USOO86481.12B2 (12) United States Patent (10) Patent No.: US 8,648,112 B2 Seeram et al. (45) Date of Patent: *Feb. 11, 2014 (54) THERAPEUTIC USES OF UROLITHINS Keratinocytes'. Photochemistry and Photobiology (Jan.-Feb. 2005), 81:38-45. Albrecht; et al., “Pomegranate Extracts Potently Suppress Prolifera (75) Inventors: Navindra P. Seeram, Kingston, RI (US); tion, Xenograft Growth, and Invasion of Human Prostate Cancer David Heber, Los Angeles, CA (US) Cells”, Journal of Medicinal Food (2004), 7(3):274-283. Cerda; et al., “Evaluation of the bioavailability and metabolism in the (73) Assignee: The Regents of the University of rat of punicalagin, an antioxidant polyphenol from pomegranate California, Oakland, CA (US) juice'. Eur, J. Nutr. (Jan. 2003), 42:18-28. Cerda; et al., “Metabolism of Antioxidant and Chemopreventive (*) Notice: Subject to any disclaimer, the term of this Ellagitannins from Strawberries, Rasberries, Walnuts, and Oak-Aged patent is extended or adjusted under 35 Wine in Humans: Identification of Biomarkers and Individual Vari ability”. J. Agric. Food Chem. (Jan. 2005), 53:227-235. U.S.C. 154(b) by 0 days. Cerda; et al., “Pomegranate juice Supplementation in chronic This patent is Subject to a terminal dis obstructive pulmonary disease: a 5-week randomized, double-blind, claimer. placebo-controlled trial”. European Journal of Clinical Nutrition (Feb. 2006): 60:245-253. Cerda; et al., “Repeated Oral Administration of High Doses of the (21) Appl. No.: 13/452,204 Pomegranate Ellagitannin Punicalagin to Rats for 37 Days Is Not Toxic”, J. Agric. Food Chem. (May 2003), 51:3493-3501. (22) Filed: Apr. 20, 2012 Cerda; et al., “The potent in vitro antioxidant elagitannins from pomegranate juice are metabolised into bioavailable but poor (65) Prior Publication Data antioxidant hydroxy-6H-dibenzopyran-6-one derivatives by the US 2012/O264819 A1 Oct. 18, 2012 colonic microflora of healthy humans”, Eur, J. Nutr. (Aug. 2004), 43:205-220. Kiss; et al., “Induction of neutral endopeptidase activity in PC-3 cells Related U.S. Application Data by an aqueous extract of Epilobium angustifolium L. and oenothein B”, Phytomedicine (Mar. 2006), 13(4):284-289. (63) Continuation of application No. 12/298,122, filed as Lansky; et al., “Possible synergistic prostate cancer Suppression by application No. PCT/US2007/010054 on Apr. 26, anatomically discrete pomegranate fractions'. Investigational New 2007, now Pat. No. 8,183,282. Drugs (Jan. 2005), 23:11-20. Larrosa; et al., “Urolithins, Ellagic Acid-Derived Metabolites Pro (60) Provisional application No. 60/745,717, filed on Apr. duced by Human Colonic Microflora, Exhibit Estrogenic and 26, 2006. Antiestrogenic Activities”. J. Agric. Food Chem. (Mar. 2006), 54:1611-1620. (51) Int. Cl. Malik, et al., “Pomegranate fruit juice for chemoprevention and A6 IK3I/366 (2006.01) chemotherapy of prostate cancer', PNAS (Oct. 2005), (52) U.S. Cl. 102(41): 14813-8. USPC .......................................................... S14/455 Sakagami H; et al., “Cytotoxic activity of hydrolyzable tannins against human oral tumor cell lines: A possible mechanism'. (58) Field of Classification Search Phytomedicine (Mar. 2000), 7(1):39-47, abstract only. None Seeram; et al., “In vitro antiproliferative, apoptotic and antioxidant See application file for complete search history. activies of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found (56) References Cited in pomegranate juice'. Journal of Nutritional Biochemistry (Jun. 2005), 16:360-367. U.S. PATENT DOCUMENTS Seeram; et al., “Pomegranate Juice Ellagitannin Metabolites Are Present in Human Plasma and Some Persist in Urine for Up to 48 7,678,549 B2 3/2010 Buxton Hours”. The Journal of Nutrition (Oct. 2006), 136:2481-2485. 8,183,282 B2 * 5/2012 Seeram et al. ................ 514,455 2005/0282781 A1* 12/2005 Ghosal ............................ 514.80 * cited by examiner OTHER PUBLICATIONS Primary Examiner — James D Anderson Adams, et al., “Pomegranate Juice, Total Pomegranate Ellagitannins, (74) Attorney, Agent, or Firm — Bozicevic, Field & Francis and Punicalagin Suppress Inflammatory Cell Signalling in Colon LLP: Pamela J. Sherwood Cancer Cells”. J. Agric. Food Chem. (Feb. 2006), 54:980-985. Afaq; et al., “Anthocyanin- and Hydrolyzable Tannin-Rich Pome (57) ABSTRACT granate Fruit Extract Modulates MAPK and NF-KappaB Pathways and Inhibits Skin Tumorigenesis in CD-1 Mice'. Int. J. Cancer (Jan. The Subject invention is drawn to elagitannin metabolites 2005), 113:423-433. (e.g., urolithin) that find use in treating or preventing a neo Afaq; et al., “Pomegranate Fruit Extract Modulates UV-B mediated plastic disease in a Subject. Phosphorylation of Mitogen-activated Protein Kinases and Activa tion of Nuclear Factor Kappa B in Normal Human Epidermal 14 Claims, 7 Drawing Sheets U.S. Patent Feb. 11, 2014 Sheet 1 of 7 US 8,648,112 B2 FIGURE Ellagic