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 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 , 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 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 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, -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., ) 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 S3. 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

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. 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 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. (2005) Int J. Cancer. 2005; 113:423-33. inhibition of tumor volume by 6 weeks after tumor injection. Pomegranate fruit extract has also been suggested to reduce PE or vehicle control was administered when tumors became prostate tumor growth and prostate serum antigen (PSA) 40 palpable 2 weeks after injection of 200,000 prostate tumor levels in athymic nude mice implanted with CWR22Rv1 cells (LAPC-4). prostate cells (Malik et al. (2005) Proc Natl Acad. Sci. 2005; FIGS. 6A-6B. (A) Concentrations of urolithin A (UA) and 102: 14813-8. UA-conjugates: methylated UA, UA sulfate, and UA-glucu Although the absorption, metabolism, distribution and ronide in plasma (ng/mL) and tissues (ng/g) of mice that excretion of pomegranate ETs in animals and in humans, have 45 received UA (0.3 mg/mouse/dose) by the oral route. Metabo been reported, their pharmacokinetic parameters remain lite levels at the 24 h time point were below the detectable uninvestigated. It is becoming clear that considerable inter limit (3 ng/mL for blood and 5 ng/g for tissues) and are individual variability occurs in polyphenol metabolism in therefore not shown. Data show the mean+SE for n=6 mice humans. As such, there is a need to establish bioavailability, per time point. (B) Concentrations of urolithin A (UA) and metabolism and pharmacokinetic parameters of pomegranate 50 UA-conjugates: methylated UA, UA sulfate, and UA-glucu ETs in human volunteers and to link ETs and/or metabolites ronide in plasma (ng/mL) and tissues (ng/g) of mice that thereof to specific beneficial properties, e.g., antioxidant, received UA (0.3 mg/mouse/dose) by the intraperitoneal cancer cell antiproliferative and pro-apoptotic effects, etc. route. Metabolite levels at the 24 h time point were below the This invention meets these and other needs. detectable limit (3 ng/mL for blood and 5 ng/g for tissues) and are therefore not shown. Data show the meantSE for n=6 SUMMARY OF THE INVENTION 55 mice per time point. The present invention is drawn to metabolites of pome DESCRIPTION OF THE SPECIFIC granate polyphenols (e.g., ellagitannins) or derivatives EMBODIMENTS thereof, which polyphenols find use in methods of treating and/or preventing a hyperproliferative disease in a subject. 60 The present invention is drawn to metabolites of pome The subjectellagitannin metabolites are shown hereinto have granate polyphenols (e.g., ellagitannins) that find use in meth an anti-proliferative and/or pro-apoptotic effect on cells, and ods of treating and/or preventing a neoplastic disease in a as such, find use in the treatment and prevention of a number Subject. The subject ellagitannin metabolites are shown of disease states, e.g. to inhibit tumor growth, to decrease herein to have an anti-proliferative and/or pro-apoptotic inflammation associated with alymphoproliferative disorder, 65 effect, and as such, find use in the treatment and prevention of to inhibit graft rejection, or neurological damage due to tissue a number of hyperproliferative disease states. Among the repair, etc. tumor cells found to be sensitive to treatment with elagitannin US 8,648,112 B2 3 4 metabolites are solid tumors, e.g. carcinomas Such as pancre Organic Chemicals, Eastman Kodak Company (Rochester atic carcinoma cells, colon carcinoma cells, etc. Treatment of N.Y.), Fisher Scientific Co. (Pittsburgh Pa.), Fisons Chemi prostate carcinoma, including androgen sensitive and insen cals (Leicestershire UK). Frontier Scientific (Logan Utah), sitive prostate cancers, is also of interest. In some embodi ICN Biomedicals, Inc. (Costa Mesa Calif.), Key Organics ments, patients are classified as metabolite producers and (Cornwall U.K.), Lancaster Synthesis (Windham N.H.), metabolite non-producers, where treatment may be tailored Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish to the status of the patient. In certain embodiments, the ella Chemical Co. (Orem Utah), Pfaltz & Bauer, Inc. (Waterbury gitannin metabolites of interest are urolithins. Conn.), Polyorganix (Houston Tex.), Pierce Chemical Co. Before the present invention is described in greater detail, (Rockford Ill.), Riedel de Haen AG (Hannover, Germany), it is to be understood that this invention is not limited to 10 Spectrum Quality Product, Inc. (New Brunswick, N.J.), TCI particular embodiments described, as Such may vary. It is also America (Portland Oreg.), Trans World Chemicals, Inc. to be understood that the terminology used herein is for the (Rockville Md.), Wako Chemicals USA, Inc. (Richmond purpose of describing particular embodiments only, and is not Va.); Molecular Probes (Eugene, Oreg.): Applied Biosys intended to be limiting, since the scope of the present inven tems, Inc. (Foster City, Calif.); and Glen Research (Sterling, tion will be limited only by the appended claims. 