US 20110105421Al (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0105421 A1 Mathee et al. (43) Pub. Date: May 5, 2011

(54) AS INHIBITORS OF (86) PCT No.: PCT/US09/37163 BACTERIAL QUORUM SENSING § 371 (00)’ (2), (4) Date: Dec. 14, 2010 (75) Inventors: Kalai Mathee, Miami, FL (US); _ _ Allison L‘ Adonizio’ Cambridge, Related US. Application Data MA (U S); Frederick AllSllbel, (60) Provisional application No. 61/036,812, ?led on Mar. Boston, MA (US); Jon Clardy, 14, 2008. Boston, MA (US); Bradley . . . . Bennett’ Miami’ FL (Us); Kelsey Publication Classi?cation DoWnum, Arlington, TX (US) (51) Illt- Cl A61K 31/70 (2006.01) A61K 31/7048 (2006.01) (73) Assignee: The Florida International A611) 31/04 (200601) UPiVeFsitY 30m‘ "f Trustees’ (52) us. Cl...... 514/29; 514/23; 514/39 M1am1, FL (US) (57) ABSTRACT (21) APPL NO; 12/922 555 Described herein are materials and methods for the inhibition ’ of bacterial QS. Methods of treating bacterial infections by administration of one or more ellagitannins in amount effec (22) PCT Filed: Mar. 13, 2009 tive to inhibit bacterial QS is also provided. Patent Application Publication May 5, 2011 Sheet 1 0f 4 US 2011/0105421 A1

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ELLAGITANNINS AS INHIBITORS OF bacterial density, the concentration of the signaling molecule BACTERIAL QUORUM SENSING reaches a critical threshold to activate virulence genes. [0006] In Gram negative bacteria, for example, the signal CROSS-REFERENCE TO RELATED molecule is an acylated homoserine lactone (AHSL), often APPLICATION referred to as the autoinducer, Which interacts With a protein of a quorum regulon. A quorum regulon includes tWo pro [0001] The present application claims the bene?t of priority teins, the autoinducer synthase (the I protein) and the regula of US. Provisional Application No. 61/036,812, ?led Mar. tor (the R protein), Which, upon binding of the autoinducer, 14, 2008, the disclosure of Which is incorporated herein by activates the transcription of numerous genes. In Pseudomo reference in its entirety. nas aeruginosa, tWo quorum regulons have been identi?ed. One quorum regulon is knoWn as the LasIR system and is FIELD OF THE INVENTION mediated by a 3-oxo-dodecanoyl homoserine lactone (3-oxo [0002] The present application is directed to the use of C12-HSL) signal molecule. The other quorum regulon is knoWn as the Rhl1R system and is mediated by a butyryl ellagitannins for the inhibition of bacterial quorum sensing. homoserine lactone (C4-HSL) signal molecule. BACKGROUND OF THE INVENTION [0007] In recent years it has become apparent that many Gram-negative bacteria employ one or more quorum sensing [0003] Many microbial pathogens cause tremendous dam systems. The quorum sensing system is an attractive anti age WorldWide, in humans as Well as in animals and crop bacterial target because it is not found in humans and is plants. The continuing emergence of multiple-drug-resistant critical for high level bacterial virulence. Bacterial quorum pathogen strains has necessitated ?nding neW compounds sensing systems comprise AHL derivatives With different that can be used in antimicrobial treatment. In general, tWo acyl side chains to regulate, in a cell-density dependent man strategies exist for controlling pathogens, either kill the ner, a Wide variety of physiological processes unique to the pathogen or attenuate its virulence such that it does not dam life-cycle of each microbe. These processes include: sWarm age the host. ing, motility, bio?lm formation, conjugation, biolumines [0004] Many bacteria use autoinducer ligands to monitor cence and/or production of pigments, antibiotics and their population densities in a phenomenon called quorum enZymes. For example, in P aeruginosa quorum sensing sensing. See Fuqua & Greenberg, Nature Reviews Molecular pathWays affect the expression of various exoenZymes, bio Cell Biology, 3:685-695, 2002; or de Kievit et al., Infection & ?lm formation and cell-cell spacing. Other bacteria react to Immunity, 68: 4839-4849, 2000, for a revieW of the Quorum quorum sensing stimulation by expressing proteases and pec sensing system in pathogenic bacteria. Bacteria use quorum tinases, expressing pili, entering stationary phase, emerging sensing to regulate a variety of phenotypes, such as bio?lm from lag phase and initiating cell division. formation, toxin production, exopolysaccharide production, [0008] Bio?lms are dense extracellular polymeric matrices virulence factor production, and motility, Which are essential in Which the bacteria embed themselves. Bio?lms alloW bac for the successful establishment of a symbiotic or pathogenic teria to create a microenvironment that attaches the bacteria to relationship With their respective eukaryotic hosts (Marketon the host surface and Which contains excreted enZymes and et al., J. Bacteriol., 185:325-331, 2003; Ohtani et al., Mol. other factors alloWing the bacteria to evade host immune Microbiol., 44:171-179, 2002; Quinones et al., Mol. Plant responses including antibodies and cellular immune Microbe Interact. 18:682-693, 2005; Rice et al., J. Bacteriol., responses. Such bio?lms can also exclude antibiotics. Fur 187:3477-3485, 2005; Suntharalingam et al., Trends Micro ther, bio?lms can be extremely resistant to removal and dis biol., 1313-6, 2005). At high cell densities, bacteria use this infection. For individuals suffering from cystic ?brosis, the chemical signaling process to sWitch from a nomadic exist formation of bio?lms by R aeruginosa is eventually fatal. ence to that of a multicellular community. This lifestyle Other bacteria also respond to quorum sensing signals by sWitch is signi?cant, as numerous pathogenic bacteria use producing bio?lms. Bio?lms are inherent in dental plaques, quorum sensing to turn on virulence pathWays and form drug and are found on surgical instruments, food processing and impervious communities called bio?lms that are the basis of agriculture equipment and Water treatment and poWer gener a myriad chronic infections. Over 80% of bacterial infections ating machinery and equipment. in humans involve the formation of bio?lms, as exempli?ed in [0009] Because of the virulence factors it triggers, the bac lung infections by Pseudomonas aeruginosa, Which is the terial quorum-sensing system offers a target for use in modu primary cause of morbidity in cystic ?brosis patients. The lating the virulence of pathogenic bacteria. All acyl-ho treatment of infections by pathogens that form bio?lms costs moserine lactone quorum-sensing systems described to date, over $1 billion/year in the US alone. Studies With animal except that of V harveyi, utiliZe AI synthases encoded by a models have shoWn that strains With inactivated quorum sens gene homologous to luxI of V?scheri. The response to the ing genes shoW reduced virulence. Disrupting quorum sens autoinducer is mediated by a transcriptional activator protein ing may interfere With the ability of bacteria to form robust encoded by a gene homologous to luxR of V?scheri (Bassler bio?lms and thus render the bacteria more sensitive to anti and Silverman, in TWo Component Signal Transduction, bacterial agents and the host’s immune response. Hoch et al., eds., Am. Soc. Microbiol. Washington DC, pp. [0005] Quorum sensing is mediated by a signal molecule 431-435,1995). that binds to a cognate transcriptional activator to cause either [0010] Gram-negative bacteria represent numerous rel upregulation or repression of genes that increase virulence evant pathogens using quorum-sensing pathWays. Besides R factors, Which include exotoxins, proteases, alginates, aeruginosa, other gram-negative quorum sensing bacteria lipopolysaccharides, pyocyanin and rhamnolipids. At loW include: Aeromonas hydrophila, A. salmonicida, Agrobacle bacterial cell density, the concentration of the signaling mol rium Zumefaciens, Burkholderia cepacia, Chromobaclerium ecule does not activate the virulence genes, While at higher violaceum, Enlerobacler agglomeran, Erwinia carolovora, US 2011/0105421A1 May 5,2011

E. chrysanlhemi, Escherichia coli, Nilrosomas europaea, malian subject, the method comprising administering to the Obesumbaclerium proleus, Panloea slewarlii, Pseudomonas subject an in an amount effective to treat the aureofaciens, P syringae, Ralslonia solanacearum, Rhiso infection. In one embodiment, the infection an infection bium elli, R. leguminosarum, Rhodobacler sphaeroides, Ser caused by a bacterium is selected from the group consisting ralia liguefaciens, S. marcescens, [?brio anguillarum, V?s of. Aeromonas hydrophila, Aeromonas salmonicida, Agro cheri, V cholerae, Xenorhabdus nemalophilus, Yersinia baclerium Zumefaciens, Burkholderia cepacia, Chromobac enlerocolilica, I’. peslis, I’. pseudoluberculosis, I’. medievalis, Zerium violaceum, Enlerobacler agglomeran, Erwinia caro and I’. ruckeri. Zovora, Erwinia chrysanlhemi, Escherichia coli, Nilrosomas [0011] In addition to their pathogenic costs, quorum sens europaea, Obesumbaclerium proleus, Panloea slewarlii, ing bacteria also have signi?cant economic impact in indus Pseudomonas aureofaciens, Pseudomonas aeruginosa, tries other than health care. For example, in agriculture, vari Pseudomonas syringae, Ralslonia solanacearum, Rhisobium ous species of the genera Rhizobium, Bradyrhizobium and Sinorhizobium are important plant symbionts helping elli, Rhisobium leguminosarum, Rhodobacler sphaeroides, legumes to ?x nitrogen, While, species of the genera Erwinia, Serralia liguefaciens, Serralia marcescens, Staphylococcus Xanlhomonas and Pseudomonas are responsible for signi? aureus, Slaphyllococcus epidermidis, [?brio anguillarum, cant food-spoilage. Other industries, such as poWer genera lflbrio?scheri, [?brio cholerae, Xenorhabdus nemalophilus, tion, paper making and Water treatment are subject to biofoul Yersinia enlerocolilica, Yersiniapeslis, Yersiniapseudoluber ing by many types of slime forming bacteria, such as culosis, Yersinia medievalis, and Yersinia ruckeri. In some Deinococcus geolhermalis. embodiments, exemplary bacterial infections include, but are [0012] are Widespread throughout the angiosperms not limited to, bacteremia, septicemia, endo- and pericarditis, (Okuda et al., Phytochem., 32:507-521, 1993), conferring sinusitis, upper respiratory tract infection, chronic bronchitis, structural bene?ts to the plant While providing protection pneumonia, cerebral and pulmonary lesions, meningitis, der through antioxidant and anti-feedant activity. Often classi?ed matitis or folliculitis, necrotiZing fascitis, cellulitis, urinary as “Waste” in natural products chemistry due to their abun tract infections, osteomylitis, enterocolitis, contact lens-asso dance and lack of protein speci?city (Zhu et al., 1997), tan ciated kerititis and conjunctivitis. In some embodiments, the nins and other polyphenolics have been previously ignored by mammalian subject to be treated is an immunocompromised the pharmaceutical industry. individual, such as a human subject, for example and Without [0013] Many polyphenolics possess antimicrobial activity limitation, having cancer or AIDS. potentially explained by inhibition of microbial enZymes, [0017] In another aspect, the present invention provides a substrate or iron deprivation, or inhibition of oxidative phos method of treating a disorder associated With bio?lm forma phorylation (Scalbert, Phytochem., 30:3875-3883, 1991). tion in a mammalian subject, the method comprising admin HoWever, the same study shoWs most bacteria are not suscep istering an ellagitannin to the subject in an amount effective to tible to ellagitannins, i.e. these compounds do not seem to disrupt bio?lm formation in the subject. In various embodi have groWth inhibition or cidal effects. Furthermore, it has ments, the disorder associated With bio?lm formation in the been shoWn that (a component of ellagitannins) subject is selected from the group consisting of cystic ?brosis, can interfere With bacterial quorum sensing (Huber et al., dental caries, periodonitis, otitis media, muscular skeletal Biosciences, 58:879-884, 2004). infections, necrotiZing fasciitis, biliary tract infection, osteo [0014] Thus, there is a need to identify and develop com myelitis, bacterial prostatitis, endocarditis, native valve pounds that are useful as inhibitors of bacterial quorum sens endocarditis, cystic ?brosis pneumonia, meloidosis, or skin mg. lesions associated With bullous impetigo, atopic dermatitis and pemphigus foliaceus or implanted device-related infec SUMMARY OF THE INVENTION tions. In some embodiments, the condition is a nosocomial [0015] The present application is based on the discovery infection, including but not limited to, pneumonia or an infec that ellagitannins, components in some medicinal plants, are tion associated With sutures, exit sites, arteriovenous sites, capable of inhibiting quorum sensing (OS) in pathogenic scleral buckles, contact lenses, urinary catheter cystitis, peri bacteria. Thus, in one aspect, the invention provides methods toneal dialysis (CAPD) peritonitis, IUDs, endotracheal tubes, of inhibiting OS in pathogenic bacteria in a mammalian sub Hickman catheters, central venous catheters, mechanical ject contacting the bacteria With an ellagitannin in an amount heart valves, vascular grafts, biliary stent blockage, and effective to inhibit OS in the bacteria. In one embodiment, the orthopedic devices. bacteria is contacted With the ellagitannin in vivo. In such [0018] Also provided is a method of modulating bio?lm embodiments, the contacting comprises administering the formation on a surface, the method comprising contacting the ellagitannin to the mammalian subject. In one embodiment, surface With an ellagitannin in an amount effective for disrupt the mammalian subject is af?icted With a bacterial infection or inhibit bio?lm formation on the surface. In one embodi associated With bacterial QS and the ellagitannin is adminis ment, the surface is an inanimate surface. Exemplary inani tered in an amount effective to treat the bacterial infection. In mate surfaces include, but are not limited to, metal, glass, another embodiment, the mammalian subject is a?llicted With plastic, Wood and stone surfaces. In another embodiment, the a disorder associated With bio?lm formation and the ellagi surface is an animate surface. Exemplary animate surfaces is administered in an amount effective to treat the include, but are not limited to, mammalian tissues, mamma disorder. In one embodiment, the mammalian subject is lian membranes, mammalian skin. human. In another embodiment, the human is immunocom [0019] As used herein, the term “pathogenic bacterium” or promised (e.g., having, for example and Without limitation, “pathogenic bacteria” refers to both gram-negative and gram cancer or AIDS). positive bacterial cells capable of infecting and causing dis [0016] Another aspect of the invention provides a method ease in a mammalian host, as Well as producing infection of treating a bacterial infection associated with OS in a mam related symptoms in the infected host, such as fever or other US 2011/0105421Al May 5,2011

