mixture contained 240 ul HEPES (10 reference to the previously observed increase in intracellular 35 mM, pH 7.4) containing 390 ul ethanol reagent, MgCl2 (40 NAD" levels caused by resveratrol. Primary human foetal mM), 100 ul ATP (12.5 mM), 50 ul ADH (0.5 mg/ml), and the astrocytes were seeded into 24 well culture plates (~10 cells/ appropriate amount of hr-NMNAT1. The reaction was started well). Each well contained 1 ml of RPMI1640 cell culture by adding NMN to a final concentration of 0.62 mM. medium supplemented with 10% foetal bovine serum (FBS) NMNAT activity was measured for resveratrol (50 uM, 100 and 0.5% Glutamix. Cultures were left to equilibrate for 24 40 uM and 200 uM). The increase in absorbance for 10 minutes hours before treatment with tannic acid (100 uM) or resvera was recorded continuously at 340 nm using the Cary 50BIO trol (100 uM) or resveratrol (100 uM)+tannic acid (100 uM). UV spectrophotometer (Varian, Sydney). Change in absor Cultures were incubated for a further 24 hours at 37°C. in 5% bance per minute was calculated from the slope of the linear CO before being washed twice in PBS, followed by sonica progress curve and the amount of NADH produced per tion in -300 uL PBS. The resulting homogenates were placed 45 minute (nM/min) was determined using an NADH standard on ice before immediate analysis of cellular NAD" levels as CUV. described in Example 1 above. These results strongly suggest that resveratrol is acting Treatment with tannic acid (24 hours, 100 uM) completely directly on the NAD salvage pathway enzyme NMNAT most abrogated the effect of resveratrol on intracellular NAD" likely through an as yet unidentified allosteric up-regulation levels (FIG. 14). Treatment with tannic acid alone (100 uM) 50 of enzyme activity. did not significantly reduce NAD" levels over the 24 hour incubation period. (FIG. 14) (p<0.05 compared to resvera Example 11 trol treatment alone. **p-0.05 compared to control). These results indicate that complete inhibition of NMNAT by tannic Clioquinol and Vitamin E Reduce NAD' Depletion acid was able to prevent the previously observed resveratrol 55 in Neuroblastoma Cells Exposed to H2O and Fe mediated increase in intracellular NAD". The increase in intracellular NAD" levels in the presence of resveratrol may Human neuroblastoma cells (SK-N-SH) were maintained therefore be best explained by an up-regulation in NMNAT in RPMI 1640 cell culture medium supplemented with 10% activity. foetal bovine serum, 2 mMI-glutamine, 1% penicillin/strep 60 tomycin, at 37° C. in a humidified atmosphere containing Example 10 95% air/5% CO. Before experimentation cells were seeded into 24 well culture plates to a density of approximately Resveratrol Increases NMNAT Activity in Human 5x10 cells and were incubated overnight before experimen Brain Astrocytes tation. On the day of the experiment, the culture medium was 65 aspirated and discarded. Cells were then washed twice with The effect of resveratrol on NMNAT activity in whole cell 500 uL DPBS before addition of 1 ml DPBS/well containing homogenates of human astrocytes is shown in (FIG. 15). 10 uM iron or copper, 10 uMascorbic acid (to maintain iron US 8,815,936 B2 27 28 in its Fe" oxidation state), and 100 uM H.O. The iron was Example 13 incubated with the chelator clioquinol (CQ) and/or 20 LM of the antioxidant vitamin E (VitE) for 5 mins before the addi Effect of U.V. Radiation on DNA Damage and tion of H2O. All combinations were incubated for 30 mins Repair in Human Skin Cells (Keratinocytes, before NAD" assays were performed as described in Example 5 Fibroblasts) 1 above. Treatment with the chelator, clioquinol, and the antioxi Human skin cells (keratinocytes and fibroblasts) were dant, Vitamin E, produced a synergistic protection against grown at a density of 5x10° in PERMANOX(R) chamber free radical induced NAD" depletion in neuroblastoma cells slides (NUNC) for 2 weeks. This method is commonly used 10 in the industry and is well known to those skilled in the art. (FIG. 18). Accordingly, there is no need to detail the method utilised in this specification. Example 12 Cells were exposed to 15 min UVB (-200 m.Jcm’). The Effect of U.V. Radiation+/-Supplementation on culture medium was then supplemented with any of the fol 15 lowing: Intracellular NAD and Extracellular LDH