15 Va.). Where a range of values is provided, it is understood that As used herein, “suitable conditions” for carrying out a each intervening value, to the tenth of the unit of the lower synthetic step are explicitly provided herein or may be dis limit unless the context clearly dictates otherwise, between cerned by reference to publications directed to methods used the upper and lower limit of that range and any other stated or in synthetic organic chemistry. The reference books and trea intervening value in that stated range, is encompassed within tise set forth above that detail the synthesis of reactants useful the invention. The upper and lower limits of these smaller in the preparation of compounds of the present invention, will ranges may independently be included in the Smaller ranges also provide Suitable conditions for carrying out a synthetic and are also encompassed within the invention, Subject to any step according to the present invention. specifically excluded limit in the stated range. Where the As used herein, “methods known to one of ordinary skill in stated range includes one or both of the limits, ranges exclud the art” may be identified though various reference books and ing either or both of those included limits are also included in 25 databases. Suitable referencebooks and treatise that detail the the invention. synthesis of reactants useful in the preparation of compounds Unless defined otherwise, all technical and scientific terms of the present invention, or provide references to articles that used herein have the same meaning as commonly understood describe the preparation, include for example, “Synthetic by one of ordinary skill in the art to which this invention Organic Chemistry”, John Wiley & Sons, Inc., New York; S. belongs. Although any methods and materials similar or 30 R. Sandler et al., “Organic Functional Group Preparations.” equivalent to those described herein can also be used in the 2nd Ed., Academic Press, New York, 1983; H. O. House, practice or testing of the present invention, representative “Modern Synthetic Reactions”, 2nd Ed., W. A. Benjamin, Inc. illustrative methods and materials are now described. Menlo Park, Calif. 1972: T. L. Gilchrist, “Heterocyclic All publications and patents cited in this specification are Chemistry, 2nd Ed., John Wiley & Sons, New York, 1992:J. herein incorporated by reference as if each individual publi March, “Advanced Organic Chemistry: Reactions, Mecha cation or patent were specifically and individually indicated 35 nisms and Structure', 4th Ed., Wiley-Interscience, New York, to be incorporated by reference and are incorporated herein 1992. Specific and analogous reactants may also be identified by reference to disclose and describe the methods and/or through the indices of known chemicals prepared by the materials in connection with which the publications are cited. Chemical Abstract Service of the American Chemical Soci The citation of any publication is for its disclosure prior to the ety, which are available in most public and university librar filing date and should not be construed as an admission that 40 ies, as well as through on-line databases (the American the present invention is not entitled to antedate Such publica Chemical Society, Washington, D.C., may be contacted for tion by virtue of prior invention. Further, the dates of publi more details). Chemicals that are known but not commer cation provided may be different from the actual publication cially available in catalogs may be prepared by custom dates which may need to be independently confirmed. chemical synthesis houses, where many of the standard It is noted that, as used herein and in the appended claims, 45 chemical Supply houses (e.g., those listed above) provide the singular forms “a”, “an', and “the include plural refer custom synthesis services. ents unless the context clearly dictates otherwise. It is further "Stable compound' and “stable structure' are meant to noted that the claims may be drafted to exclude any optional indicate a compound that is sufficiently robust to survive element. As such, this statement is intended to serve as ante isolation to a useful degree of purity from a reaction mixture, cedent basis for use of such exclusive terminology as “solely.” 50 and formulation into an efficacious therapeutic agent. “only' and the like in connection with the recitation of claim “Optional' or “optionally’ means that the subsequently elements, or use of a “negative limitation. described event of circumstances may or may not occur, and As will be apparent to those of skill in the art upon reading that the description includes instances where said event or this disclosure, each of the individual embodiments described circumstance occurs and instances in which it does not. For and illustrated herein has discrete components and features example, “optionally substituted aryl' means that the aryl which may be readily separated from or combined with the 55 radical may or may not be substituted and that the description features of any of the other several embodiments without includes both substituted aryl radicals and aryl radicals hav departing from the scope or spirit of the present invention. ing no substitution. The term lower alkyl will be used herein Any recited method can be carried out in the order of events as known in the art to refer to an alkyl, Straight, branched or recited or in any other order which is logically possible. cyclic, of from about 1 to 6 carbons. As used herein, compounds which are “commercially 60 "Pharmaceutically acceptable carrier, diluent or excipient' available may be obtained from standard commercial includes without limitation any adjuvant, carrier, excipient, Sources including Acros Organics (Pittsburgh Pa.), Aldrich glidant, Sweetening agent, diluent, preservative, dye? colo Chemical (Milwaukee Wis., including Sigma Chemical and rant, flavor enhancer, Surfactant, wetting agent, dispersing Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado agent, Suspending agent, stabilizer, isotonic agent, solvent, or Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), 65 emulsifier which has been approved by the United States Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester Food and Drug Administration as being acceptable for use in Pa.), Crescent Chemical Co. (Hauppauge N.Y.), Eastman humans or domestic animals.