signs of in?ammation, intestinal symptoms, respiratory Daptomycin, Ramoplanin), ARBELIC (TD-6424) (Thera symptoms, dehydration, and the like. vance), TD-6424 (Theravance), isoniaZid (INN), rifampin [0020] In some embodiments, and Without limitation, the (RIF), pyraZinamide (PZA), Ethambutol (EMB), Capreomy bacteria is of a genus selected from the group consisting of cin, cycloserine, ethionamide (ETH), kanamycun, andp-ami Aeromonas, Agrobaclerium, Burkholderia, Chromobacle nosalicylic acid (PAS). rium, Enlerobacler, Erwinia, Escherichia, Nilrosomas, Obe [0023] Combination therapy comprising an ellagitannin sumbaclerium, Panloea, Pseudomonas, Ralslonia, Rhiso and a standard of care anti-bacterial therapeutic described bium, Rhodobacler, Serralia, Slaphyllococcus, Vibl’iO, herein for the treatment of a bacterial infection associated Xenorhabdus, and Yersinia. For example, in some embodi With QS is speci?cally contemplated. For example, in one ments and Without limitation, the bacteria is of a species embodiment, the invention provides a method of treating a selected from the group consisting of Aeromonas hydrophila, Aeromonas salmonicida, Agrobaclerium Zumefaciens, bacterial infection associated With bacterial QS in a mamma Burkholderia cepacia, Chromobaclerium violaceum, Emera lian subject in need of treatment comprising administering to bacler agglomeran, Erwinia carolovora, Erwinia chrysan the subject a therapeutically-effective amount of a combina Zhemi, Escherichia coli, Nilrosomas europaea, Obesumbac tion therapy comprising (a) an ellagitannin and (b) a standard Zerium proleus, Panloea slewarlii, Pseudomonas of care anti -bacterial therapeutic. In another embodiment, the aureofaciens, Pseudomonas aeruginosa, Pseudomonas invention provides a method of treating a disorder associated syringae, Ralslonia solanacearum, Rhisobium elli, Rhiso With bio?lm formation in a mammalian subject comprising bium leguminosarum, Rhodobacler sphaeroides, Serralia administering to the subject a therapeutically-effective liguefaciens, Serralia marcescens, Staphylococcus aureus, amount of a combination therapy comprising (a) an ellagitan Slaphyllococcus epidermidis, [?brio anguillarum, [?brio?s nin and (b) a standard of care anti-bacterial therapeutic. In cheri, [?brio cholerae, Xenorhabdus nemalophilus, Yersinia another embodiment, the invention provides a method of enlerocolilica, Yersinia peslis, Yersinia pseudoluberculosis, treating a disorder associated With bacterial QS (or bio?lm Yersinia medievalis, and Yersinia ruckeri. formation) in a mammalian subject comprising administering [0021] Also provided is a method of treating a disorder to the subject a therapeutically-effective amount of a combi associated With QS in a mammalian subject resistant to treat nation therapy comprising (a) an ellagitannin and (b) a stan ment With a standard of care anti-bacterial therapeutic com dard of care additional/ second agent as described herein. prising administering to the subject an ellagitannin in an [0024] Such combination therapy Would be provided in a amount effective to inhibit QS in the bacteria causing the combined amount effective to inhibit QS in the bacteria and/ infection. or treat the bacterial infection and/or treat the disorder asso ciated With bio?lm formation. This process involves admin [0022] In some embodiments, the methods described herein further comprise the step of administering a standard istering to a subject in need thereof an ellagitannin and a of care anti-bacterial therapeutic to the subject in need of standard of care anti-bacterial therapeutic at the same time, treatment. In the context of methods of the invention, “stan Which may be achieved by administering a single composi dard of care” refers to a treatment that is generally accepted tion or pharmacological formulation that includes both an by clinicians for a certain type of patient diagnosed With a ellagitannin and a standard of care therapeutic, or by admin type of illness. For cardiac disorders, for example, an aspect istering tWo distinct compositions or formulations, at the of the invention is to improve standard of care therapy With same time, Wherein one composition includes an ellagitannin co-therapy With one or more ellagitannins described herein. and the other includes a standard of care anti-bacterial thera Exemplary standard of care anti-bacterial therapeutics peutic. In another embodiment, the combination therapy include, but are not limited to, colloidal silver, penicillin, involves administering to a subject in need thereof an ellagi penicillin G, erythromycin, polymyxin B, viomycin, chloro tannin and a standard of care anti-bacterial therapeutic at mycetin, streptomycins, cefaZolin, ampicillin, methicillin, different times, Which may be achieved by administering tWo oxacillin, nafcillin, cloxacillin, dicloxacillin aZactam, tobra distinct compositions or formulations, at different time inter mycin, cephalosporins (including cephalothin, cefaZolin, vals, Wherein one composition includes an ellagitannin and cephalexin, cephradine, cefamandole, cefoxitin, and 3rd-gen the other includes a standard of care anti-bacterial therapeu eration cephalosporins), Carbapenems (including imipenem, tic. meropenem, Biapenem), bacitracin, tetracycline, doxycy [0025] In some embodiments, the ellagitannin is selected cline, gentamycin, quinolines, neomycin, clindamycin, kana from the group consisting of vescalagin, , punica mycin, metronidaZole, treptogramins (including Quinupris lin, rhoipteleanin H, rhoipteleanin I, rhoipteleanin J, tellima tin/dalfopristun (SynercidTM)), Streptomycin, Ceftriaxone, grandin I, tellimagrandin II (eugeniin), pterocaryanin C, san Cefotaxime, Rifampin, Glycopeptides (including vancomy guin H-4, sanguin H-5, , potentillin, hemicetal cin, teicoplanin, LY-333328 (Ortivancin)), Macrolides (in congener , davidiin, , , carpi cluding erythromycin, clarithromycin, aZithromycin, linco nusin, , , elaeocarpusin, mycin, and clindamycun), Ketolides (including repandusinic acid A, repandusinin, stachyurin, , Telithromycin, ABT-773), Tetracyclines, Glycylcyclines (in pedunculagin, 5-desgalloyl-stachyurin, casuariin, roburin A, cluding Terbutyl-minocycline (GAR-936)), Aminoglyco roburin D, cercidininA, cercidinin B, cuspinin, platycaryanin sides, Chloramphenicol, Imipenem-cilastatin, Glycopeptides D, nupharin A, sanguiin H-6, grandinin, coriariin, agrimo (including oritavancin, LY-333328, dalbavancin), Fluoroqui niin, rugosin D, oenothein B, Woodfordin C, and nolones (including o?oxacin, spar?oxacin, gemi?oxacin, trapanin B. In one embodiment, the ellagitannin is vescalagin. cina?oxacun (DU-6859a)) and other topoisomerase inhibi In another embodiment, the ellagitannin is castalagin. tors, Trimethoprim-sulfamethoxaZole (TMP-SMX), Ciprof [0026] Compositions comprising the ellagitannin and a loxacin, topical mupirocin, OxaZolidinones (including AZD pharmaceutically-acceptable carrier, diluent or excipient are 2563, LineZolid (ZyvoxTM)), Lipopeptides (including also contemplated. US 2011/0105421 A1 May 5,2011

[0027] Also provided is the use of an ellagitannin in the activity of this plant on the bacterial QS system may explain manufacture of a medicament for the treatment of a disorder its traditional use for these diseases. The data presented associated With bacterial OS (or for treatment of a disorder herein identi?ed tWo hydrolyZable tannins, vescalagin and associated With bio?lm formation). castalagin, to be responsible for anti-QS activity in C. ereclus. [0028] The foregoing summary is not intended to de?ne Thus, the use of ellagitannins as an inhibitor of OS activity is every aspect of the invention, and additional aspects are speci?cally contemplated. described in other sections, such as the Detailed Description. I. Ellagitannins The entire document is intended to be related as a uni?ed [0038] disclosure, and it should be understood that all combinations [0039] The term “ellagitannin” as used herein means a of features described herein may be contemplated, even if the compound having a polyol core that is esteri?ed With at least combination of features are not found together in the same tWo galloyl moieties, Wherein at least tWo of the galloyl sentence, or paragraph, or section of this document. moieties are oxidatively carbon-carbon coupled to each other. [0029] In addition to the foregoing, the invention includes, In one embodiment, the polyol core is a carbohydrate. In as an additional aspect, all embodiments of the invention another embodiment, the polyol core is glucose. In another narroWer in scope in any Way than the variations de?ned by embodiment, the polyol core is D-glucose. In yet another speci?c paragraphs herein. For example, certain aspects of embodiment, the polyol core is an open-chain D-glucose. the invention that are described as a genus, and it should be When the ellagitannin comprises a carbohydrate polyol core, understood that every member of a genus is, individually, an the anomeric carbon can form a C- or O-glycosidic bond With aspect of the invention. Also, aspects described as a genus or a galloyl moiety. In one embodiment, the ellagitannin forms a selecting a member of a genus, should be understood to C-glycosidic bond With the galloyl moiety. embrace combinations of tWo or more members of the genus. [0040] In some embodiments, the carbon-carbon coupled [0030] It should be understood that While various embodi galloyl moieties are 4,6-hexahydroxybiphenoyl (HHBP or ments in the speci?cation are presented using “comprising” castalagin) and/or 2,3,5-nonahydroxyterphenoyl (NHTP or language, under various circumstances, a related embodi vescalagin). ment may also be described using “consisting of’ or “con sisting essentially of’ language. It is to be noted that the term “a” or “an”, refers to one or more, for example, “an ellagitan nin,” is understood to represent one or more ellagitannins. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

BRIEF DESCRIPTION OF THE FIGURES [0031] FIG. 1 shoWs thin layer chromatography (TLC) of HO OH C. ereclus crude extract and the visualization of phenolic anti-QS activity. OH [0032] FIG. 2 is a schematic of fractionation of C. ereclus galloyl moiety crude extract. OH [0033] FIG. 3 shoWs the results of an anti-QS bioassay of fractionation products of C. ereclus. [0034] FIG. 4 shoWs the HPLC separation of Fraction A.

DETAILED DESCRIPTION OF THE INVENTION [0035] Many bacterial phenotypic traits are modulated in response to bacterial density that is detected by OS. These phenotypes have important health consequences in patho genic bacteria and include virulence, carbapenem antibiotic production, bio?lm formation, enZyme synthesis and second ary metabolite synthesis. Modulation or interruption of these signaling pathWays can alter the life-cycle of quorum-sensing bacteria and thereby alter their virulence. [0036] A number of medicinal plants, including Conocar pus ereclus, have been found to be effective in inhibiting the pathogenicity of R aeruginosa via attenuation of the OS system (AdoniZio et al., 2008a; AdoniZio et al., 2008b; AdoniZio et al., 2006, the disclosures of Which are incorpo rated herein by reference in their entireties), but prior to the ?ling of the present application, the active components responsible for the inhibition of OS Was not knoWn. [0037] Commonly knoWn as buttonWood, C. ereclus has been used throughout the Caribbean, Puerto Rico, and parts of Africa against catarrh, conjunctivitis, diarrhea, syphilis, NHTP Unit and gonorrhea (Melendez, 1982, the disclosure of Which is incorporated herein by reference in their entireties). The US 2011/0105421A1 May 5,2011