Activity in 100 uM Clioquinol (C), 100 uM Picolinamide (P), 100 uM Cultures of Human Skin Cells (Keratinocytes, Melatonin (M) or 100 uM Resveratrol (R) as single Fibroblasts) therapy; 10 uM(R)--50 uM (M), 10 uM(R)+0.1 uM (C) or 90 uM Human skin cells (keratinocytes) were grown at a density (R)+0.08 uM (P) as dual therapy; or of 5x10 in PERMANOX(R) chamber-slides (NUNC) for 2 0.65 uM (P)+33 uM (C)+5uM (R) or 0.3 uM (P)+33 uM weeks. This method is commonly used in the industry and is (C)+5uM(R) as triple therapy. well known to those skilled in the art. Accordingly, there is no All doses used for single therapy were equivalent to ICoo need to detail the method utilised in this specification. for each therapeutic, whilst optimum dose combinations used Cells were either left unexposed or exposed to 15 min UVB 25 for dual therapy were {ICso (R)+ICso (M)}, {ICo (R)+ICoo (-200 mljcm?). The culture medium was then supplemented (C)}, {ICoo (R)+IClo (P) and the optimum dose combina with either 0.1-100 uM Clioquinol (CLO) (FIG. 19a), Picoli tions for the triple therapy were IC (R)+IC (M)+IC namide (PAM) (FIG. 19b), Melatonin (MEL) (FIG. 19C) or (C)}, {IC (R)+IC (M)+IC (P)}. Resveratrol (RES) (FIG. 19d) as single therapy for 24 hrs. Cells were left to incubate for 24 hrs before analysis of Cells were left to incubate for 24 hrs before analysis of 30 DNA damage (see FIGS. 22a and 22b for Keratinocytes and intracellular NAD (FIG. 19a-d to FIG. 21) and extracellular FIGS. 23a and 23b for Fibroblasts). LDH (FIG. 21). FIGS. 19a-d show the intracellular NAD For Immunocytochemistry visualisation and semi quanti concentrations--/-UV exposure, +/-Supplementation. FIGS. tation, cells were fixed with acetone/methanol (vol/vol) for 20 19e-h show supernatant LDH activity+/-UVE exposure, minat -20°C. Cells were then rinsed 3 times with PBS and a 35 gentle membranous permeabilization was performed by incu +/-Supplementation. bation with 0.025% Triton X 100 in PBS for 10 min at room FIGS. 19a-d show that NAD levels are significantly temperature. After washing, cells were incubated with 5% reduced, and LDH activity in the Supernatant (indicating cell normal goat serum (NGS) in PBS for 45 min at room tem death, FIGS. 19e-h) is correspondingly increased in human perature, rinsed twice with PBS and incubated for 1 h at 37° fibroblasts exposed to UVB radiation. Minimum effective 40 C. with the primary antibody 6-4 PP mAb (Cosmo Bio Ltd, concentrations (MEC) for each therapeutic were: 0.1 uM Japan) diluted in 5% NGS. Cells were then washed with 5% MEL, 0.1 uM CLQ, 10 uM MEL and 50 uM RES, (N=3 for NGS solution and incubated for 1 h at 37°C. with the appro each treatment group). priate labelled secondary antibodies (goat anti-mouse IgG FIGS. 20a and 20b show that, in general, dual therapies coupled with Alexa 594). Nuclear staining was performed containing RES are much more Successful in regenerating 45 using DAPI at 1 lug/ml for 5 min at room temperature. After intracellular NAD and minimising overall cell death (LDH several washings with PBS at 37° C., the cover slips were activity) compared any other dual combination. Importantly quickly mounted on glass slides with Fluoromount-G, and selected dual therapy combinations were able to return NAD examined using an Olympus BX60 fluorescence microscope levels and reduce cell death (LDH activity) to no-UV treat associated with a digital SensiCam. The following three con ment control levels. FIG.20a shows the effect of dual supple 50 trols were performed for each labelling experiment: 1) iso mentation on NAD concentrations following treatment with typic antibody controls for mAbs and serum control for p Abs, RES+/-PAM+/-CLQ+/-MEL on NAD recovery 24 hr after 2) incubation with only the secondary labelled antibodies, 3) UVB exposure, in human primary Keratinocytes. The lighter estimation of auto-fluorescence of unlabelled cells. Intensity shade bars (in greyscale) represent treatment containing RES. has been quantified (semi-quantification) using Image.J 10.2. FIG. 20b shows the effect of dual supplementation on LDH 55 Three individual microscopic fields were analysed for each release (cell death) following treatment with RES+MEL or treatment and the SEM for the data was determined to be RES+CLQ or RES+PAM for 24 hr after UVB exposure, in <5%. human