US 8,648,112 B2 8 conventional, additives Such as solubilizers, isotonic agents, Suspending agents, emulsifying agents, stabilizers and pre servatives. The compounds can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable pro pellants such as dichlorodifluoromethane, propane, nitrogen and the like. Furthermore, the compounds can be made into Suppositories by mixing with a variety of bases Such as emul 10 sifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a Supposi tory. The Suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at OR body temperature, yet are solidified at room temperature. 15 Unit dosage forms for oral or rectal administration Such as where R is H, OH, OR where R is a C to Co lower alkyl, syrups, elixirs, and Suspensions may be provided wherein NH, NHR, SH, SCH. F. OCN, O(CH)nNH, O(CH.) each dosage unit, for example, teaspoonful, tablespoonful, nCH where n is from 1 to about 10; C to Co lower alkyl, tablet or Suppository, contains a predetermined amount of the substituted lower alkyl, alkaryl or aralkyl, Cl, Br, CN, CF, composition containing one or more compounds of the OCF O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH, SO; present invention. Similarly, unit dosage forms for injection CH: ONO; NO; N; and or intravenous administration may comprise the compound of R is H, or C to Co lower alkyl. the present invention in a composition as a solution in sterile The ET metabolites or derivatives thereof may be admin water, normal saline or another pharmaceutically acceptable istered to a Subject (e.g., mammal) in a variety of ways. For carrier. example, the ET metabolite(s) can be administered orally, 25 Implants for Sustained release formulations are well rectally, intravenously, intramuscularly, intraperitoneally, known in the art. Implants are formulated as microspheres; intra-cerobrospinally, Subcutaneously, intra-articularly, intra slabs, etc. with biodegradable or non-biodegradable poly synovially, intrathecally, topically, or by inhalation. As such, mers. For example, polymers of lactic acid and/or glycolic the form of the ET metabolite dose can be in a variety of acid form an erodible polymer that is well-tolerated by the forms, including microcapsules, nano-capsules, liposomes, 30 host. The implant containing the inhibitory compounds may plasters, inhalation forms, nose sprays, Sublingual tablets, be placed in proximity to the site of a tumor, so that the local and Sustained-release preparations. concentration of active agentis increased relative to the rest of The compounds of this invention can be incorporated into the body. a variety of formulations fortherapeutic administration. More The term “unit dosage form', as used herein, refers to particularly, the compounds of the present invention can be physically discrete units Suitable as unitary dosages for formulated into pharmaceutical compositions by combina 35 human and animal Subjects, each unit containing a predeter tion with appropriate pharmaceutically acceptable carriers or mined quantity of compounds of the present invention calcu diluents, and may be formulated into preparations in Solid, lated in an amount sufficient to produce the desired effect in semi-solid, liquid orgaseous forms, such as tablets, capsules, association with a pharmaceutically acceptable diluent, car powders, granules, ointments, solutions, Suppositories, injec rier or vehicle. The specifications for the novel unit dosage tions, inhalants, gels, microspheres, and aerosols. As such, 40 forms of the present invention depend on the particular com administration of the compounds can be achieved in various pound employed and the effect to, be achieved, and the phar ways, including oral, buccal, rectal, parenteral, intraperito macodynamics associated with each compound in the host. neal, intradermal, transdermal, intracheal, etc., administra The pharmaceutically acceptable excipients, such as tion. The active agent may be systemic after administration or vehicles, adjuvants, carriers or diluents, are readily available may be localized by the use of regional administration, intra 45 to the public. Moreover, pharmaceutically acceptable auxil mural administration, or use of an implant that acts to retain iary Substances, such as pH adjusting and buffering agents, the active dose at the site of implantation. tonicity adjusting agents, stabilizers, wetting agents and the In pharmaceutical dosage forms, the compounds may be like, are readily available to the public. administered in the form of their pharmaceutically acceptable The combined