[0041] In one embodiment, the ellagitannin comprises a malian subject comprising administering to the subject one or C-glycosidic, open-chain D-glucose core coupled to HHBP more ellagitannins in an amount effective to inhibit QS in the and NHTP (e.g. castalagin and vescalagin, respectively). bacteria. [0042] Castalagin and vescalagin (Mayer et al., 1967; [0048] In yet another aspect, the invention provides a Mayer et al., 1970) belong to a sub-class of hydrolyZable method of treating a disorder associated With bio?lm forma tannins knoWn as C-glycosidic ellagitannins derived from tion in a mammalian subject are also provided. Such methods gallic acid metabolism (Quideau & Feldman, 1996). Casta comprise administering one or more ellagitannins to the sub ject in an amount effective to disrupt bio?lm formation in the lagin and vescalagin are highly Water-soluble compounds subject. featuring an open-chain glucose core esteri?ed to numerous oxidatively coupled galloyl moieties (speci?cally a 4,6 [0049] In one embodiment, the disorder associated With hexahydroxybiphenoyl (HHBP) unit and a 2,3,5-nonahy bio?lm formation in the subject is selected from the group droxyterphenoyl (NHTP) unit) (Khanbabaee & van Ree, consisting of cystic ?brosis, dental caries, periodonitis, otitis 2001). These complex structural units confer stereochemical media, muscular skeletal infections, necrotiZing fasciitis, bil rigidity to the molecule, and in fact, C-glycosidic ellagitan iary tract infection, osteomyelitis, bacterial prostatitis, nins Would seem to have a higher tendency for selective endocarditis, native valve endocarditis, cystic ?brosis pneu protein interaction than other classes of polyphenolics monia, meloidosis, or skin lesions associated With bullous impetigo, atopic dermatitis and pemphigus foliaceus or (Haslam, 1996; Zhu et al., 1997). implanted device-related inventions. In another embodiment, [0043] Ellagitannins knoWn in the art, other than castalagin the condition is a nosocomial infection, including but not and vescalagin, are also contemplated for use in the methods limited to, pneumonia or an infection associated With sutures, described herein. Such ellagitannins include, but are not lim exit sites, arteriovenous sites, scleral buckles, contact lenses, ited to, [4,6-(S-S)-gallagyl-D-glucopyranose], urinary catheter cystitis, peritoneal dialysis (CAPD) perito Rhoipteleanin H, Rhoipteleanin I, Rhoipteleanin J, tellima nitis, IUDs, endotracheal tubes, Hickman catheters, central grandin I, tellimagrandin II (eugeniin), pterocaryanin C, san venous catheters, mechanical heart valves, vascular grafts, guine H-4, sanguine H-5, casuarictin, potentillin, hemicetal biliary stent blockage, and orthopedic devices. congener pedunculagin, davidiin, corilagin, geraniin, carpi nusin, chebulinic acid, chebulagic acid, elaeocarpusin, [0050] In some embodiments and Without limitation, the repandusinic acid A, repandusinin, stachyurin, casuarinin, bacteria is of a genus selected from the group consisting of pedunculagin, 5-desgalloyl-stachyurin, casuariin, roburin A, Aeromonas, Agrobaclerium, Burkholderia, Chromobacle roburin D, cercidininA, cercidinin B, cuspinin, platycaryanin rium, Enlerobacler, Erwinia, Escherichia, Nilrosomas, Obe D, nupharin A, sanguiin H-6, grandinin, coriariin, agrimo sumbaclerium, Panloea, Pseudomonas, Ralslonia, Rhiso niin, rugosin D, oenothein B, Woodfordin C, strictinin and bium, Rhodobacler, Serralia, Staphylococcus, Vibl’iO, Xenorhabdus, and Yersinia. For example, in some embodi trapanin B. For a revieW of ellagitannins, see Quideau et al., ments and Without limitation, the bacteria is of a species Chem. Rev., 96:475-504, 1996 and Khanbabaee et al., Nat. selected from the group consisting of Aeromonas hydrophila, Prod. Rep., 18:641-649, 2001). Aeromonas salmonicida, Agrobaclerium Zumefaciens, [0044] II. Therapeutic Uses of Ellagitannins Burkholderia cepacia, Chromobaclerium violaceum, Enlero [0045] The invention provides in one aspect a method of bacler agglomeran, Erwinia carolovora, Erwinia chrysan inhibiting bacterial QS comprising contacting the bacteria Zhemi, Escherichia coli, Nilrosomas europaea, Obesumbac With an ellagitannin in an amount effective to inhibit QS in the Zerium proleus, Panloea slewarlii, Pseudomonas bacteria. In one embodiment, the bacteria is contacted With aureofaciens, Pseudomonas aeruginosa, Pseudomonas the ellagitannin in vivo. In such embodiments, the contacting syringae, Ralslonia solanacearum, Rhisobium elli, Rhiso comprises administering the ellagitannin to the mammalian bium leguminosarum, Rhodobacler sphaeroides, Serralia subject. In one embodiment, the mammalian subject is liguefaciens, Serralia marcescens, Slaphyllococcus aureus, a?licted With a bacterial infection associated With bacterial [?brio anguillarum, [?brio ?scheri, Vibrio cholerae, QS and the ellagitannin is administered in an amount effec Xenorhabdus nemalophilus, Yersinia enlerocolilica, Yersinia tive to treat the bacterial infection. In another embodiment, peslis, Yersinia pseudoluberculosis, Yersinia medievalis, and the mammalian subject is a?licted With a disorder associated Yersinia ruckeri. With bio?lm formation and the ellagitannin is administered in [0051] In various embodiments, an ellagitannin for use in an amount effective to treat the disorder. In one embodiment, the methods described is selected from the group consisting the mammalian subject is human. In another embodiment, the of vescalagin, castalagin, punicalin, Rhoipteleanin H, human is immunocompromised (e.g., having, for example Rhoipteleanin I, Rhoipteleanin J, , tellima and Without limitation, cancer or AIDS). Practice of methods grandin II (eugeniin), pterocaryanin C, sanguine H-4, san of the invention in other mammalian subjects, especially guine H-5, casuarictin, potentillin, hemicetal congener mammals that are conventionally used as models for demon pedunculagin, davidiin, corilagin, geraniin, carpinusin, che strating therapeutic e?icacy in humans (e.g., primate, por bulinic acid, chebulagic acid, elaeocarpusin, repandusinic cine, canine, or rabbit animals), is also contemplated. acid A, repandusinin, stachyurin, casuarinin, pedunculagin, [0046] In another embodiment, the bacteria is contacted 5-desgalloyl-stachyurin, casuariin, roburin A, roburin D, cer With the ellagitannin ex vivo. In such an embodiment, for cidinin A, cercidinin B, cuspinin, platycaryanin D, nupharin example, the contacting comprises administering the ellagi A, sanguiin H-6, grandinin, coriariin, agrimoniin, rugosin D, tannin to a surface in an amount effective to inhibit bio?lm oenothein B, Woodfordin C, trapanin B and any combination formation associated With bacterial quorum sensing on sur thereof. In one embodiment, the ellagitannin is castalagin. In face (including Without limitation, a medical device). another embodiment, the ellagitannin is vescalagin. [0047] In another aspect, the invention provides a method [0052] In one embodiment, the methods described herein of treating a bacterial infection associated With QS in a mam further comprise the step of administering a standard of care US 2011/0105421Al May 5,2011

anti-bacterial therapeutic to the subject in need of treatment. moZyme®), CTFR-correcting drugs (including but not lim In the context of methods of the invention, “standard of care” ited to, gentamicin), anti-in?ammatory agents, NSAIDS, refers to a treatment that is generally accepted by clinicians aldosterone antagonists, anti-bacterial agents, a COX-2 for a certain type of patient diagnosed With a type of illness. inhibitors, an ot-adrenergic antagonist, an [3-adrenergic For bacterial infections associated With bacterial QS, for antagonist, an anti-allergic compound, an anti-diabetic com example, an aspect of the invention is to improve standard of pounds, an anti-hyperlipidemic compound, an anti-tussive care therapy With co -therapy With one or more ellagitannins. compound, an angiotensin II antagonist, an angiotensin con Exemplary standard of care anti-bacterial therapeutics verting enZyme (ACE) inhibitor, a bronchodilator, an anti include, but are not limited to, colloidal silver, penicillin, sense nucleotide, anti-thrombotic and vasodilator compound, penicillin G, erythromycin, polymyxin B, viomycin, chloro an antithrombogenic agent, a phosphodiesterase inhibitor, a mycetin, streptomycins, cefaZolin, ampicillin, methicillin, tissue plasminogen activator, a thrombolytic agent, a ?brin oxacillin, nafcillin, cloxacillin, dicloxacillin aZactam, tobra olytic agent, a vasospasm inhibitor, an endothelin antagonist, mycin, cephalosporins (including cephalothin, cefaZolin, an expectorant, an H2 receptor antagonist, a neural endopep cephalexin, cephradine, cefamandole, cefoxitin, and 3rd-gen tidase inhibitor, a calcium channel blocker, a potassium chan eration cephalosporins), carbapenems (including imipenem, nel blocker, a nitrate, a nitric oxide promoter, a vasodilator, an meropenem, Biapenem), bacitracin, tetracycline, doxycy antimicrobial agent, an antibiotic, a platelet reducing agent, a cline, gentamycin, quinolines, neomycin, clindamycin, kana proton pump inhibitor, a rennin inhibitor, a steroid, an anti mycin, metronidaZole, treptogramins (including Quinupris mitotic, a microtubule inhibitor, an actin inhibitor, a remod tin/dalfopristun (SynercidTM)), Streptomycin, Ceftriaxone, eling inhibitor, an agent for molecular genetic intervention, a Cefotaxime, Rifampin, glycopeptides (including vancomy cell cycle inhibitor, an inhibitor of the surface glycoprotein cin, teicoplanin, LY-333328 (Ortivancin), dalbavancin), mac receptor, an anti-metabolite, an anti-proliferative agent, a rolides (including erythromycin, clarithromycin, aZithromy chemotherapeutic agent, an anti-in?ammatory steroid, an cin, lincomycin, and clindamycun), ketolides (including immunosuppressive agent, an antibiotic, a radiotherapeutic Telithromycin, ABT-773), tetracyclines, glycylcyclines (in agent, iodine-containing compounds, barium-containing cluding Terbutyl-minocycline (GAR-936)), aminoglyco compounds, a heavy metal functioning as a radiopaque agent, sides, chloramphenicol, Imipenem-cilastatin, ?uoroquinolo an extracellular matrix component, a cellular component, a nes (including o?oxacin, spar?oxacin, gemi?oxacin, biologic agent, ascorbic acid, a free radical scavenger, an iron cina?oxacun (DU-6859a)) and other topoisomerase inhibi chelator, an antioxidant, a radiolabelled form or other radio tors, Trimethoprim-sulfamethoxaZole (TMP-SMX), Ciprof labelled forrn of any of the foregoing, or a mixture of any of loxacin, topical mupirocin, OxaZolidinones (including AZD these. 2563, LineZolid (ZyvoxTM)), Lipopeptides (including [0057] Exemplary antifungal agents including nystatin, Daptomycin, Ramoplanin), ARBELIC (TD-6424) (Thera liposomal nystatin, amorol?ne, butena?na, nafti?ne, terbin vance), TD-6424 (Theravance), isoniaZid (INN), rifampin a?ne, ?ucytosine, ?uconaZole, itraconasole, ketoconaZole, (RIF), pyraZinamide (PZA), Ethambutol (EMB), Capreomy posaconaZole, ravuconaZole, voriconaZole, clotrimaZole, cin, cycloserine, ethionamide (ETH), kanamycun, and p-ami econasole, miconaZole, oxiconaZole, sulconaZole, tercona nosalicylic acid (PAS). Zole, ticonaZole, nikkomycin Z, caspofungin, micafungin, [0053] Also provided is a method of modulating bio?lm amphotericin B (AmB), AmB lipid complex, AmB colloidal formation on a surface, the method comprising contacting the dispersion, pimaricin, griseofulvin, ciclopirox olamine, halo surface With an ellagitannin in an amount effective for affect progin, tolnaftate, undecylrnate. ing bio?lm formation on the surface. In one embodiment, the [0058] Exemplary antiviral agents include, but are not lim surface is an inanimate surface. Exemplary inanimate sur ited to, acyclovir, docosanol, ribarivin, interferons, cellulose faces include, but are not limited to, metal, glass, plastic, acetate, carbopol, carrageenan (CAS No. 9000-07-1), plecon Wood and stone surfaces. In another embodiment, the surface aril, amantidine, rimantidine, fomivirsen, Zidovudine, lami is an animate surface. Exemplary animate surfaces include vudine, Zanamivir, oseltamivir, brivudine, abacavir, adefovir, mammalian tissues, mammalian membranes, mammalian amprenavir, arbidol, ataZanavir, atripla, cidofovir, combivir, skin. edoxudine, efavirenZ, emtricitabine, enfuvirtide, entecavir, [0054] A. Combination Therapy famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, [0055] Combination therapy comprising one or more ella ganciclovir, gardasil, ibacitabine, immunovir, idoxuridine, gitannins and a standard of care anti-bacterial therapeutic imiquimod, indinavir, inosine, integrase inhibitor, lamivu described herein for the treatment of a bacterial infection dine, lopinavir, loviride, mk-05l8, maraviroc, moroxydine, associated With QS is speci?cally contemplated. For nel?navir, nevirapine, nexavir, nucleoside analogues, oselta example, in one aspect, the invention provides a method of mivir, penciclovir, peramivir, pleconaril, podophyllotoxin, treating a bacterial infection associated With bacterial QS in a ribavirin, rimantadine, ritonavir, saquinavir, stavudine, teno mammalian subject in need of treatment comprising admin fovir, tenofovir disoproxil, tipranavir, tri?uridine, triZivir, tro istering to the subject a therapeutically-effective amount of a mantadine, truvada, valaciclovir, valganciclovir, vicriviroc, combination therapy comprising (a) one or more ellagitan vidarabine, viramidine, Zalcitabine, Zanamivir and Zidovu nins and (b) a standard of care anti-bacterial therapeutic. dine; [0056] In other embodiments, the combination of an ella [0059] Exemplary anti-microbial agents, include, but are gitannin With one or more additional therapeutics/second not limited to, acediasulfone, aceturate, acetyl sulfameto agents in methods of the invention may reduce the amount of ssipiraZine, acetyl sulfamethoxypyraZine, acranil, albenda either agent needed as a therapeutically effective dosage, and Zole, alexidine, amatadine, ambaZone, amdinocillin, amika thereby reduce any negative side effects the agents may cin, p-aminosalicylic acid, p-aminosalicylic acid hydraZine, induce in vivo. Exemplary additional therapeutic/second amoxicillin, ampicillin, anisomycin, apalcillin, apicyclin, agents include, but are not limited to, dornase alfa (Pul apramycin, arbekacin, argininsa, aspoxicillin, aZidamfenicol, US 2011/0105421A1 May 5,2011