primary Keratinocytes. FIG. 21a also shows that triple FIGS. 22a and 23a show intense staining for DNA damage therapy with RES/MEL/PIC was able to return NAD levels (middle column) for UV treated cells without supplementa and reduce cell death (LDH activity) to no-UV treatment 60 tion. The intensity of the DNA damage signal was slightly control levels. FIG. 21 shows the effect of triple supplemen weaker for single therapy, weaker still for dual therapy and no tation (selected combinations) on NAD concentrations fol different to -ve control (i.e. no UV damage) in cells treated lowing treatment with RES+MEL+CLQ or RES+MEL+ with triple therapy (N=3, Keratinocytes, N=1 Fibroblasts for PAM on NAD recovery 24 hr after UVB exposure, in human each treatment group). Note that maximum effective single primary Keratinocytes. For each therapeutic Supplement a 65 therapies (ICoo) were not as effective as ICoo equivalent concentration equal to its EC was used for the triple com dual therapies that were in turn not as effective as ICoo bination. equivalent triple therapies. US 8,815,936 B2 29 30 FIGS. 22b and 23b show the semiquantitation of fluores weeks. This method is commonly used in the industry and is cent intensities for each of the ICC stained sections indicating well known to those skilled in the art. Accordingly, there is no strong graphical evidence for triple therapy producing the need to detail the method utilised in this specification. most effective DNA repair. Cells were either left unexposed or exposed to 15 min UVB (~200 mljcm?). The culture medium was then supplemented Example 14 with either 100 uM Clioquinol (CLO), Picolinamide (PAM), Effect of U.V. Radiation+/-Supplementation on Melatonin (MEL) or Resveratrol (RES) as single therapy for DNA Repair in Cultures of Human Skin Cells 24 hrs. (Fibroblasts) Cells were left to incubate for 24 hrs before analysis of 10 intracellular residual DNA damage (dsDNA breaks) (FIG. Human skin cells (Fibroblasts) were grown at a density of 27). When residual dsDNA breaks is less than that for the UV 5x10 in PERMANOX(R) chamber-slides (NUNC) for 2 treatment control this indicates efficiency of DNA repair. weeks. This method is commonly used in the industry and is FIG. 27 shows that the amount of dsDNA breaks is signifi well known to those skilled in the art. Accordingly, there is no cantly reduced following mono therapy with RES, PAM, need to detail the method utilised in this specification. 15 CLO or MEL. With the greatest apparent improvement Cells were either left unexposed or exposed to 15 min UVB observed for the MEL treated group with ~30% reduction in (-200 mljcm?). The culture medium was then supplemented damaged DNA. FIG. 27 shows the effect of mono therapy with either 100 uM Clioquinol (CLO), Picolinamide (PAM), supplementation on DNA repair following treatment with Melatonin (MEL) or Resveratrol (RES) as single therapy for RES (100 uM), MEL (100 uM), CLQ (0.1 uM) or PAM (100 24 hrs. uM) for 24 hr after UVB exposure, in human primary Kera Cells were left to incubate for 24 hrs before analysis of tinocytes. The concentrations used were equivalent to EC intracellular residual DNA damage (dsDNA breaks) (FIG. for each therapeutic. 24). When residual dsDNA breaks is less than that for the UV FIG. 28 shows that the amount of dsDNA breaks is also treatment control this indicates efficiency of DNA repair. FIG. 24 shows the effect of monotherapy supplementation on significantly reduced following dual therapy with RES+ DNA repair following treatment with RES (100 uM), MEL 25 MEL RES+CLQ, RES+PAM. With the greatest improve (100 uM), CLQ (0.1 uM) or PAM (100 uM) for 24 hr after ment observed for the MEL treated group with ~40% reduc UVB exposure, in human primary Fibroblasts. The concen tion in damaged DNA. FIG. 28 shows the effect of dual tration used were equivalent to ECoo for each therapeutic. therapy Supplementation on DNA repair following treatment FIG. 24 shows that the amount of dsDNA breaks is signifi with RES+MEL RES+CLQ, RES+PAM for 24 hr after UVB cantly reduced following mono therapy with RES, PAM, 30 exposure, in human primary Keratinocytes. Concentrations CLO or MEL. With the greatest apparent improvement used were equivalent to ECs/ECs. EC/ECo EC/EC observed for the MEL treated group with ~40% reduction