use of the provided compounds of the salts. They may also be used in appropriate association with 50 present invention and other cytotoxic agents has the advan other pharmaceutically active compounds. The following tages that the required dosages for the individual drugs is methods and excipients are merely exemplary and are in no lower, and the effect of the different drugs complementary. way limiting. Depending on the patient and condition being treated and on For oral preparations, the compounds can be used alone or the administration route, the Subject compounds may be in combination with appropriate additives to make tablets, administered in dosages of at least about or around about 0.1 powders, granules or capsules, for example, with conven 55 pg/kg body weight, at least about or around about 1 ng/kg tional additives, such as lactose, mannitol, corn Starch or body weight, at least about or around about 1 ug/kg body potato starch; with binders, such as crystalline cellulose, cel weight, at least about or around about 1 mg/kg body weight, lulose derivatives, acacia, corn starch or gelatins; with disin at least about or around about 10 mg/kg body weight, per day tegrators, such as corn starch, potato starch or sodium car or even higher values, and usually not more than about 100 boxymethylcellulose; with lubricants, such as talc or 60 mg/kg body weight per day. The range is broad, since in magnesium Stearate; and if desired, with diluents, buffering general the efficacy of a therapeutic effect for different mam agents, moistening agents, preservatives and flavoring agents. mals varies widely with doses typically being 20, 30 or even The compounds can be formulated into preparations for 40 times Smaller (per unit body weight) in manthan in the rat. injections by dissolving, Suspending or emulsifying them in Similarly the mode of administration can have a large effect an aqueous or nonaqueous solvent, such as Vegetable or other 65 on dosage. Thus for example oral dosages in the rat maybe ten similar oils, synthetic aliphatic acid glycerides, esters of times the injection dose. Lower doses maybe used for local higheraliphatic acids or propylene glycol; and if desired, with ized routes of delivery. US 8,648,112 B2 10 A typical dosage may be a solution Suitable for intravenous is preferably at least about 10%, at least about 20%, at least administration; a tablet taken from two to six times daily, or about 30%, at least about 40%, at least about 50%, at least one time-release capsule or tablet taken once a day and con about 60%, at least about 70%, at least about 80%, at least taining a proportionally higher content of active ingredient, about 90%, and ideally 100%. etc. The time-release effect may be obtained by capsule mate The following examples are put forth so as to provide those rials that dissolve at different pH values, by capsules that of ordinary skill in the art with a complete disclosure and release slowly by osmotic pressure, or by any other known description of how to make and use the present invention, and means of controlled release. are not intended to limit the scope of what the inventors regard Those of skill will readily appreciate that dose levels can as their invention nor are they intended to represent that the vary as a function of the specific compound, the severity of the 10 experiments below are all or the only experiments performed. symptoms and the susceptibility of the subject to side effects. Efforts have been made to ensure accuracy with respect to Some of the specific compounds are more potent than others. numbers used (e.g. amounts, temperature, etc.) but some Preferred dosages for a given compound are readily deter experimental errors and deviations should be accounted for. minable by those of skill in the art by a variety of means. A Unless indicated otherwise, parts are parts by weight, preferred means is to measure the physiological potency of a 15 molecular weight is weight average molecular weight, tem given compound. perature is in degrees Centigrade, and pressure is at or near For use in the Subject methods, the Subject compounds may atmospheric. beformulated with other pharmaceutically active agents, par ticularly other anti-metastatic, antitumor or anti-angiogenic All publications and patent applications cited in this speci agents. Angiostatic compounds of interest include angiosta fication are herein incorporated by reference as if each indi tin, endostatin, carboxy terminal peptides of collagen alpha vidual publication or patent application were specifically