aZidocillin, aZithromycin, aZlocillin, aZtreonam, bacampicil 4-sulfanilamido salicylic acid, 4-4'-sulfanilylbenZylamine, lin, benZoylpas, benZyl penicillin acid, benZyl sulfamide, p-sulfanilylbenZylamine, 2-p-sul?nylanilinoethanol, sulfa bicoZamycin, bipenam, brodimoprim, capreomycin, carbeni nilylurea, sulfoniaZide, sulfaperine, sulfaphenaZole, sul cillin, carbomycin, cafaZedone, carindacillin, carumonam, faproxyline, sulfapyraZine, sulfapyridine, sulfathiaZole, sul cefcapene piVoxil, cefaclor, cefadroxil, cefafroxil, cefaman faethidole, sulfathiourea, sul?somidine, sulfasomiZole, dole, cefatamet, cefatriZine, cefaZedone, cefaZolin, ceihu sulfasymaZine, sul?soxaZole, 4,4'-sul?nyldianiline, N4-sul peraZone, cefclidin, cefdinir, cefditoren, ce?xime, fanilylsulfanilamide, N-sulfanilyl-3,4-xylamide, sultamicil ce?nenoxime, cefmetaZole, cefminox, cefodiZime, lin, talampicillin, tambutol, taurolidine, teiclplanin, temocil cefonicid, cefoperaZone, ceforanide, cefotaxime, cefotetan, lin, tetracycline, tetroxoprim, thiabendaZole, thiaZolsulfone, tibeZonium iodide, ticarcillin, tigemonam, tinidaZole, tobra cefotiam, cefoxitin, cefoZopran, cefpimiZole, cefpiramide, mycin, tosu?oxacin, trimethoprim, troleandromycin, tros cefpirome, cefpodoxime proxetil, cefproZil, cefroxadine, cef pectomycin, trova?oxacin, tubercidine, miokamycin, olean sulodin, ceftaZidime, cefteram, cefteZole, ceftibuten, ceftio domycin, troleandromycin, Vancomycin, VeraZide, Viomycin, fur, ceftiZoxime, ceftriaxone, cefuroxime, cefuZonam, ceph Virginiamycin and Zalcitabine. acetrile sodium, cephadrine, cephalexin, cephaloglycin, cephaloridine, cephalosporin C, cephalothin, cephapirin [0060] Exemplary aldosterone antagonists include, but are sodium, cephradine, chloramphenicol, chlorotetracycline, not limited to, canrenone, potassium canrenoate, dro cinoxacin, cipro?oxacin, claritromycin, clavulanic acid, spirenone, spironolactone, eplerenone (INSPRA®), clina?oxacin, clindamycin, clofaZimine, clofoctal, clometo epoxymexrenone, fadroZole, pregn-4-ene-7,21-dicarboxylic cillin, clomocycline, cloxacillin, cloxyquin, cyclacilline, acid, 9,1 1-epoxy-17-hydroxy-3-oxo, .gamma.-lactone, cycloserine, dano?axcin, dapsone, deoxycycline, deoxydihy methyl ester, (70.,1 1(X,17[3.)-; pregn-4-ene-7,21-dicarboxylic drostreptomycin, dibekacin, dicloxacillin, di?oxacin, dihy acid, 9,11-epoxy-17-hydroxy-3-oxo-dimethyl ester, (7a, drostreptomycin, dimetridaZole, diminaZene, dirirtomycin, 1 10,176)‘; 3'H-cyclopropa(6,7)pregna-4,6-diene-21-car doripenam, e?omithine, enoxacin, enro?oxacin, enviomy boxylic acid, 9,1 1-epoxy-6,7-dihydro-17-hydroxy-3 -oxo-, cin, epicillin, erythromycin, etacillin, ethambutol, ethiona .gamma.-lactone, (70.,1 1(X,17[3)-, pregn-4-ene-7,21-dicar mide, famciclovir, fenbecillin, ?eroxacin, ?omoxef, ?oxacil boxylic acid, 9,1 1-epoxy-17-hydroxy-3-oxo-, 7-(1-methyl lin, ?umequine, furonaZide, fortimycin, furaZolium chloride, ethyl) ester, monopotassium salt, (7(X,11(X,17[3.)-, pregn-4 gentamycin, glyconiaZide, grepa?oxacin, guamecycline, ene-7,21-dicarboxylic acid, 9,1 1-epoxy-17-hydroxy-3 -oxo-, halofuginone, hetacillin, homidium, hydroxyl-stilbamidine, 7-methyl ester, monopotassium salt, (7(X,11(X,17[3)-, 3'H-cy ibostamycin, imidocarb, imipenam, ipronidazole, isoniazide, clopropa(6,7) pregna-1,4,6-triene-21-carboxylic acid, 9,1 1 iosamycin, inosine, lauroguadine, lenampicillin, levo?oxin, epoxy-6,7-dihydro-17-hydroxy-3-oxo-y-lactone, (66,76, lincomycin, lome?oxacin, loracarbef, lymecyclin, mafenide, 1 lot)-; 3'H-cyclopropa(6,7)pregna-4,6-diene-21-carboxylic mebendaZole, meclocyclin, meropenem, metampicillin, met acid, 9,1 1 -epoxy-6,7-dihydro-17-hydroxy-3-oxo-, methyl acicline, methacycline, methicillin sodium, metronidaZole, ester, (66,76,110t, 176)-; 3'H-cyclopropa (6,7)pregna-4,6-di 4'-(methylsulfamoyl) sulfanilanilide, meZlocillin, meZiocil ene-21-carboxylic acid, 9,11-epoxy-6,7-dihydro-17-hy lin, micronomycin, midecamycin A1, minocycline, mioca droxy-3-oxo-, monopotassium salt, (66,76,110t, 176)-; 3'H mycin, miokamycin, morfaZinamide, moxalactam, mupiro cyclopropa(6,7)pregna-1,4,6-triene-21-carboxylic acid, cin, myxin, nadi?oxacin, nalidixic acid, negamycin, 9,1 1-epoxy-6,7-dihydro-17-hydroxy-3-oxo-y-lactone, (66, neomycin, netlimycin, nifurfoline, nifurpirinol, nifurpraZine, 76,110t, 176)-; pregn-4-ene-7,21-dicarboxylic acid, 9,11-ep nimoraZole, nitroxoline, nor?oxacin, novobiocin, o?oxacin, oxy-17-hydroxy-3-oxo-, .gamma.-lactone, ethyl ester, (7a, oleandomycin, opiniaZide, oxacillin, oxophenarsine, oxo 11(>t,176)-; pregn-4-ene-7,21-dicarboxylic acid, 9,11-epoxy linic acid, oxytetracycline, panipenam, paromycin, paZu 17-hydroxy-3-oxo-, y-lactone, 1-methylethyl ester, (70.,1 10., ?oxacin, pe?oxacin, penicillin G potassium salt, penicillin N, 176)-; RU-28318, and the like penicillin O, penicillinV, penethamate hydroiodide, pentami [0061] Exemplary ot-adrenergic receptor antagonists dine, phenamidine, phenethicillin potassium salt, phenyl receptor antagonists, include, but are not limited to, phento aminosalicyclate, pipacycline, pipemidic acid, piperacillin, lamine, tolaZoline, idaZoxan, deriglidole, RX 821002, BRL pirlimycin, piromidic acid, pivampicillin, pivcefalexin, pro 44408, BRL 44409, BAM 1303, labetelol, ifenprodil, rau ?romycin, propamidine, propicillin, protionamide, puralta Wolscine, corynathine, raubascine, tetrahydroalstonine, done, puromycin, pyraZinamide, pyrimethamine, quinacillin, apoyohimbine, akuammigine, .beta.-yohimbine, yohimbol, quinacrine, quinapyramine, quintine, ribostamycin, rifabu yohimbine, pseudoyohimbine, epi-3.alpha.-yohimbine, tine, rifamide, rifampin, rifamycin, rifanpin, rifapentine, rif 10-hydroxy-yohimbine, 1 1 -hydroxy-yohimbine, tamsulosin, axymine, ritipenem, rokitamycin, rolitetracycline, rosamy benoxathian, atipameZole, BE 2254, WB 4101, HU-723, cin, ru?oxacin, salaZosulfadimidine, salinaZid, sancycline, tedisamil, mirtaZipine, setiptiline, reboxitine, delequamine, sara?oxacin, sedacamycin, secnidaZole, sisomycin, spar naftopil, saterinone, SL 89.0591, ARC 239, urapidil, 5-me ?oxacin, spectinomycin, spiramycin, spiramycin I, spiramy thylurapidil, monatepi, haloperidol, indoramin, SB 216469, cin ll, spiramycin Ill, stilbamidine, streptomycin, streptoni moxisylyte, traZodone, dapiproZole, efaroxan, Recordati ciZid, sulbactam, sulbenicillin, succisulfone, sulfanilamide, 15/2739, SNAP 1069, SNAP 5089, SNAP 5272, RS 17053, sulfabenZamide, sulfacetamide, sulfachloropyridaZine, sul SL 89.0591, KMD 3213, spiperone, AH 11110A, chloroeth fachrysoidine, sulfacytine, sulfadiaZine, sulfadicramide, sul ylclonidine, BMY 7378, niguldipine, and the like. fadimethoxine, sulfadoxine, sulfadraZine, sulfaetidol, sul [0062] Exemplary 6-adrenergic antagonists include, but fafenaZol, sulfaguanidine, sulfaguanole, sulfalene, are not limited to, acebutolol, alprenolol, amosulalol, aroti sulfameraZine, sulfameter, sulfamethaZine, sulfamethiZole, nolol, atenolol, befunolol, betaxolol, bevantolol, bisoprolol, sulfamethomidine, sulfamethoxaZole, sulfamethoxypy bopindolol, bucindolol, bucumolol, bufetolol, bufuralol, ridaZine, sulfamethylthiaZol, sulfamethylthiaZole, sulfame bunitrolol, bupranolol, buto?lolol, caraZolol, capsinolol, car trole, sulfamidochrysoidine, sulfamoxole, sulfanilamide, teolol, carvedilol (COREG®), celiprolol, cetamolol, cin US 2011/0105421A1 May 5,2011