in respectively. damaged DNA. However, FIG. 29 shows that the amount of dsDNA breaks FIG. 25 shows that the amount of dsDNA breaks is also is even more significantly reduced following triple therapy significantly reduced following dual therapy with RES+ 35 with RES+MEL+CLQ or RES+MEL+PAM. The greatest MEL RES+CLQ, RES+PAM. With the greatest improve improvement in DNA repair was observed for the RES+ ment observed for the MEL treated group with >60% reduc MEL+PAM treated group with ~60% reduction in damaged tion in damaged DNA. FIG. 25 shows the effect of dual DNA. FIG. 29 shows the effect of triple therapy supplemen therapy Supplementation on DNA repair following treatment tation on DNA repair following treatment with RES+MEL+ with RES+MEL RES+CLQ, RES+PAM for 24hr after UVB 40 CLQ or RES+MEL+PAM for 24 hr after UVB exposure, in exposure, inhuman primary Fibroblasts. Concentrations used human primary Keratinocytes. were equivalent to ECso/ECso, EC/ECo EC/EC Taken together, this data clearly shows that, dual therapies respectively. are much more Successful in enhancing DNA repair (as was However, FIG. 26 shows that the amount of dsDNA breaks is even more significantly reduced following triple therapy previously observed for regenerating intracellular NAD) with RES+MEL+CLQ or RES+MEL+PAM. The greatest 45 compared to any mono therapy (even at EC concentra improvement in DNA repair was observed for the RES+ tions). It also shows that triple therapies are even more effec MEL+PAM treated group with >80% reduction in damaged tive than dual therapies (even using EC equivalent combi DNA. FIG. 26 shows the effect of triple therapy supplemen nations) at enhancing DNA repair. tation on DNA repair following treatment with RES+MEL+ CLQ or RES+MEL+PAM for 24 hr after UVB exposure, in 50 Example 16 human primary Fibroblasts. Taken together, this data clearly shows that, dual therapies Synergistic Compositions containing RES are much more successful in enhancing DNA repair (as was previously observed for regenerating intracel Human primary human foetal astrocytes were cultured as lular NAD) compared any monotherapy (even at ECo con 55 described in Guillemin et al. J. Neurochem. (2001) 78(4): centrations). It also shows that triple therapies were even 842-853. Intracellular NAD" levels were measured using the more effective than dual therapies (even using ECoo equiva method described by Grant and Kapoor.J. Neurochem. (1998) lent combinations) at enhancing DNA repair. 70(4): 1759-1763. Intracellular NAD" concentration was adjusted for variations in cell number between cultures by Example 15 60 measuring the amount of protein in the cell homogenate using the Bradford protein assay is derived by Bradford Anal. Bio Effect of U.V. Radiation+/-Supplementation on chem. (1976) 53: 452-458. DNA Repair in Cultures of Human Skin Cells Approximately 1x10 cells were seeded into 24 well cul (Keratinocytes) ture plates and maintained in culture for 24 hours as previ 65 ously described in Guillemin et al. J. Neurochem. (2001) Human skin cells (Keratinocytes) were grown at a density 78(4): pp. 842-853. Selected concentrations of individual of 5x10 in PERMANOX(R) chamber-slides (NUNC) for 2 drug or drug combinations (Clioquinol 10 uM, Melatonin 5 US 8,815,936 B2 31 32 uM, resveratrol 100LLM) were added in 10 LILaliquots to 1 mL disease or condition is characterized by the presence of excess of medium. After 24 hours culture medium was removed and oxidative compounds in the Subject, the method consisting of washed twice with replaced with phosphate buffered saline administering to the Subject a composition consisting of a (PBS). 300 uL of PBS containing 100 uM HO, was then synergistic composition of: added to each culture well and left to incubate at 37°C. in 5% 5 100 uM of resveratrol or a functionally equivalent ana CO for 15 minutes. The PBS was then aspirated from each logue or derivative thereof effective to promote culture and replaced with fresh homogenate mixture. Cells NAD+ synthesis in the subject; were then sonicated and the homogenate analysed for NAD+ 10 uM of clioquinol to reduce production of additional (H) and total protein. oxidative compounds in the Subject; and FIG. 30 shows the effect of 24 hour pre-treatment with 10 5uM of melatonin to minimize the oxidative activity