and (XV), etc. Cytotoxic and cytostatic agents of interest include individually indicated to be incorporated by reference. adriamycin, alkeran, Ara-C, BICNU, busulfan, CNNU, cis The present invention has been described in terms of par platinum, cytoxan, daunorubicin, DTIC, 5-FU, hydrea, ifos ticular embodiments found or proposed by the present inven famide, methotrexate, mithramycin, mitomycin, mitox tor to comprise preferred modes for the practice of the inven antrone, nitrogen mustard, Velban, Vincristine, vinblastine, 25 tion. It will be appreciated by those of skill in the art that, in VP-16, carboplatinum, fludarabine, gemcitabine, idarubicin, light of the present disclosure, numerous modifications and irinotecan, leustatin, navelbine, taxol, taxotere, topotecan, changes can be made in the particular embodiments exempli etc. fied without departing from the intended scope of the inven The urolithin compounds are useful for prophylactic or tion. All such modifications are intended to be included therapeutic purposes. As used herein, the term “treating is 30 within the scope of the appended claims. used to refer to both prevention of disease, and treatment of pre-existing conditions. The prevention of proliferation is EXPERIMENTAL accomplished by administration of the Subject compounds prior to development of overt disease, e.g., to prevent the Example 1 regrowth of tumors, prevent metastatic growth, diminish res 35 tenosis associated with cardiovascular Surgery, etc. Alterna tively the compounds are used to treat ongoing disease, by Subjects, Materials and Methods stabilizing or improving the clinical symptoms of the patient. The host, or patient, may be from any mammalian species, Reagents and Instruments. e.g., primate sp., particularly humans; rodents, including All solvents were HPLC grade and purchased from Fisher mice, rats and hamsters; rabbits; equines, bovines, canines, 40 Scientific Co. (Tustin, Calif.). Ellagic, gallic, formic, phos felines; etc. Animal models are of interest for experimental phoric, 2-bromo-benzoic, 2-bromo-5-methoxybenzoic and investigations, providing a model for treatment of human acetic acids, resorcinol, potassium dihydrogen phosphate, disease. ABTS (2,2-azinobis-3-ethylbenzothiazoline-6-sulphonic The susceptibility of a particular cell to treatment with the acid diammonium salt), manganese dioxide, Trolox (6-hy Subject compounds may be determined by in vitro testing. 45 droxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), mag Typically a culture of the cell is combined with a subject nesium chloride, Tris HCL, sodium acetate, diethylenetri compound at varying concentrations for a. period of time amine pentaacetic acid (DTPA), deoxyguanosine (dG), B-D- sufficient to allow the active agents to induce cell death or glucuronidase (type X-A from Escherichia coli) and inhibit migration, usually between about one hour and one arylsulfatase (type VIII from abalone entrails) were pur week. For in vitro testing, cultured cells from a biopsy sample 50 chased from Sigma-Aldrich (St. Louis, Mo.). The high per may be used. The viable cells left after treatment are then formance liquid chromatography with ultraviolet detection counted. (HPLC-UV) analyses were carried out on a Waters Alliance The dose will vary depending on the specific compound 2690 system equipped with a photo diode array (PDA) detec utilized, specific disorder, patient status, etc. Typically a tor (Waters Corp., Milford, Mass.) and data handling was therapeutic dose will be sufficient to substantially decrease with Waters Millenium V 3.02 software. The high perfor the undesirable cell population in the targeted tissue, while 55 mance liquid chromatography with electrochemical detec maintaining patient viability. Treatment will generally be tion (HPLC-ECD) system consisted of an Agilent Technolo continued until there is a Substantial reduction, e.g., at least gies 1100 quaternary pump, temperature regulated about 50%, decrease in the cell burden, and may be continued autosampler controlled by Chemistation Software 9.01 (Agi until there are essentially none of the undesirable cells lent Technology, Wilmington, Del.), an ESA 5600A coular detected in the body. The reduction is preferably at least about 60 rray electrochemical detector (ESA, Bedford, Mass.). The 10%, at least about 20%, at least about 30%, at least about high performance liquid chromatography with mass spectros 40%, at least about 50%, at least about 60%, at least about copy (HPLC-MS) system consisted of an LCQ Classic Finni 70%, at