acid (2) MI (arolithin B U.S. Patent Feb. 11, 2014 Sheet 2 of 7 US 8,648,112 B2 s es s &xé 5 S. : 3 - 5 g 3 : 3 & 8 (oiluoore dues) (oluoofed ues) sisodody s sododw O CD soF 3.S 5 5 5 3 s E &r 8 3 as (ouluoo?e dues) (ouoo?e dues) s sodod W sisodody U.S. Patent Feb. 11, 2014 Sheet 3 of 7 US 8,648,112 B2 Colon Cancer HCT-116 2 OMSO Ellagic acid Urolithin A Methyl Urolithin-3 Urolithin-A Samples (100ppm) FIGURE 3 U.S. Patent Feb. 11, 2014 Sheet 4 of 7 US 8,648,112 B2 Pancreatic Cells ASPC-1 2O OO 8 O 2 O O OMSO Elagic acid Urolithin A Methyl Urolithin 3 Urolithin A Samples (100ppm) A Pancreatic Cells BXPC-3 OMSO Elagic acid Urolithin-A Methyl Unlithin 3 troithin-A Samples (100ppm) B FIGURE 4 U.S. Patent Feb. 11, 2014 Sheet 7 Of 7 US 8,648,112 B2 s . s: is x: : 8 : . : : is o so o & 3. is s is & s & : 3. si. s x : : s: s x : : & it e e --->8 as & 8 . : * :: 8 . & 3 & . it . is is: . all e de Au?fu "fifu epuounongf-wn & 8 . de A upfu f/6u (wn US 8,648,112 B2 1. 2 THERAPEUTIC USES OF UROLITHINS In one embodiment of the invention, a pharmaceutical composition is provided, comprising an effective dose of an FEDERALLY SPONSORED RESEARCH AND ellagitannin metabolite and a pharmaceutically acceptable DEVELOPMENT excipient. The effective dose will usually be effective for 5 inhibiting the proliferation of a cell, including tumor cells, This invention was made with Government support of e.g. solid tumor cells, such as carcinomas, e.g. pancreatic Grant No. AT000151 awarded by the National Institutes of carcinomas, prostate carcinomas, and the like. In certain Health. The Government has certain rights in this invention. embodiments, the ellagitannin metabolites of interest are urolithins and/or derivatives thereof. INTRODUCTION 10 In another embodiment of the invention, methods are pro vided for inhibiting undesirable cell proliferation, the method Ellagitannins (ETs) are polymeric polyphenols abundant comprising contacting the cells with an effective dose of an in Some fruits and nuts such as pomegranates, raspberries, ellagitannin metabolite and a pharmaceutically acceptable strawberries, black raspberries, walnuts and almonds. excipient. Cells of interest include, without limitation, tumor Despite numerous reports of the biological properties and cells, e.g. solid tumor cells, such as carcinomas, e.g. pancre human health benefits of ETs, knowledge of their bioavail 15 atic carcinomas, prostate carcinomas, and the like. In certain ability, pharmacokinetics, disposition and metabolic fate in embodiments, the ellagitannin metabolites of interest are humans is limited. urolithins and/or derivatives thereof. Pomegranate (Punica granatum L.) fruits are widely con In some embodiments of the invention, patients are classi Sumed fresh and as beverages such as juice (PJ). In commer fied as metabolite producers and metabolite non-producers, cial juice processing methods, ETs which are abundant in the where treatment may be tailored to the status of the patient. fruit peels, are extracted in large quantities into the juice. Punicalagin 2.3 hexahydroxydiphenoyl-4,6-gallagylglu BRIEF DESCRIPTION OF THE DRAWINGS cose), which occurs as isomers (FIG. 1), is the predominant ET present in PJ as a result of this process. ETs belong to the FIG. 1: Chemical structures of punicalagin isomers (1), the chemical class of hydrolyzable tannins, which release ellagic 25 major ellagitannin (ET) present in pomegranate juice, and its acid (EA) on hydrolysis. In addition, PJ contains other hydrolysis product, ellagic acid (EA) (2). polyphenols such as anthocyanins, which are present in the FIG. 2: Pro-apototic activity of urolithin B against four fruitarils, imparting its brilliant ruby-red color. human prostate cancer cell lines: A=22Rv1; B=PC3; The potent antioxidant properties of PJ have been attrib C-LNCaP; D=LNCaP-AR. Statistical significance are: uted to its high content of punicalagin isomers which can 30 *p=0.05 and **p=0.01. reach levels.>2 g/Ljuice. ETs have also been identified as the FIG. 3: Anti-proliferative activity of urolithins against a active anti-atherogenic compounds in PJ. It has been sug human colon cancer cell line HCT-116. gested that pomegranate ETs and pomegranate fruit extracts FIGS. 4A and 4.B. Anti-proliferative activity of urolithins inhibit the proliferation of human cancer cells and modulate against human pancreatic cell lines ASPC-1 and BXPC-3. inflammatory sub-cellular signaling pathways and apoptosis, FIG. 5: Inhibition of tumor xenograft (LAPC-4) growth by see, for example, Seerametal. (2005).J Nutr Biochem. 2005; 35 PE in SCID mice. Inhibition of growth was significant begin 16:360-7: Adams et al. (2006) JAgric Food Chem. 2006, 54, ning at two weeks after initiation of PE administration (0.8 980-85; Afaq et al. (2005) Photochem Photobiol. 2005; mg/mouse/dose) orally (p<0.05) with greater than 50 percent 81:38-45; Afaq et al.