in Melatonin-5 uM (antioxidant) and/or Clioquinol-10 uM the subject. (Fe"/Cut Chelator) and/or resveratrol 100 uM (NMNAT 2. The method according to claim 1, wherein the disease or enzyme activator) on intracellular NAD" levels following 15 condition includes a neurodegenerative disorder, accelerated min of HO, (100 uM) induced oxidative stress. NAD" levels aging of the skin, and/or DNA damage. were significantly higher in cells treated with triple therapy 15 3. The method according to claim 1, wherein the neurode compared to treatment with any single agent or combination generative disorder is Alzheimer's disease or Parkinson's of any two agents. disease. HO. Treatment 4. The method according to claim 2, wherein the DNA FIG. 30 shows that cells treated with HO results in a damage includes UV-induced DNA damage in the subjects significant decrease in intracellular NAD levels. This result is skin cells. due to hydroxyl radical (Fenton chemistry) induced DNA 5. The method according to claim 4, wherein the skin cells damage and resultant overactivation of the DNA repair are keratinocytes and fibroblasts. enzyme PARP which uses NAD as a substrate. 6. The method according to claim 2, wherein the disease or Mono Drug Therapy condition is cancer. Pre-treatment with the potent antioxidant melatonin or the 25 7. The method according to claim 2, wherein the disease or Fe"/Cut chelator clioquinol significantly reduced NAD" condition results from exposure to ultra-violet light, ionizing depletion (FIG. 30). However pre-treatment with resveratrol radiation, exposure to chemical agents, infection, inflamma alone did not show a significant change in intracellular NAD" tion, reduced mitochondrial efficiency, or a combination compared to oxidative (H2O) insult alone. thereof. Dual Drug Therapy 30 8. The method according to claim 1, wherein the resvera Pre-treatment with any two of clioquinol, melatonin or trol or functionally equivalent analogue or derivative is a resveratrol preserved NAD" levels following oxidative functionally equivalent analogue selected from the group (HO) insult to a significantly greater degree that any mono consisting of hydroxylated resveratrol analogues, methoxy drug therapy (FIG. 19). Dual therapy containing resveratol lated resveratrol analogues, cis-resveratrol glucoside (cis preserved NAD" levels to a significantly greater degree that 35 piceid), and trans-resveratrol-3-O-B-glucoside (trans-pi therapies that did not contain resveratrol (FIG. 19), providing ceid). indication of a synergistic effect. 9. The method according to claim 1, wherein the resvera Triple Drug Therapy trol or a functionally equivalent analogue orderivative thereof Pre-treatment with all three of clioquinol plus melatonin promotes NAD+ synthesis by upregulating nicotinamide plus resveratrol preserved NAD" levels following oxidative 40 mononucleotide adenylyl transferase (NMNAT) activity in (H2O) insult to a significantly greater degree that any single the Subject, thereby increasing the metabolism of nicotina or dual combination drug therapy (FIG. 19) (p<0.05, mide (NAM). **p<0.05 compared to H2O2 alone, ***p<0.05 compared to 10. The method according to claim 9, wherein the increase each dual therapy treatment). in the metabolism NAD" increases PARP enzyme activity, 45 thereby further promoting DNA repair in the subject, and also INDUSTRIAL APPLICABILITY providing a correlated increase in mammalian sirtuin enzyme The present invention can be utilised in respect of methods activity, thereby promoting cell viability and longevity in the and compositions for inducing DNA repair and NAD" syn Subject. thesis in a subject. Particularly, the invention can be utilised 50 11. The method according to claim 10, wherein the PARP with respect to methods and pharmaceutical compositions for enzymes are selected from PARP-1 or PARP-2. the prevention and treatment of conditions and diseases asso 12. The method according to claim 10, wherein the mam ciated with oxidative stress and/or DNA damage. malian sirtuin enzyme family members are selected from the The invention claimed is: group consisting of SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, 1. A method of treating a disease or condition associated SIRT6, and SIRT7. with oxidative stress or DNA damage in a subject, wherein the