least about 80%, at least about 90%, and ideally gan system (ThermoFinnigan, San Jose, Calif.), equipped 100%. with a HP 1100 series HPLC system consisting of an As used herein, "prevention of a condition (e.g., a neo 65 autosampler/injector, quaternary pump, column heater, and plastic disease) means that the occurrence of the condition diode array detector (DAD) with Xcalibur 1.2 software and/or its symptoms are reduced or eliminated. The reduction (Finnigan Corp.). US 8,648,112 B2 11 12 HPLC-MS/MS Analyses. are expressed as percentage of untreated cells (i.e. treatment Conditions for detection of ET-metabolites were as fol value-blank/vehicle value-blank), meantSE for three repli lows: Column, Symmetery C-18, 100 mmx2.1 i.d., 3.5 um, cations. (Waters Corp., Milford, Mass.): Solvent A) 2% HCOOH/ Assessment of apoptosis. Apoptosis was assessed utilizing H2O, B) 2% HCOOH/MeOH: gradient% A: initial: 99%, 30 the Cell Death Detection ELISAPLUS Assay (Boehringer min: 80%, 45 min: 60%, 60 min: 5%; run time 60 min; flow Mannheim, Indianapolis, Ind.). This assay is a photometric rate 0.15 mL/min: injection volume 20 LL; MS parameters: enzyme-linked immunoassay that quantitatively measures Ionization mode, electron spray ionization (ESI) in both posi the internucleosomal degradation of DNA, which occurs dur tive and negative modes (see FIG. 3); scan range: 120-1500 ing apoptosis. Specifically, the assay detects histone associ amu: Scan rate: 1 scan/sec; cone Voltage: 17 eV. Peak identi 10 ated mono- and oligonucleosomes, which are indicators of ties were obtained by matching their molecular ions (M-H+) apoptosis. Cells (22RV1, PC-3, LNCaP, LNCaP-AR), were or (M+H+) obtained by ESI/MS and MS/MS with the plated in 60 mm dishes at a density of 100,000 cells/dish and expected theoretical molecular weights from literature data allowed to attach for 24 hours. Cells were treated with vehicle (4, 5, 13, 15, 18). control (100% DMSO, 0.3% final concentration), or urolithin Cell Culture Materials. The 22Rv1, DU145, LNCaP and 15 B (0-50 ug/mL or 0-236 uM) for 48 hours. Following treat PC3 prostate cancer cell lines were obtained from American ments, non-adherent cells were collected and pelleted at Type Culture Collection (ATCC, Rockville, Md.). The 200xg for ten minutes. The supernatant was discarded; the LNCaP-AR and Hi-Myc cell lines were a kind donation by Dr. C. Sawyers (David Geffen School of Medicine, UCLA, cell pellet was washed with cold CMF-PBS and re-centri Los Angeles, Calif.). DU145, 22Rv1, LNCaP LNCaP-AR, fuged. Adherent cells were washed with cold calcium mag and PC3 cells were grown in RPMI 1640; Hi-Myc cells were nesium free-phosphate buffered saline (CMF-PBS, 137 grown in Isecove’s Modified Dulbecco's Medium. All media mmol/L Sodium chloride, 1.5 mmol/L potassium phosphate, contained 10% fetal bovine serum (FBS) in the presence of 7.2 mmol/L Sodium phosphate, 2.7 mmol/L potassium chlo 100 U/mL penicillin and 0.1 g/L streptomycin. RWPE-1 ride, pH 7.4), trypsinized, collected and combined with non prostate cells were grown in Defined Keratinocyte Serum adherent cells into a total of 1 mL DMEM. Both live and dead Free Medium (DKSFM) containing epidermal growth factor 25 cells were then counted via trypan blue exclusion (Pierce, (EGF), insulin and fibroblast growth factor (FGF). Cells were Rockford, Ill.) and equal numbers of cells were added to the incubated at 37°C. with 95% air and 5% CO2. All cells were microtiter plate for all treatment groups and apoptosis assay maintained below passage 20 and used in experiments during was performed according to the manufacturers instructions. the linear phase of growth. Data are expressed as absorbance at 405 nm of each sample Syntheses of urolithin B and methyl-urolithin A. Urolithin 30 over vehicle controls as follows=treatment value-blank/ve (dibenzob.dpyran-6-one) derivatives were synthesized hicle value-blank. according to the published method (21). Briefly, 2-bromo Results benzoic acid (5 g) and resorcinol (5 g) were suspended in Antiproliferative Activity. aqueous NaOH solution (2 g/25 mL water), and refluxed for EA and EA-metabolites (gallic and gallagic acids, uroli 30 min. After adding 5% aqueous CuSO solution (15 mL), 35 thin B and methylated-urolithin A) were evaluated for anti the mixture was refluxed for an additional 10 min. On cool ing, urolithin B (3-hydroxy-6H-dibenzob.dpyran-6-one) proliferative activity againstapanel of human prostate cancer precipitated as a pale white powder and was re-crystallized cell lines (22Rv1, PC3, DU145, LNCaP, LNCaP-AR) and from MeOH-glacial acetic acid (10:1) solution as needles. one mouse prostate cancer cell line (Hi-Myc). The IC50 val Similarly, 8-methoxyl-urolithin A (3-hydroxy-8-methoxyl ues are shown in Table 1. The cell lines showed different 6H-dibenzob.dpyran-6-one), was synthesized from 40 levels of sensitivity towards specific metabolites as follows. 