dolol, cloranolol, dileValol, diprafenone, epanolol, ersentil [0066] Exemplary antitussive compounds, include, but are ide, esmolol, esprolol, hydroxalol, indenolol, labetalol, not limited to, dextromethorphan, carbetapentane, carami landiolol, laniolol, levobunolol, mepindolol, methylpranol, phen, diphenylhydramine, hydrocodene, codeine and the metindol, metipranolol, metriZoranolol, metoprolol, like. moprolol, nadolol, nadoxolol, nebiVolol, nifenalol, [0067] Exemplary angiotensin II antagonists include, but nipradilol, oxprenolol, penbutolol, pindolol, practolol, prone are not limited to, angiotensin, abitesartan, candesartan, can thalol, propranolol, sotalol, sotalolnadolol, sul?nalol, talip desartan cilexetil, elisartan, embusartan, enoltasosartan, rolol, talinolol, tertatolol, tilisolol, timolol, toliprolol, eprosartan, fonsartan, forasartan, glycyllosartan, irbesartan, tomalolol, trimepranol, xamoterol, xibenolol, 2-(3-(1,1-dim losartan, olmesartan, milfasartan, medoxomil, ripisartan, pra ethylethyl)-amino-2-hydroxypropoxy)-3-pyridenecarboni tosartan, saprisartan, saralasin, sarmesin, tasosartan, telmis trilHCl-, 1-butylamino-3-(2,5 -dichlorophenoxy)-2-pro artan, Valsartan, Zolasartan, 3-(2'(tetraZole-5-yl)-1,1'-biphen panol, 1 -isopropylamino -3 -(4-(2 -cyclopropylmethoxyethyl) 4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imi-daZo(4,5-b) phenoxy)-2-propanol, 3 -isopropylamino-1 -(7 -methylindan pyridine, antibodies to angiotensin II, A-81282, A-81988, 4-yloxy)-2-butanol, 2-(3-t-butylamino-2-hydroxy BAY 106734, BlBR-363, BIBS-39, BIBS-222, BMS propylthio)-4-(5-carbamoyl-2-thienyl)thiaZol, 7-(2 180560, BMS-184698, BMS-346567, CGP-38560A, CGP hydroxy-3 -t -butylaminpropoxy)phthalide, Acc 93 69, AMO 42112A, CGP-48369, CGP-49870, CGP-63170, Cl-996, 140, BIB-16S, CP-331684, Fr-172516, lSV-208, L-653328, CP-148130, CL-329167, CV-11194, DA-2079, DE-3489, LM-2616, SB-226552, SR-58894A, SR-59230A, TZC-5665, DMP-811, DuP-167, DuP-532, DuP-753, E-1477, E-4177, UK-1745, YM-430, and the like. E-4188, EMD-66397, EMD-666R4, EMD-73495, EMD [0063] Exemplary anti-allergic compounds include, but are 66684, EXP-063, EXP-929, EXP-3174, EXP-6155, EXP 6803, EXP-7711, EXP-9270, EXP-9954, FK-739, FRI not limited to, acrivastine, allociamide, amlexanox, bromex 153332, GA-0050, GA-0056, HN-65021, HOE-720, ine, cetiriZine, clobenZepam, chromoglycate, chromolyn, HR-720, lCl-D6888, lCl-D7155, lCl-D8731, KRl-1177, deslortidine, emedastine, epinastine, fexofenadine, formot KT3-671, KT-3579, KW-3433, L-158809, L-158978, erol, hydroxyZine, ketotifen, loratadine, levocabastine, L-159282, L-159689, L-159874, L-161177, L-162154, lodoxamide, mabuterol, montelukast, nedocromil, repirinast, L-162234, L-162441, L-163007, L-163017, LF-70156, salmeterol, seratrodast, suplatast tosylate, terfenadine, tiara LRB-057, LRB-081, LRB-087, LY-235656, LY-266099, mide, and the like. LY-285434, LY-301875, LY-302289, LY-315995, ME-3221, [0064] Exemplary anti-diabetic compounds include, but MK-954, PD-123177, PD-123319, PD-126055, PD-150304, are not limited to, acarbose, acetohexamide, buformin, carb RG-13647, RWJ-38970, RWJ-46458, S-8307, S-8308, utamide, chlorpropamide, glibomuride, gliclaZide, glime SC-51757, SC-54629, SC-52458, SC-52459, SK 1080, piride, glipiZide, gliquidone, glisoxepid, glyburide, gly SL-910102, SR-47436, TAK-536, UP-2696, U-96849, buthiaZol(e), glybuZole, glyhexamide, glymidine, U-97018, UK-77778, UP-275-22, WAY-126227, WK-1260, glypinamide, insulin, metformin, miglitol, nateglinide, phen WK-1360, WK-1492, WY 126227, YH-1498, YM-358, butamide, phenformin, pioglitaZone, repaglinide, rosiglita YM-31472, X-6803, XH-148, XR-510, ZD-6888, ZD-7155, Zone, tolaZamide, tolbutamide, tolcyclamide, troglitaZone, ZD-8731, ZD 8131, the compounds of ACS registry numbers Voglibose, and the like. 124750-92-1, 133240-46-7, 135070-05-2, 139958-16-0, 145160-84-5, 147403-03-0, 153806-29-2, 439904-54-8P, [0065] Exemplary anti-hyperlipidemic compounds 439904-55-9P, 439904-56-0P, 439904-57-1P, 439904-58-2P, include, but are not limited to, statins or HMG-CoA reductase 155918-60-8P,155918-61-9R 272438-16-1P, 272446-75-0P, inhibitors, such as, for example, atorvastatin (LIPITOR®), 223926-77-0P, 169281-89-4, 439904-65-1P, 165113-01-9P, bervastatin, cerivastatin (BAYCOL®), dalvastatin, ?uin 165113-02-0P,165113-03-1R165113-03-2R165113-05-3P, dostatin (SandoZ XU-62-320), ?uvastatin, glenvastatin, lov 165113-06-4P,165113-07-5R165113-08-6R165113-09-7P, astatin (MEVACOR®), mevastatin, pravastatin (PRAVA 165113-10-0P,165113-11-1R165113-12-2R165113-17-7P, CHOL®), rosuvastatin (CRESTRO®), simVastatin 165113-18-8P,165113-19-9R165113-20-2R165113-13-3P, (ZOCOR®), Velostatin (also knoWn as synvinolin), 165113-14-4P,165113-15-5R165113-16-6R165113-21-3P, VYTORINTM (eZetimibe/simVastatin), GR-95030, SQ 165113-22-4P,165113-23-5R165113-24-6R165113-25-7P, 33,600, BMY 22089, BMY 22,566, Cl-980, and the like; 165113-26-8P,165113-27-9R165113-28-0R165113-29-1P, gem?broZil, cholystyramine, colestipol, niacin, nicotinic 165113-30-4P,165113-31-5R165113-32-6R165113-33-7P, acid, bile acid sequestrants, such as, for example, 165113-34-8P,165113-35-9R165113-36-0R165113-37-1P, cholestyramine, colesevelam, colestipol, poly(methyl-(3-tri 165113-38-2P,165113-39-3R165113-40-6R165113-41-7P, methylaminopropyl) imino-trimethylene dihalide) and the 165113-42-8P,165113-43-9R165113-44-0R165113-45-1P, like; probucol; ?bric acid agents or ?brates, such as, for 165113-46-2P,165113-47-3R165113-48-4R165113-49-5P, example, beZa?brate (BeZalipTM), beclobrate, bini?brate, 165113-50-8P,165113-51-9R165113-52-0R165113-53-1P, cipro?brate, clino?brate, clo?brate, eto?brate, feno?brate 165113-54-2P,165113-55-3R165113-56-4R165113-57-5P, (LipidilTM, Lipidil MicroTM), gem?broZil (LopidTM), nico? 165113-58-6P,165113-59-7R165113-60-0R165113-61-1P, brate, piri?brate, roni?brate, sim?brate, theo?brate and the 165113-62-2P,165113-63-3R165113-64-4R165113-65-5P, like; cholesterol ester transfer protein (CETP) inhibitors, such 165113-66-6P,165113-67-7R165113-68-8R165113-69-9P, as for example, CGS 25159, CP-529414 (torcetrapid), JTT 165113-70-2P,165113-71-3R165113-72-4R165113-73-5P, 705, substituted N-[3-(1,1,2,2-tetra?uoroethoxy)benZyl]-N 165113-74-6P, 114798-27-5, 114798-28-6, 114798-29-7, (3-phenoxyphenyl)-tri?uoro-3-a-mino-2-propanols, N,N 124749-82-2, 114798-28-6, 124749-84-4, 124750-88-5, disubstituted tri?uoro-3-amino-2-propanols, PD 140195 124750-91-0, 124750-93-2, 161946-65-2P, 161947-47-3P, (4-phenyl-5 -tridecyl-4H-1,2,4-triaZole-3 -thiol), SC-794, 161947-48-4P,161947-51-9R161947-52-0R 161947-55-3P, SC-795, SCH 58149, and the like. 161947-56-4P,161947-60-0R161947-61-1R 161947-68-8P, US 2011/0105421A1 May 5,2011

161947-69-9P, 161947-70-2P, 161947-71-3P, 161947-72-4P, lamine, papaveroline, reviparin sodium salt, ridogrel, suloc 161947-74-6P, 161947-75-7P, 161947-81-5P, 161947-82-6P, tidil, tinofedrine, tinZaparin, trifusal, vintoperol, xanthinal 161947-83-7P, 161947-84-8P, 161947-85-9P, 161947-86-0P, niacinate, and the like. 161947-87-1P, 161947-88-2P, 161947-89-3P, 161947-90-6P, [0071] Exemplary bronchodilators include, but are not lim 161947-91-7P, 161947-92-8P, 161947-93-9P, 161947-94-0P, ited to, ambroxol, atropine, bevonium methyl sulfate, 161947-95-1P, 161947-96-2P, 161947-97-3P, 161947-98-4P, bethanechol, chlorprenaline, cyclodrine, daiphenacine, 161947-99-5P,161948-00-1R 161948-01-2P,161948-02-3P, N-desethyl-oxybutynin, dicyclomine, emepronium, ephe 168686-32-6P, 167301-42-0P, 166813-82-7P, 166961-56-4P, drine, epinephrine, etafredine, ethylnorepinephrine, ?avox 166961-58-6P, 158872-96-9P, 158872-97-0P, 158807-14-8P, ate, ?utoprium bromide, hexoprenaline, 2-hydroxy-2,2 158807-15-9P,158807-16-0R 158807-17-1P,158807-18-2P, diphenyl-N-( 1 ,2 ,3 ,6 -tetra hydro -pyridin-4-ylmethyl) acetamide, ipratropium bromide, isoetharine, NS 21, 158807-19-3P, 158807-20-6P, 155884-08-5P, 154749-99-2, oxybutynin, oxitropium bromide, propanthelin, propiverine, 167371-59-7P, 244126-99-6P, 177848-35-0P and 141309 rispenZepine, terbutaline, 1-teobromine actetic acid, 82-2P, and the like. terodiline, tiotropium bromide, tolterodine, trospium, vami [0068] Exemplary angiotensin-converting enzyme inhibi camide, Zamiphenacine, and the like. tors (ACE inhibitors) include, but are not limited to, alacepril, [0072] Exemplary calcium channel blockers include, but benaZepril (LOTENSIN®, CIBACEN®), benaZeprilat, cap are not limited to, amlodipine (NORVASC®), anipamil, topril, ceronapril, cilaZapril, delapril, duinapril, enalapril, aranidipine, aminone, aZelnidipine, barnidipine, bencyclane, enalaprilat, fasidotril, fosinopril, fosinoprilat, gemopatrilat, benidipine, bepridil, cilnidipine, cinnariZine, clentiaZem, dil glycopril, idrapril, imidapril, lisinopril, moexipril, movel tiaZem, dotariZine, efonidipine, elgodipine, fantofarone, felo tipril, naphthopidil, omapatrilat, pentopril, perindopril, per dipine, fendiline, ?unariZine, ?uspirilene, fumidipine, gallo indoprilat, quinapril, quinaprilat, ramipril, ramiprilat, rent pamil, ipenoxaZone, isradipine, lacidipine, lemildipine, ipril, saralasin acetate, spirapril, temocapril, trandolapril, lercanidipine, lomeriZine, manidipine, mibefradil, trandolaprilat, urapidil, Zofenopril, acylmercapto and mer monatepil, nicardipine, nifedipine, niguldipine, niludipine, captoalkalnoyl pralines, carboxyalkyl dipeptides, carboxy nilvadipine, nimodipine, nisoldipine, nitrendipine, nival alkyl dipeptide, phosphinylalkanoyl pralines, registry no. dipine, oxodipine, perhexylene, phenyloin, phenylpreny 796406, AVE 7688, BP1.137, CHF 1514, E 4030, ER 3295, lamine, pranidipine, ranolaZine, ryosidine, semotiadil, tamo FPL-66564, MDL 100240, RL 6134, RL 6207, RL 6893, SA lariZine, temiverine hydrochloride, terodiline, tiapamil, 760, S-5590, Z 13752A, and the like. vatanidipine hydrochloride, verapamil, Ziconotide, AE-0047, [0069] Exemplary antioxidants include, but are not limited CAI, JTV-519, CHE-1521, L-651582, NS-7, NW-1015, to, small-molecule antioxidants and antioxidant enZymes. RO-2933, SB-237376, SL-34.0829-08, S-312d, SD-3212, Suitable small-molecule antioxidants include, but are not lim TA-993, YM-430, and the like. ited to, hydralaZine compounds, glutathione, vitamin C, vita [0073] Exemplary endothelin antagonists include, but are min E, cysteine, N-acetyl-cysteine, .beta.-carotene, not limited to, atrasentan, bosentan, darusentan, endothelin, ubiquinone, ubiquinol-10, tocopherols, coenZyme Q, super enrasentan, sitaxsentan, sulfonamide endothelin antagonists, oxide dismutase mimetics, such as, for example, 2,2,6,6 teZosentan, BMS 193884, BQ-123, SQ 28608, and the like. tetramethyl- 1 -piperidinyloxy (TEMPO), DOXYL, PROXYL [0074] Exemplary expectorants include, but are not limited nitroxide compounds; 4-hydroxy-2,2,6,6-tetramethyl-1-pip to, ambroxol, domiodol, erdosteine, guaiacol, guaifenesin, eridinyloxy (Tempol), M-40401, M-40403, M-40407, iodinated glycerol, letosteine, mensa, sobrerol, strepronine, M-40419, M-40484, M-40587, M-40588, and the like, Suit terpin, tiopronin, and the like. able antioxidant enZymes include, but are not limited to, [0075] Exemplary H2 receptor antagonists include, but are superoxide dismutase, catalase, glutathione peroxidase, not limited to, burimamide, cimetidine, ebrotidin, famoti NADPH oxidase inhibitors, such as, for example, apocynin, dine, niZatidine, roxatidine, rantidine, tiotidine, and the like. aminoguanidine, ONO 1714, S17834 (benZo(b)pyran-4-one [0076] Exemplary neutral endopeptidase inhibitors derivative), and the like; xanthine oxidase inhibitors, such as, include, but are not limited to, atrial natriuretic peptides, for example, allopurinol, oxypurinol, am?utiZole, dieth diaZapins, aZepinones, ecadotril, fasidotril, fasidotrilat, oma yldithiocarbamate, 2-styrylchromones, chrysin, luteolin, patrilat, sampatrilat, BMS 189,921, Z 13752 A, and the like. kaempferol, quercetin, myricetin, isorhamnetin, benZophe [0077] Exemplary NSAlDs include, but are not limited to, nones such as 2,2',4,4'-tetrahydroxybenZophenone, 3,4,5,2', acetaminophen, acemetacin, aceclofenac, alminoprofen, 3',4'-hexahydroxybenZophenone and 4,4'-dihydroxyben amfenac, bendaZac, benoxaprofen, bromfenac, bucloxic acid, Zophenone; benZothiaZinone analogues such as 2-amino-4H butibufen, carprofen, cinmetacin, clopirac, diclofenac, etod 1,3-benZothiaZine-4-one, 2-guanidino-4H-1,3-benZothiaZin olac, felbinac, fencloZic acid, fenbufen, fenoprofen, fen 4-one and rhodanine; N-hydroxyguanidine derivative such tiaZac, ?unoxaprofen, ?urbiprofen, ibufenac, ibuprofen, as, PR5 (1-(3,4-dimethoxy-2-chlorobenZylideneamino-3 indomethacin, isofeZolac, isoxepac, indoprofen, ketoprofen, hydroxyguanidine); 6-formylpterin, and the like lonaZolac, loxoprofen, metiaZinic acid, mofeZolac, miropro [0070] Exemplary antithrombotic and vasodilator com fen, naproxen, oxaproZin, piroZolac, pirprofen, pranoprofen, pounds include, but are not limited to, abciximab, acetorphan, protiZinic acid, salicylamide, sulindac, suprofen, suxibuZone, acetylsalicylic acid, argatroban, bamethan, benfurodil, ben tiaprofenic acid, tolmetin, xenbucin, ximoprofen, Zaltopro Ziodarone, betahistine, bisaramil, brovincamine, bufeniode, fen, Zomepirac, aspirin, acemetcin, bumadiZon, carprofenac, citicoline, clobenfurol, clopidogrel, cyclandelate, dalteparin, clidanac, di?unisal, enfenamic acid, fendosal, ?ufenamic dipyridamol, droprenilamine, enoxaparin, fendiline, ifen acid, ?unixin, gentisic acid, ketorolac, meclofenamic acid, prodil, iloprost, indobufen, isobogrel, isoxsuprine, heparin, mefenamic acid, mesalamine, prodrugs thereof, and the like. lami?ban, midrodine, nadroparin, nicotinoyl alcohol, nylid [0078] Exemplary phosphodiesterase inhibitors, include rin, oZagrel, perhexyline, phenylpropanolamine, preny but are not limited to, ?laminast, piclamilast, rolipram, Org US 2011/0105421A1 May 5,2011

20241, MCI-154, ro?umilast, toborinone, posicar, lixaZi sulotroban, ticlopidine, tiro?ban, trapidil, ticlopidine, none, Zaprinast, sildena?l, pyraZolopyrimidinones, motapi trifenagrel, trilinolein, 3-substituted 5,6-bis(4-methoxyphe Zone, pimobendan, Zardaverine, siguaZodan, Cl-930, EMD nyl)-1,2,4-triaZines; antibodies to glycoprotein IIb/IIIa; anti 53998, imaZodan, saterinone, loprinone hydrochloride, 3-py serotonin drugs, such as, for example, clopridogrel; sul?n ridinecarbonitrile derivatives, acefylline, albifylline, bami pyraZone and the like; aspirin; dipyridamole; clo?brate; fylline, denbufyllene, diphylline, doxofylline, etofylline, tor pyridinol carbamate; glucagon, caffeine; theophyllin pen bafylline, theophylline, nanterinone, pentoxofylline, toxifyllin; ticlopidine, and the like. proxyphylline, cilostaZol, cilostamide, MS 857, piroximone, [0081] Exemplary proton pump inhibitors include, but are milrinone, aminone, tolafentrine, dipyridamole, papavero not limited to, disulpraZole, esomepraZole, lansopraZole, line, E4021, thienopyrimidine derivatives, tri?usal, ICOS leminopraZole, omepraZole, pantopraZole, rabepraZole, timo 3 51, tetrahydropiperaZino(1,2-b)beta-carboline-1,4-dione praZole, tenatopraZole, 2-(2-benZimidaZolyl)-pyridine, tricy derivatives, carboline derivatives, 2-pyraZolin-5-one deriva clic imidaZole, thienopydidine benZimidaZole, ?uoroalkoxy tives, fused pyridaZine derivatives, quinaZoline derivatives, substituted benZimidaZole, dialkoxy benZimidaZole, N-sub anthranilic acid derivatives, imidaZoquinaZoline derivatives, stituted 2-(pyridylalkenesul?nyl)benZimidaZole, cyclohep tadala?l and vardena?l. tenepyridine, 5-pyrrolyl-2-pyridylmethylsul?nyl benZimida [0079] Exemplary potassium channel blockers include, but Zole, alkylsul?nyl benZimidaZole, ?uoro are not limited to, nicorandil, pinacidil, cromakalim (BRL pyridylmethylsul?nyl benZimidaZole, imidaZo(4,5-b) 34915), aprikalim, bimakalim, emakalim, lemakalim, pydridine, RO 18-5362, IY 81149, 4-amino-3-carbonyl minoxidil, diaZoxide, 9-chloro-7-(2-chlorophenyl)-5H-py quinoline, 4-amino-3-acylnaphthyride, 4-aminoquinoline, rimido(5,4-d)(2)-benZaZepine, Ribi, CPG-11952, CGS 4-amino-3 -acylquinoline, 3-butyryl-4-(2-methylpheny 9896, ZD 6169, diaZixide, Bay X 9227, P1075, Bay X 9228, lamino)-8-(2-hydroxyethoxy)quinoline, quinaZoline, tet SDZ PCO 400, WAY-120,491, WAY-120,129, Ro 31-6930, rahydroisoquinolin-2-yl pyrimidine, YH 1885, 3-substituted SR 44869, BRL 38226, S 0121, SR 46142A, CGP 42500, SR 1,2,4-thiadiaZolo(4,5-a) benZimidaZole, 3-substituted imi 44994, artilide fumarate, loraZepam, temaZepam, ril daZo(1,2-d)-thiadiaZole, 2-sul?nylnicotinamide, pyridyl maZafone, nimetaZepam, midaZolam, lormetaZepam, lopra sul?nylbenZ imidaZole, pyridylsul?nyl thieno imidaZole, Zolam, ibutilide fumarate, haloxaZolam, ?unitraZepam, esta theinoimidaZole-toluidine, 4,5-dihydrooxaZole, thienoimi Zolam, doxefaZepam, clonaZepam, cinolaZepam, brotiZolam, daZole-toluidine, Hoe-731, imidaZo(1,2-a)pyridine, pyrrolo and the like. (2,3-b)pyridine, and the like. [0080] Exemplary platelet reducing agents include, but are [0082] Exemplary renin inhibitors include, but are not lim not limited to, ?brinolytic agents such as for example, ancrod, ited to, aldosterone, aliskiren (SPP-100), ditekiren, enalkrein anistreplase, bisobrin lactate, brinolase, Hageman factor (i.e. (A-64662), medullipin, terlkiren, tonin, Zankiren, RO factor X11) fragments, plasminogen activators such as, for 42-5892 (remikiren), A 62198, A 64662, A 65317, A 69729, example, streptokinase, tissue plasmino gen activators (TPA), A 72517 (Zankiren), A 74273, CP 80794, CGP 29287, CGP urokinase, pro-urokinase, recombinant TPA, plasmin, plas 38560A, EMD 47942, ES 305, ES 1005, ES 8891, FK 906, minogen, and the like; anti-coagulant agents including but are FK 744, H 113, H-142, KRI 1314, pepstatin A, RO 44-9375 not limited to, inhibitors of factor Xa, factor TFPI, factor (ciprokiren), RO 42-5892, RO 66-1132, RO 66-1168, SP 500, Vlla, factor lXc, factor Va, factor Vllla, inhibitors of other SP 800, SR-43845, SQ 34017, U 71038, YM-21095, coagulation factors, and the like; vitamin K antagonists, such YM-26365, urea derivatives of peptides, amino acids con as, for example, coumarin, coumarin derivatives (e.g., War nected by nonpeptide bonds, di- and tri-peptide derivatives farin sodium); glycosoaminoglycans such as, for example, (e.g., Act-A, Act-B, Act-C, ACT-D, and the like), amino acids heparins both in unfractionated form and in loW molecular and derivatives thereof, diol sulfonamides and sul?nyls, Weight form; ardeparin sodium, bivalirudin, bromindione, modi?ed peptides, peptidyl beta-aminoacyl aminodiol car coumarin, dalteparin sodium, danaparoid sodium; daZoxiben bamates, monoclonal antibodies to renin. hydrochloride, desirudin, dicumarol, efegatran sulfate, enox [0083] Exemplary COX-2 inhibitors include, but are not aparin sodium, ifetroban, ifetroban sodium, lyapolate limited to, nimesulide, celecoxib (CELEBREX®), etoricoxib sodium, nafamo stat mesylate, phenprocoumon, sulfatide, tin (ARCOXIA®), ?osulide, lumiracoxib (PREXIG®, COX Zaparin sodium, retaplase; trifenagrel, Warfarin, dextrans and 189), parecoxib (DYNSTAT®), rofecoxib (V IOXX®), tira the like; abciximab, acadesine, anipamil, argatroban, aspirin, coxib (JTE-522), valdecoxib (BEXTRA®), ABT 963, BMS clopidogrel, diadeno sine 5',5"'-P1,P4-tetraphosphate 347070, CS 502, DuP 697, GW-406381, NS-386, SC-57666, (Ap4A) analogs, di?brotide, dilaZep dihydrochloride, dipy SC-58125, SC-58635, and the like, and combinations oftWo ridamole, dopamine, 3-methoxytyramine, glucagon, glyco or more thereof. protein antagonists, such as, for example, Ro-43-8857, [0084] Exemplary steroids include, but are not limited to, L-700,462, iloprost, isocarbacyclin methyl ester, itaZigrel, 21-acetoxypregnenolone, alcolometasone, algestone, amci ketanserin, BM-13.177, lami?ban, lifariZine, molsidomine, nonide, beclomethasone, betamethasone, budesonide, chlor nifedipine, oxagrelate, prostaglandins, platelet activating fac prednisone, clobetasol, clobentasone, clocortolone, clopred tor antagonists such as, for example, lexipafant, prostacy nol, corticosterone, cortisine, corticaZol (cortivatol), clins, pyraZines, pyridinol carbamate, ReoPro (i.e., abcix de?aZacort, desonide, desoximetasone, dexamethasone, imab), sul?npyraZone, synthetic compounds BN-50727, di?orasone, di?ucortolone, di?uprednate, enoxolone, ?uZa BN-52021, CV-4151, E-5510, FK-409, GU-7, KB-2796, cort, ?ucloronide, ?umethasone, ?unisolide, ?ucinolone KBT-3022, KC-404, KF-4939, OP-41483, TRK-100, acetonide, ?uocininide, ?uocortin butyl, ?uocortolone, ?uo TA-3090, TFC-612, ZK-36374, 2,4,5,7-tetrathiaoctane, 2,4, rometholone, ?uperolone acetate, ?uprednidene acetate, ?u 5,7-tetrathiaoctane 2,2-dioxide, 2,4,5-trithiahexane, theo prednisolone, ?urandrenolide, ?uticasone propionate, ?uti phyllin pentoxifyllin, thromboxane and thromboxane syn casone propionate, formocortal, halcinonide, halobetasol thetase inhibitors such as, for example, picotamide, propionate, halometasone, haloprednone acetate, hydrocor US 2011/0105421A1 May 5,2011

tamate, hydrocortisone and its derivatives (such as phosphate, tannin in an amount effective to inhibit bio?lm formation. 21 -sodium succinate and the like), hydrocortisone terbutate, Percutaneous devices (such as catheters) and implanted iso?upredone, loteprednol etabonate, maZipredone, medical devices (including, but not limited to, pacemakers, medrysone, meprednisone, methylprednisolone, mometa vascular grafts, stents, and heart valves) commonly serve as sone furoate, paremethasone, prednicarbate, prednisolone foci for bacterial infection. The tendency of some microor and its derivatives (such as 21-stearoylglycolate, sodium ganisms to adhere to and coloniZe the surface of the device phosphate and the like), prednisone, prednival, prednylidene promotes such infections, Which increase the morbidity and and its derivatives (such as 21-diethylaminoactetate and the mortality associated With use of the devices. like), rimexolone, tixocortol, trimcinolone and its derivatives [0089] For example, one or more ellagitannins is used to (such as acetonide, benetonide and the like), and the like. inhibit bio?lm formation on substrates used to manufacture [0085] The combination therapies described herein is pro medical devices associated With non-invasive and invasive vided in a combined amount effective to inhibit QS in the medical procedures. Such substrates include, Without limita bacteria (and/ or treat a bacterial infection associated With tion, tubular, sheet, rod and articles of proper shape for use in bacterial QS and/or treat a disorder associated With bio?lm a number of medical devices such as vascular grafts, aortic formation). This process may involve administering to a sub grafts, arterial, venous, or vascular tubing, vascular stents, ject in need thereof one or more ellagitannins and a standard dialysis membranes, tubing or connectors, blood oxygenator of care anti -bacterial therapeutic (and/ or additional therapeu tubing or membranes, surgical instruments, ultra?ltration tic/ second agent) at the same time, Which may be achieved by membranes, intra-aortic balloons, stents, blood bags, cath administering a single composition or pharmacological for eters, sutures, soft or hard tissue prostheses, synthetic pros mulation that includes both an ellagitannin and a standard of theses, prosthetic heart valves, tissue adhesives, cardiac pace care therapeutic, or by administering tWo distinct composi maker leads, arti?cial organs, endotracheal tubes, lenses for tions or formulations, at the same time, Wherein one compo the eye such as contact or intraocular lenses, blood handling sition includes an ellagitannin and the other includes a stan equipment, apheresis equipment, diagnostic and monitoring dard of care anti-bacterial therapeutic. In another catheters and sensors, biosensors, dental devices, drug deliv embodiment, the combination therapy involves administer ery systems, or bodily implants of any kind. For example, ing to a subject in need thereof an ellagitannin and a standard arthroscopic surgery is routinely performed With use of medi of care anti -bacterial therapeutic (and/ or additional therapeu cal devices that minimiZe the invasiveness of the procedure. tic/second agent) at different times, Which may be achieved Such devices include, for example and Without limitation, by administering tWo distinct compositions or formulations, ultrathin micro?beroptic endoscopes that offer the laryngolo at different time intervals, Wherein one composition includes gist unique access to the limited spaces of the temporal bone an ellagitannin and the other includes a standard of care and skull base. In another example, a stent supplemented With anti-bacterial therapeutic (and/ or additional therapeutic/sec one or more ellagitannins can be constructed. Stents are used ond agent). to maintain an open lumen in tissues including the tracheo [0086] Alternatively, the treatment With the ellagitannin(s) bronchial system, the biliary hepatic system, the esophageal may precede or folloW the treatment With the standard of care boWel system, and the urinary tract system. anti-bacterial therapeutic (and/ or additional therapeutic/sec [0090] III. Routes of Administration and Dosage ond agent) by intervals ranging from minutes to Weeks. In [0091] Ellagitannin(s) either alone or in combination With a embodiments Where the ellagitannin(s) and the standard of standard of care anti-bacterial therapeutic as described herein care anti-bacterial therapeutic (and/ or additional therapeutic/ are administered by any route that delivers an effective do sage second agent) are administered separately (either in separate to the desired site of action, With acceptable (preferably mini compositions administered simultaneously or in separate mal) side-effects. Numerous routes of administration are compositions administered at different time intervals), one knoWn, including for example, oral, rectal, vaginal, transmu Would generally ensure that a signi?cant period of time did cosal, buccal or intestinal administration; parenteral delivery, not expire betWeen the times of each delivery, such that the including intraperitoneal, intramuscular, subcutaneous, further therapeutic agent and the ellagitannin Would still be intramedullary injections, as Well as intrathecal, cutaneous or able to exert an advantageously combined effect. In such intraderrnal injections; respiratory or inhalation, nasal, pul instances, it is contemplated that one Would administer both monary and topical application, including ocular and trans modalities Within about 1, about 2, about 3, about 4, about 5, dermal applications. about 5, about 6, about 7, about 8, about 9, about 10, about 11, [0092] When used in the above or other treatments, a about 12, about 13, about 14, about 15, about 16, about 17, "therapeutically-effective amount” or an “effective amount” about 18, about 19, about 20, about 21, about 22, about 23, of an ellagitannin or a composition comprising an ellagitan about 24, about 36, about 48, or about 72 hours of each other. nin means a su?icient amount of the ellagitannin is provided In one embodiment, both modalities are administered Within to treat disorders or to achieve a desired result. It Will be about 6-12 hours of each other. In some situations, it may be understood, hoWever, that the total daily usage of the ellagi desirable to extend the time period for treatment signi?cantly. tannin in a therapeutic method described herein Will be Exemplary routes of administration of the peptides or com decided by the attending physician Within the scope of sound positions described herein include, but are not limited to, medical judgment. The speci?c therapeutically effective dose intraderrnal, intramuscular, intraperitoneal, intraocular, intra level for any particular patient Will depend upon a variety of venous, subcutaneous, topical, oral and intranasal adminis factors including the disorderbeing treated and the severity of tration. the disorder; activity of the speci?c compound employed; the [0087] C. Medical Devices speci?c composition employed; the age, body Weight, gen [0088] In another embodiment, one or more ellagitannins is eral health, sex and diet of the patient; the time of adminis used to inhibit bio?lm formation associated With bacterial QS tration, route of administration, and rate of excretion of the on a medical device by contacting the device With an ellagi speci?c compound employed; the duration of the treatment; US 2011/0105421Al May 5,2011