2-bromo-5-methoxybenzoic acid (1 g), resorcinol (1 g) and Urolithin B was most effective against LNCaP-AR cells with 8% aqueous NaOH solution (2g/25 mL water; 10 mL). 8-me an IC50 of 28.5 uM whereas the methylated derivative of thyl-urolithin Aprecipitated as a pale yellow powder from the urolithin A showed the greatest effect against this same cell solution and was re-crystallized from MeOH-glacial acetic line but at a much higher concentration with an IC50 of 150.7 acid solution (10:1) as needles. The LC-MS/MS data of the 45 uM. EA was most effective against the Hi-Myc cells with an urolithin derivatives corresponded to published reports (5. IC-50 of 1.7 LM, gallic acid against 22RV1 with an IC50 of 13-15). 18.6 uM and gallagic acid against LNCaP with an IC50 of Antiproliferative Cell Assay. 71.4 uM. Proliferation was measured utilizing the CelTiter-Glo(R) TABLE 1 Luminescent Cell Viability Assay (Technical Bulletin #288, 50 Promega Corp., Madison, Wis.). When added to cells, the IC50 values (IM) of EA and EA-metabolites against prostate cancer assay reagent produces luminescence in the presence of ATP cell lines. from viable cells. Cells were plated in 96-well plates at a density of 10,000 cells/well and incubated for 24 hours. Test 8-methyl- Ellagic Gallagic Urolithin B Urolithin A acid Gallic acid acid samples were solubilized in DMSO by sonication, filter ster 55 ilized and diluted with media to the desired treatment con 22 Rv1 117.6 127.0 41.7 18.5 1661 centration. Cells were treated with 100 uLcontrol media, DU145 59.4 466.5 96.O 29.4 197.7 vehicle control (DMSO<0.2% of total) or test samples and HiMyc 59.9 S86.2 1.7 73.5 82.7 incubated for 48 h drug exposure duration at 6.25, 12.5, 25, 50 LNCaP 90.5 2O6.4 2O.S 32.3 71.4 and 100 g/mL concentrations. At the end of 48 h, plates were LNCap-AR 28.5 150.7 82.6 29.2 82.4 equilibrated at room temperature for 30 min; 100 uL of the 60 PC3 127.2 526.4 83.6 110.9 83.6 assay reagent was added to each well and cell-lysis was induced on an orbital shaker for 2 min. Plates were incubated Pro-Apoptotic Activity. at room temperature for 10 minto stabilize the luminescence Urolithin B was screened for ability to induce apoptosis of signal and results were read on an Orion Microplate Lumi the 22Rv1, PC3, LNCaP and LNCaP-AR cell lines at con nometer (Bertholds Detection Systems, Pforzheim, Ger 65 centrations ranging from 0 to 236 uM (0 to 50 ug/mL) as many). All plates had control wells containing medium with shown in FIGS. 5A-D. Urolithin B showed statistically sig out cells to obtain a value forbackground luminescence. Data nificant pro-apoptotic effects against different cell lines as US 8,648,112 B2 13 14 follows: 59 uMagainst 22Rv1 (p=0.05), 29.5uMagainst PC3 were more sensitive towards urolithin B than the LNCaP cells (p=0.05), 118 uM against LNCaP (p=0.01) and 59 uMagan (FIG.5). Concentrations of urolithin B that induced apoptosis inst LNCaP-AR (p=0.01). against these cells lines were 29.5, 59, 59 and 118 uM for Discussion PC3, 22Rv1 mLNCaP-AR and LNCaP cells respectively. As The biological properties attributed to pomegranates and 5 with the proliferation data, the LNCaP-AR cell line was more PJ has been related to its phenolic constituents and in particu sensitive to the effects of urolithin B than the LNCaP parent lar to its major ET, punicalagin (3, 6-9). We have previously cell line Suggesting that pomegranate consumption may play investigated the anti-proliferative activity of a number of a role in the progression of prostate cancer. phenolic acids produced by the action of colonic bacteria on A significant interindividual variability of urolithin pro flavonoids from tea, soy and citrus and we found that activity 10 duction was noted among the study Subjects leading to a was specific to a particular metabolite (32). In our current potential classification into metabolite producers and non study we evaluated a number of potential and putative producers. Similar interindividual variability has been metabolites from pomegranate ETs, including elagic, gallic observed for other polyphenols. The urolithins are produced and gallagic acids, urolithin Band methylated-urolithin A for as the result of metabolic