drugs used in combination or coincidental With the speci?c Waxes, and natural and synthetic gums, e.g., acacia sodium compound employed; and like factors Well knoWn in the alginate, polyvinylpyrrolidone, cellulosic polymers (includ medical arts. For example, it is Well Within the skill of the art ing hydroxypropyl cellulose, hydroxypropyl methylcellu to start doses of the compound at levels loWer than required to lose, methyl cellulose, ethyl cellulose, hydroxyethyl cellu achieve the desired therapeutic effect and to gradually lose, and the like), and Veegum. Diluents are typically increase the dosage until the desired effect is achieved. necessary to increase bulk so that a practical siZe tablet is [0093] The dose of ellagitannin administered to a mamma ultimately provided. Suitable diluents include dicalcium lian subject range from about 10 pg to about 400 mg/day. In phosphate, calcium sulfate, lactose, cellulose, kaolin, manni some embodiments, the dose is about 10 ug/day, about 25 tol, sodium chloride, dry starch and poWdered sugar. Lubri ug/day, about 50 ug/day, about 75 ug/day, about 100 ug/day, cants are used to facilitate tablet manufacture; examples of about 125 ug/day, about 150 ug/day, about 175 ug/day, about suitable lubricants include, for example, vegetable oils such 200 ug/day, about 225 ug/day, about 250 ug/day, about 275 as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, ug/day, about 300 ug/ day, about 325 ug/day, about 350 and oil of theobroma, glycerin, magnesium stearate, calcium ug/day, about 375 ug/ day, about 400 ug/day, about 425 stearate, and stearic acid. Disintegrants are used to facilitate ug/day, about 450 ug/ day, about 475 ug/day, about 500 disintegration of the tablet, and are generally starches, clays, ug/day, about 750 ug/day, about 1 mg/day, about 5 mg/day, celluloses, algins, gums or crosslinked polymers. Fillers about 10 mg/ day, about 25 mg/ day, about 30 mg/day, about 40 include, for example, materials such as silicon dioxide, tita mg/day, about 45 mg/day, about 50 mg/ day, about 60 mg/day, nium dioxide, alumina, talc, kaolin, poWdered cellulose and about 70 mg/day, about 80 mg/day, about 90 mg/day, about microcrystalline cellulose, as Well as soluble materials such 100 mg/ day, about 150 mg/ day, about 200 mg/day, about 250 as mannitol, urea, sucrose, lactose, dextrose, sodium chloride mg/day, about 300 mg/day, about 350 mg/day or about 400 and sorbitol. StabiliZers are used to inhibit or retard drug mg/day. In some embodiments, the maximum dosage is about decomposition reactions that include, by Way of example, 200 mg/ day. In some embodiments, the maximum dosage is oxidative reactions. Surfactants may be anionic, cationic, about 300 mg/day. If desired, the effective daily dose is amphoteric or nonionic surface active agents. divided into multiple doses for purposes of administration; [0097] The dosage form also includes a capsule, in Which consequently, single dose compositions may contain such case the ellagitannin-containing composition is in one aspect amounts or submultiples thereof to make up the daily dose. encapsulated in the form of a liquid or solid (including par The dosage regimen of an ellagitannin composition alone or ticulates such as granules, beads, poWders or pellets). Suit in combination as described herein to be used in treatment of able capsules may be either hard or soft, and are generally bacterial infections (orbio?lm formation) associated With QS made of gelatin, starch, or a cellulosic material, With gelatin Will be determined by the attending physician considering capsules preferred. TWo-piece hard gelatin capsules are pref various factors Which modify the action of the ellagitannin, erably sealed, such as With gelatin bands or the like. (See, for e.g., the patient’s age, sex, and diet, the severity of any infec e.g., Remington: The Science and Practice of Pharmacy, tion, time of administration and other clinical factors. supra), Which describes materials and methods for preparing [0094] Oral dosage forms include tablets, capsules, caplets, encapsulated pharmaceuticals. solutions, suspensions and/ or syrups, and may also comprise [0098] Solid dosage forms, Whether tablets, capsules, a plurality of granules, beads, poWders or pellets that may or caplets, or particulates, are, if desired, coated so as to provide may not be encapsulated. Such dosage forms are prepared for delayed release. Dosage forms With delayed release coat using conventional methods knoWn to those in the ?eld of ings are in one aspect manufactured using standard coating pharmaceutical formulation and described in the pertinent procedures and equipment. Such procedures are knoWn to texts, e.g., in Remington: The Science and Practice of Phar those skilled in the art and described in the pertinent texts macy, supra). Tablets and capsules represent the most conve (See, for e.g., Remington: The Science and Practice of Phar nient oral dosage forms, in Which case solid pharmaceutical macy, supra). In one aspect, after preparation of the solid carriers are employed. dosage form, a delayed release coating composition is applied [0095] Tablets include those manufactured using standard using a coating pan, an airless spray technique, ?uidized bed tablet processing procedures and equipment. One method for coating equipment, or the like. Delayed release coating com forming tablets is by direct compression of a poWdered, crys positions comprise in various aspects a polymeric material, talline or granular composition containing the active agent(s), e.g., cellulose butyrate phthalate, cellulose hydrogen phtha alone or in combination With one or more carriers, additives, late, cellulose proprionate phthalate, polyvinyl acetate phtha or the like. As an alternative to direct compression, tablets can late, cellulose acetate phthalate, cellulose acetate trimellitate, be prepared using Wet-granulation or dry-granulation pro hydroxypropyl methylcellulose phthalate, hydroxypropyl cesses. Tablets are also molded rather than compressed, start methylcellulose acetate, dioxypropyl methylcellulose succi ing With a moist or otherWise tractable material. nate, carboxymethyl ethylcellulose, hydroxypropyl methyl [0096] In addition to the ellagitannin either alone or in cellulose acetate succinate, polymers and copolymers formed combination as described herein, tablets prepared for oral from acrylic acid, methacrylic acid, and/ or esters thereof. administration Will in one aspect contain other materials such [0099] Sustained release dosage forms provide for drug as binders, diluents, lubricants, disintegrants, ?llers, stabiliZ release over an extended time period, and optionally are ers, surfactants, preservatives, coloring agents, ?avoring delayed release. As Will be appreciated by those of ordinary agents and the like. Binders are used to impart cohesive skill in the art, sustained release dosage forms are formulated qualities to a tablet, and thus ensure that the tablet remains in various aspects by dispersing a drug Within a matrix of a intact after compression. Suitable binder materials include, gradually bioerodible (hydrolyZable) material such as, for but are not limited to, starch (including corn starch and prege example, an insoluble plastic, a hydrophilic polymer, or a latiniZed starch), gelatin, sugars (including sucrose, glucose, fatty compound, or by coating a solid, drug-containing dos dextrose and lactose), polyethylene glycol, propylene glycol, age forrn With such a material. Insoluble plastic matrices are US 2011/0105421A1 May 5,2011

in certain aspects comprised of, for example, polyvinyl chlo ments to enhance their acceptability for various usages, ride or polyethylene. Hydrophilic polymers useful for pro provided that the additives do not deleteriously react With the viding a sustained release coating or matrix cellulosic poly ellagitannin material in the composition. mers include, Without limitation: cellulosic polymers such as [0102] Also suitable for topical application are sprayable hydroxypropyl cellulose, hydroxyethyl cellulose, hydrox aerosol preparations Wherein the ellagitannin, preferably in ypropyl methyl cellulose, methyl cellulose, ethyl cellulose, combination With a solid or liquid inert carrier material, is cellulose acetate, cellulose acetate phthalate, cellulose packaged in a squeeze bottle or in admixture With a pressur acetate trimellitate, hydroxypropylmethyl cellulose phtha iZed volatile, normally gaseous propellant, e. g., a Freon late, hydroxypropylcellulose phthalate, cellulose hexahydro (chloro?uorocarbon) or environmentally acceptable volatile phthalate, cellulose acetate hexahydrophthalate, and car propellant. Such compositions are used for in one aspect, boxymethylcellulose sodium; acrylic acid polymers and application to environmental surfaces, e. g., examining tables, copolymers, preferably formed from acrylic acid, meth toilet seats and the like, and/ or for application to the skin or to acrylic acid, acrylic acid alkyl esters, methacrylic acid alkyl mucous membranes. The aerosol or spray preparations esters, and the like, eg and Without limitation copolymers of optionally contain solvents, buffers, surfactants, perfumes, acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, and/or antioxidants in addition to the ellagitannin. methyl methacrylate and/or ethyl methacrylate, With a ter [0103] The compositions are in certain aspects employed in polymer of ethyl acrylate, methyl methacrylate and trimethy mixture With conventional excipients, i.e., pharmaceutically lammonioethyl methacrylate chloride (sold under the trade acceptable organic or inorganic carrier substances, suitable name Eudragit RS) preferred; vinyl polymers and for topical application Which do not deleteriously react With copolymers such as polyvinyl pyrrolidone, polyvinyl acetate, the ellagitannin in the composition. The compositions of the polyvinylacetate phthalate, vinylacetate crotonic acid invention optionally include diluents, ?llers, salts, buffers, copolymer, and ethylene-vinyl acetate copolymers; Zein; and stabiliZers, solubiliZers, and other materials Well knoWn in the shellac, ammoniated shellac, shellac-acetyl alcohol, and shel art (Remington’s Pharmaceutical Sciences 16th edition, lac n-butyl stearate. Fatty compounds for use as a sustained Osol, A. Ed. (1980). release matrix material include, but are not limited to, Waxes [0104] IV. Kits and Unit Doses generally (e.g., carnauba Wax) and glyceryl tristearate. [0105] In related variations of the preceding embodiments, [0100] Although compositions described herein are in one a composition comprising an ellagitannin as described herein aspect administered orally, other modes of administration are packaged alone, e.g., in a kit or package or unit dose, or is contemplated as Well. Exemplary modes of administration optionally arranged to permit co-administration With one or include transmucosal (e.g., US. Pat. Nos. 5,288,498; 6,248, more other therapeutic agents as described herein, but the 760; 6,355,248; 6,548,490, the disclosures of Which are ellagitannin and the agent are not in admixture. In an altema incorporated herein by reference in their entireties), transure tive variation, the ellagitannin and the agent are in admixture. thral (e.g., e.g., US. Pat. Nos. 5,919,474 and 5,925,629, the In some embodiments, the tWo components to the kit/unit disclosures of Which are incorporated herein by reference in dose are packaged With instructions for administering the tWo their entireties), vaginal or perivaginal (e.g., US. Pat. Nos. agents to a human subject for treatment of one of the above 4,211,679; 5,491,171 and 6,576,250, the disclosures ofWhich indicated disorders and diseases. The kit may comprise a are incorporated herein by reference in their entireties) and composition described herein in combination With a vehicle intranasal or inhalation (e.g., US. Pat. Nos. 4,800,878; 5,112, in a cream or gel base, as a pump-spray, as an aerosol, on an 804; 5,179,079; 6,017,963; 6,391,318 and 6,815,424, the dis impregnated bandage, or in a dropper. closures of Which are incorporated herein by reference in their EXAMPLES entireties). One of skill in the art Would be able to modify a composition comprising an ellagitannin either alone or in Example 1 combination With a standard of care anti -bacterial therapeutic as described herein to be used in any of the modes of admin Ellagitannins Inhibited Bacterial QS istration described herein. [0106] The present Example describes the isolation and [0101] Compositions comprising an ellagitannin either veri?cation of tWo C-glycosidic ellagitannins, castalagin and alone or in combination as described herein are be used as a vescalagin, from C. ereclus and the con?rmation of anti-QS topical agent. The topical agent is a solution, that is, in one activity of these compounds. aspect, a liquid formulation comprising the ellagitannin and a [0107] Materials and Methods: carrier. Other suitable forms include semi-solid or solid forms [0108] Plant extraction: Leaves of the medicinal plant C. comprising a carrier indigenous to topical application and ereclus (Combretaceae), Were collected and processed having a dynamic viscosity preferably greater than that of according to methods described previously (AdoniZio et al., Water, provided that the carrier does not deleteriously react 2006). Brie?y, pulverized plant material Was extracted into With the ellagitannin in the composition. Suitable formula boiling Water, freeZe-dried using a lyophiliZer, and stored at tions include, but are not limited to, lip balms, suspensions, —200 C. until needed. emulsions, creams, ointments, poWders, liniments, salves and [0109] Bioassay-guided fractionation: Anti-QS activity the like. If desired, these compositions may be steriliZed or Was con?rmed in the crude extract (AdoniZio et al., 2006) and mixed With auxiliary agents, including but not limited to, folloWed throughout the separation process using the R preservatives, stabiliZers, Wetting agents, buffers or salts for aeruginosa PAOl-derived biomonitor strains pPCS1001 and in?uencing osmotic pressure and the like Well knoWn in the pPCS1002 (Pesci et al., 1997). These strains harbor lacZ art. Preferred vehicles for semi-solid or solid forms topical fusions to the QS gene promoter regions enabling blue/White preparations include ointment bases, conventional oph selection for QS activity. Bioassays Were carried out as pre thalmic vehicles; creams; and gels. These topical prepara viously described (AdoniZio et al., 2006) With some modi? tions optionally contain emollients, perfumes, and/or pig cation. Brie?y, LB agar plates Were seeded With a laWn of one US 2011/0105421A1 May 5,2011

of the biomonitor strains and allowed to dry for 1 hour. Small [0114] NMR spectroscopy: NMR spectra Were recorded Wells Were then cut and aspirated from the agar, and 10 [1.1 using a Varian Inova 600 MHZ FT-NMR spectrometer With aliquots of each fraction Were pipetted into each Well. Wells D20 acidi?ed With d-TFA or D20:[D6]-acetone (8:2) as sol Were checked at 18 h and 24 h for Zones of QS inhibition. vents. Proton spectra Were obtained using standard param [0110] Preliminary thin layer chromatography: Approxi eters. The structure Was elucidated using COSY, HMQC, and mately 1 [1.1 of crude extract Was spotted to reverse phase thin HMBC, and through comparison With the standard spectra of layer chromatography (TLC) plates (tWo spots run in tan vescalagin and castalagin. dem). Adequate separation for visualiZation of three distinct [0115] Biological assays: Assays for AHL production, QS bands from each spot Was achieved With an 80:20 acetonitrile/ gene activity, and virulence factor production (LasA, LasB, H20 mobile phase containing 0.1% formic acid (FIG. 1, lane and pyoverdin) Were carried out as detailed in our previous A). A 1% ferric chloride stain Was applied to one-half of the Work (AdoniZio et al., 2008b). Samples Were tested at the plate as a phenolic indicator (FIG. 1, lane B). The other side folloWing concentrations: 1 mg/ml crude extract of C. erec Was overlaid With agar containing one of the aforementioned Zus, 40 ug/ml crude extract, 40 ug/ml vescalagin, or 40 ug/ml PAO1 biomonitor strains to indicate anti-QS activity (FIG. 1, castalagin. These additions Were compared to a media-only lane C). control for the reduction of QS. Prototypic R aeruginosa [0111] Fractionation methods: The fractionation of crude strain PAO1 (HolloWay & Morgan, 1986), and its promoter aqueous extract prior to HPLC separation is illustrated in fusion derivatives PZaSR-lacZ (pPCS1001), and PrhlR-lacZ FIG. 2. Separation Was patterned after methods developed for (pPCS1002), (AdoniZio et al., 2008b; Pesci et al., 1997) Were Wine polyphenolics (Sun et al., 2006). A CI8 PrepSep column used throughout this study. In addition, Staphylococcus (Fisher Scienti?c 11-131-1 15 g/20 ml) Was conditioned With aureus (ATCC # 12600) Was used in the LasA staphylolytic 200 ml methanol folloWed by 200 ml Water at pH 7. The How assay. rate of approximately 2 ml/min Was controlled through posi tive pressure applied via syringe. The crude aqueous extract [0116] Results of C. ereclus (0.25 g) Was resuspended in 5 ml Water, adjusted [0117] The data presented herein demonstrate the isolation to pH 7 With sodium hydroxide, and added to the column. and veri?cation of tWo C-glycosidic ellagitannins, castalagin FractionA Was eluted With approximately 1 50 ml Water at pH and vescalagin, from C. ereclus and the con?rmation of anti 7. The column Was then Washed With 100 ml Water and dried QS activity of these compounds. The isolation procedure Was under vacuum for several seconds. Fraction B Was then eluted largely directed by anti-QS bioassays using R aeruginosa With approximately 400 ml ethyl acetate. The column Was strains containing a lacZ fusion to the QS gene lasR or rhlR Washed With ethyl acetate and dried under vacuum before (AdoniZio et al., 2008b; Pesci et al., 1997). Each fraction Was elution of fraction C With 200 ml of acidi?ed methanol. The pipetted into a small Well in an agar plate seeded With one of presence of phenolics in each fraction Was monitored by these biomonitor strains and the appropriate reagents for periodically spotting to TLC plates coated With ferric chlo visualiZation of lacZ activity. Active fractions resulted in a ride reagent. Each fraction Was evaporated to dryness before change of color in the biomonitor strain from blue to off bioassay at 1 mg/ml concentration. White in the area surrounding the Well indicating anti-QS activity. Only fractions With activity Were subjected to further [0112] HPLC Separation: Fraction A Was separated on an Agilent 1200 series LC system (Agilent Technologies, USA) separation. using an Altima C18 column (51.i, 10><250 mm; 1004 injec [0118] TLC reveals a phenolic compound responsible for tion volume). A Water-acetonitrile mobile phase With 0.1% anti-QS activity. Prior to column chromatography, separation formic acid Was used With a ?oWrate of 1.5 ml/min. Condi Was attempted With various mobile phases on thin layer chro tions Were as folloWs: 0-2 min, 0% acetonitrile; 2-47 min, matography (TLC) plates. Reverse phase TLC of crude 0-40% acetonitrile; 47-48 min, 40-100% acetonitrile; 48-50 extract of C. ereclus using an acidi?ed acetonitrile/Water min, 100% acetonitrile; 50-51 min, 100-0% acetonitrile. mobile phase revealed tWo long-Wave UV-reactive bands and Fractions Were collected manually based on absorbance at one chromatic band, the latter being broWn in color and 313 nm and tested for anti-QS activity. slightly tailing (FIG. 1A). [0113] Mass spectrometric analysis: Direct injection elec [0119] The plates Were reacted With a number of different trospray ioniZation mass spectroscopy and MS-MS analysis agents, the most notable being ferric chloride, a stain for (Esquire 3000+, Ion Trap Mass Spectrometer Bruker Dalton phenolic compounds. Ferric chloride staining resulted in the ics, Germany) Were used for mass identi?cation of vescalagin chromatic band tuming dark blue, indicating phenolic com and castalagin. The isolated peaks 3 and 4 (FIG. 4) Were pounds in this region (FIG. 1B). An unstained portion of the injected directly into the ESI source With a syringe pump at a TLC plate Was overlaid With agar containing an anti-QS How rate of 0.2 ml/min. NebuliZer gas Was maintained at 7 psi. biomonitor strain derived from P aeruginosa (PlasR-lacZ Capillary temperature Was set at 3000 C. With a voltage of 3.5 (pPCS1001)) (AdoniZio et al., 2008b; Pesci et al., 1997). kV. Spectra Were obtained in negative ion mode With a scan Anti-QS activity Was visualiZed as a loss of blue color over ning range of 100-1000 m/ Z. MS-MS of compounds Were also the phenolic band due to reduced lasR expression and linked acquired in negative ion mode. HoWever, the conditions Were [3-galactosidase activity (FIG. 1C). modi?ed by increasing capillary temperature and voltage to [0120] Anti-QS activity against the tested lasR biomonitor 3250 C. and 4 kV respectively. Trap rolling and smart frag strain correlates With our previous data on the reduction of mentation settings Were activated, and the instrument Was set lasR gene expression seen With the crude extract of C. ereclus to scan from 50-1000 m/Z. Exact mass measurements Were (AdoniZio et al., 2008b). The localiZation of this activity made With a Waters Q-T of 2 using reserpine as a lock mass. indicates phenolic compounds are responsible for the anti-QS Samples Were introduced via LC How and reserpine ?oW activity seen in this species. Phenolic compounds have been from a syringe pump Was T-ed in. Spectra Were obtained in the previously shoWn to interfere With bacterial QS (Huber et al., positive ion mode With a scanning range from 100-1000 m/Z. 2004). US 2011/0105421A1 May 5,2011

[0121] Fractionation of polyphenolics in crude extract. Since the TLC staining and overlay procedure revealed the phenolic band to contain the anti-QS activity in C. ereclus, larger-scale fractionation Was then tailored to separation of these compounds. We adopted a method based on the resolu tion of Wine (Sun et al., 2006). A schematic of fractionation can be seen in FIG. 2. [0122] Crude aqueous extract of C. ereclus Was separated into three fractions based on solvent polarity. Fraction A eluted With Water as a bright yelloW liquid believed to contain phenolic acids and hydrolyZable tannins according to prior Work on polyphenolic separation (OsZmianski et al., 1988; Sun et al., 2006). Fraction B, Which eluted With ethyl acetate, Was colorless to pale yelloW and likely contained colorless proanthocyanins, ?avanols, and some monomer and oligomer phenolic acids (OsZmianski et al., 1988; Sun et al., 2006). Fraction C, Which eluted With acidi?ed methanol, Was dark broWn indicating the presence of complex tannins, pigmented proanthocyanidins, and pyranoanthocyanins (OsZmianski et al., 1988; Sun et al., 2006). The presence of phenolics in each fraction Was monitored by periodically spotting to TLC plates coated With ferric chloride reagent. Each fraction Was tested 1 castalagin R1 I H, R2 I OH for anti-QS effect revealing FractionA to contain the majority 2 vescalagin R1 I OH, R2 I H of activity (FIG. 3, (Panel 1)). [0123] Fraction A is the most polar fraction and thus con [0127] NMR data Were compared With spectra from the tains phenolic acids and hydrolyZable tannins. Prior Work on knoWn compounds (Glabasnia & Hofmann, 7. Agric. Food Chem., 54:3380-3390, 2006; Tang & Hancock, 1995), thus ellagic acid and EGCG (a hydrolyZable tannin) indicated that con?rming the identity of Fractions 3 and 4 as vescalagin and these compounds outperformed complex tannins (such as castalagin, respectively (FIG. 5). To avoid confusion, these those found in Fraction C) in the inhibition of Q8 (Huber et fractions Will hereafter be referred to by their compound al., 2004). names. [0124] HPLC separation revealed tWo fractions With anti [0128] Bioassays onP aeruginosa con?rm anti-QS activity QS activity. The separation of fractionA via HPLC resulted in of ellagitannins. The anti-QS activity of the ellagitannins six major peaks designated 1 to 6, and a number of minor vescalagin and castalagin is corroborated by prior Work on the peaks (FIG. 4). The detection Wavelength Was set at 313 nm QS-inhibiting properties of ellagic acid (Huber et al., 2004). HoWever, speci?c bioassays Were necessary to elaborate the based on Work by OsZmianski et al (OsZmianski et al., 1988). precise effect on P aeruginosa QS and virulence. Here, We Fractions 1 through 6 eluted at approximately 24, 26.8, 29, compare the effect of the puri?ed compounds to that of the 31.7, 33.8, and 36.4 minutes, respectively. Each fraction Was crude extract of C. ereclus examined in our previous Works collected and tested With the anti-QS biomonitor strains (AdoniZio et al., 2008a). revealing activity in Fractions 3 and 4 (FIG. 3, (Panel 11)). [0129] Polyphenolics have been shoWn to act as QS-inhibi [0125] Vescalagin and castalagin elucidated as active com tors at a concentration range of 20-60 ug/ml (Huber et al., pounds. Fractions 3 and 4 Were checked for purity by TLC 2004) thus a 40 ug/ml concentration Was chosen for the puri and behaved as pure compounds (single, non-tailing bands). ?ed ellagitannins. For comparison, We tested 40 mg/ml and 1 Both Fractions 3 and 4 When subjected to mass spectrometric ug/ml concentrations of crude extract, the latter being the Working concentration for our previous studies on of C. erec analysis produced a strong peak at m/Z 933 [M-HI and a Zus (AdoniZto et al., 2008a; AdoniZio et al., 2008b). smaller fragment peak at m/2Z 466 [M—H]2 [0130] LasA protease activity is reduced in the presence of [0126] MS-MS of compounds 3 and 4 revealed strong ellagitannins. LasA belongs to the p-lytic endopeptidase class peaks at m/ Z 915 and 613. The former being simply the parent of proteases (Kessler, 1995) and plays a major role in host compound minus Water, and the latter indicating the loss of tissue degradation (KharaZmi, 1989; Morihara & Homma, ellagic acid (302 daltons), a fragment regarded as diagnostic 1985). LasA protease activity Was determined by measuring of ellagitannins (Tang & Hancock, 1995). A literature revieW the ability of culture supematants to lyse boiled S. aureus suggested that these compounds may be the ellagitannins cells (Kong et al., Int. J. Med. Microbiol., 296:133-139, 2006). There Was a signi?cant decrease in LasA activity vescalagin and castalagin (Okuda et al., 1993; Tang & Han compared to that of the control When strain PAO1 Was groWn cock, 1995) as they both have a molecular Weight of 934 and in the presence of C. ereclus crude extract at 1 mg/ml (91% contain ellagic acid components. Exact mass measurements decrease), and a lesser effect at 40 pg/ml (26% decrease) revealed a mass of 935.0811 [M+1] for Fraction 3 and 935. (Table 1). Puri?ed vescalagin and castalagin affected signi? 0794 for Fraction 4 [M+1], a difference of 2.8 and 1.0 ppm, cant reductions in LasA activity as Well, With decreases of respectively. The calculated exact mass for the [M+1] ion of 73% and 80%, respectively (Table 1). The signi?cant effect both vescalagin and castalagin is: 935.0785 for C4lH27O26+. on LasA protease production is in agreement With our previ