transformations carried out by antiproliferative activity against a panel of human prostate 15 colonic microflora on EA and related compounds. Urolithins cancer cell lines (22Rv1, PC3, DU145, LNCaPLNCaP-AR) have been found in human urine collected 24 h after PJ and one mouse prostate cancer cell line (Hi-Myc) (Table 1). ingestion and in raturine, 4 days after pomegranate ET inges Although gallic acid has not been reported from bioavailabil ity studies with pomegranate polyphenols, it may be present tion, Suggesting that they are produced by colonic bacteria. in a free state in pomegranates and/or released from the EA and EA-metabolites (dimethyl ellagic acid-glucuronide, hydrolysis of gallotannins, known to be present in pomegran urolithin A-glucuronide and urolithin B-glucuronide) were ates. Gallic acid has one of the largest bioavailabilities among detected in 24 h urine samples of fifteen of the nineteen food phenolics (38). Due to the unavailability of a pure stan Subjects. Glucuronidation and methylation are part of the dard of urolithin A, it was not screened in the anti-cancer hepatic phase II metabolism which serves to increase water assayS. solubility and facilitate excretion. Metabolites that were the The characteristics of the cell lines are described as fol 25 most prevalent in the Subjects included dimethyl ellagic acid lows. The 22Rv1 is a human prostate carcinoma epithelial cell glucuronide and urolithin B-glucuronide found in thirteen line with a weak response to androgen, expression of prostate Volunteers. serum antigen (PSA) and androgen receptor (AR). DU145 is Potent antiproliferative activity against cancer cells has a human metastatic prostate carcinoma isolated from the been demonstrated for many flavonoids but only a limited brain tissue, it does not respond to androgen, does not express 30 number of colonic metabolites have been tested. Urolithins PSA or the AR. PC3 is a bone metastasis from a patient with have previously been evaluated for antioxidantandestrogenic metastatic prostate carcinoma with a weak response to andro activities. Our current study is the first to evaluate the anti gen, does not express PSA northe AR. The LNCaP cell line is proliferative activity of urolithins against a variety of human a human metastatic prostate carcinoma isolated from the prostate cancer cell lines, 22Rv1, PC3, DU145, LNCaP lymph node. In contrast to the aforementioned three cell lines, (Table 1). EA exhibited the strongestantiproliferative activity the LNCaP cells are androgen responsive and express PSA 35 in all cell lines (Table 1) with androgen-independent cells and the AR. The LNCaP-AR cell line is the LNCaP cell line (DU145 and PC3) being more resistant. In the antiprolifera with the AR stably overexpressed. All five cell lines are tum tive assays urolithin B proved to be two to ten times more origenic in nude mice. The Hi-Myc is a murine cell line potent than methyl urolithin A in all cell lines tested. developed to overexpress the c-myc oncogene. The pro-apoptotic effects of urolithins have also not been The pomegranate ET-metabolites significantly inhibited 40 previously studied. In the apoptosis assay, the PC3 and 22RV1 prostate cell proliferation in a dose dependent manner in cell cells were more sensitive towards urolithin B than the LNCaP lines tested (ps0.01) and IC50 values are shown in Table 1. cells (FIGS. 4A-C). Our results provide data as to the ability of these compounds In summary, we have identified ET metabolites that have to inhibit the growth of different prostate cell lines. anti-proliferative and pro-apoptotic activity towards cancer In the antiproliferative assays, Urolithin B proved to be two 45 cells. As such, these metabolites find use as therapeutic agents to tentimes more potent than methylated urolithin A in all cell for the treatment of neoplastic diseases. In addition, these lines tested, dependent upon the cell line. The cell lines were findings make it possible to Screen elagitannin-containing sensitive to urolithin B in the following order: LNCaP compositions (e.g., food products) for their ability to produce AR

In the Specification

Column 1, lines 6-8 Please replace paragraph 01 on page 1 with the following rewritten paragraph:

-- This invention was made with Government support under Grant No. AT000151, awarded by the National Institutes of Health. The Government has certain rights in the invention. --

Signed and Sealed this Ninth Day of September, 2014 74-4-04- 2% 4 Michelle K. Lee Deputy Director of the United States Patent and Trademark Office