US 201303 17094A1

(19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0317094 A1

Mathee et al. (43) Pub. Date: NOV. 28, 2013

(54) USE OF ELLAGITANNINS AS INHIBITORS (60) Provisional application No. 61/036,812, ?led on Mar. OF BACTERIAL QUORUM SENSING 14, 2008_

(71) Applicant: THE FLORIDA INTERNATIONAL UNIVERSITY BOARD OF TRUSTEES’ Miami, FL (Us) Publication Classi?cation

(72) Inventors: Kalai Mathee, Miami, FL (US); Allison (51) Int- Cl L. Adonizio, Cambridge, MA (US); A01N 43/24 (2006.01) Frederick Ausubel, Boston, MA (US); A61K 31/357 (2006.01) Jon Clardy, Boston, MA (US); Bradley A61K 45/06 (2006.01) Bennett, Miami, FL (US); Kelsey (52) us CL Downum, Arhngwn, TX (US) CPC .............. .. A01N 43/24 (2013 .01); A61K 45/06

(73) Assignee: THE FLORIDA INTERNATIONAL (2013-01); A61K 31/35 7 (2013-01) UNIVERSITY BOARD OF USPC ........................................................ .. 514/450

TRUSTEES, Miami, FL (US)

(21) Appl. No.: 13/893,916 (57) ABSTRACT (22) Filed: May 14, 2013

Related US‘ Application Data Described herein are materials and methods for the inhibition

(63) Continuation of application No, 12/9225 5 5, ?led on of bacterial QS. Methods of treating bacterial infections by Dec. 14, 2010, noW abandoned, ?led as application No. PCT/US2009/037163 on Mar. 13, 2009.

administration of one or more ellagitannins in amount effec tive to inhibit bacterial QS is also provided.

Patent Application Publication Nov. 28, 2013 Sheet 1 0f 4 US 2013/0317094 A1

Figure. 1

Patent Application Publication Nov. 28, 2013 Sheet 2 0f 4 US 2013/0317094 A1

Sap-315k. C" 1 E? (13H

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Figure 2

Patent Application Publication Nov. 28, 2013 Sheet 3 0f 4 US 2013/0317094 A1

Panel I Penal‘ II

Figure 3

Patent Application Publication Nov. 28, 2013 Sheet 4 0f 4 US 2013/0317094 A1

mAU

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500

250

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Figure 4

US 2013/0317094 A1

USE OF ELLAGITANNINS AS INHIBITORS OF BACTERIAL QUORUM SENSING

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims the bene?t of priority of US. Provisional Application No. 61/036,812, ?led Mar. 14, 2008, the disclosure of Which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present application is directed to the use of ellagitannins for the inhibition of bacterial quorum sensing.

BACKGROUND OF THE INVENTION

[0003] Many microbial pathogens cause tremendous dam age WorldWide, in humans as Well as in animals and crop plants. The continuing emergence of multiple-drug-resistant pathogen strains has necessitated ?nding neW compounds that can be used in antimicrobial treatment. In general, tWo strategies exist for controlling pathogens, either kill the pathogen or attenuate its virulence such that it does not dam age the host.

[0004] Many bacteria use autoinducer ligands to monitor their population densities in a phenomenon called quorum sensing. See Fuqua & Greenberg, Nature Reviews Molecular Cell Biology, 31685-695, 2002; or de Kievit et al., Infection & Immunity, 68: 4839-4849, 2000, for a revieW of the Quorum sensing system in pathogenic bacteria. Bacteria use quorum sensing to regulate a variety of phenotypes, such as bio?lm formation, toxin production, exopolysaccharide production, virulence factor production, and motility, Which are essential for the successful establishment of a symbiotic or pathogenic relationship With their respective eukaryotic hosts (Marketon et al., J. Bacteriol., 1851325-331, 2003; Ohtani et al., Mol. Microbiol., 441171-179, 2002; Quinones et al., Mol. Plant Microbe Interact, 181682-693, 2005; Rice et al., J. Bacteriol., 18713477-3485, 2005; Suntharalingam et al., Trends Micro biol., 1313-6, 2005). At high cell densities, bacteria use this chemical signaling process to sWitch from a nomadic exist ence to that of a multicellular community. This lifestyle sWitch is signi?cant, as numerous pathogenic bacteria use quorum sensing to turn on virulence pathWays and form drug impervious communities called bio?lms that are the basis of a myriad chronic infections. Over 80% of bacterial infections in humans involve the formation of bio?lms, as exempli?ed in lung infections by Pseudomonas aeruginosa, Which is the primary cause of morbidity in cystic ?brosis patients. The treatment of infections by pathogens that form bio?lms costs over $1 billion/year in the US alone. Studies With animal models have shoWn that strains With inactivated quorum sens ing genes shoW reduced virulence. Disrupting quorum sens ing may interfere With the ability of bacteria to form robust bio?lms and thus render the bacteria more sensitive to anti bacterial agents and the host’s immune response.

[0005] Quorum sensing is mediated by a signal molecule that binds to a cognate transcriptional activator to cause either upregulation or repression of genes that increase virulence factors, Which include exotoxins, proteases, alginates, lipopolysaccharides, pyocyanin and rhamnolipids. At loW bacterial cell density, the concentration of the signaling mol ecule does not activate the virulence genes, While at higher

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bacterial density, the concentration of the signaling molecule reaches a critical threshold to activate virulence genes.

[0006] In Gram negative bacteria, for example, the signal molecule is an acylated homoserine lactone (AHSL), often referred to as the autoinducer, Which interacts With a protein of a quorum regulon. A quorum regulon includes tWo pro teins, the autoinducer synthase (the I protein) and the regula tor (the R protein), Which, upon binding of the autoinducer, activates the transcription of numerous genes. In Pseudomo nas aeruginosa, tWo quorum regulons have been identi?ed. One quorum regulon is knoWn as the LasIR system and is mediated by a 3-oxo-dodecanoyl homoserine lactone (3-oxo C12-HSL) signal molecule. The other quorum regulon is knoWn as the RhlIR system and is mediated by a butyryl homoserine lactone (C4-HSL) signal molecule. [0007] In recent years it has become apparent that many Gram-negative bacteria employ one or more quorum sensing systems. The quorum sensing system is an attractive anti bacterial target because it is not found in humans and is critical for high level bacterial virulence. Bacterial quorum sensing systems comprise AHL derivatives With different acyl side chains to regulate, in a cell-density dependent man ner, a Wide variety of physiological processes unique to the life-cycle of each microbe. These processes include: sWarm ing, motility, bio?lm formation, conjugation, biolumines cence and/or production of pigments, antibiotics and enZymes. For example, in P aerugniosa quorum sensing pathWays affect the expression of various exoenZymes, bio ?lm formation and cell-cell spacing. Other bacteria react to quorum sensing stimulation by expressing proteases and pec tinases, expressing pili, entering stationary phase, emerging from lag phase and initiating cell division. [0008] Bio?lms are dense extracellular polymeric matrices in Which the bacteria embed themselves. Bio?lms alloW bac teria to create a microenvironment that attaches the bacteria to the host surface and Which contains excreted enZymes and other factors alloWing the bacteria to evade host immune responses including antibodies and cellular immune responses. Such bio?lms can also exclude antibiotics. Fur ther, bio?lms can be extremely resistant to removal and dis infection. For individuals suffering from cystic ?brosis, the formation of bio?lms by R aerugniosa is eventually fatal. Other bacteria also respond to quorum sensing signals by producing bio?lms. Bio?lms are inherent in dental plaques, and are found on surgical instruments, food processing and agriculture equipment and Water treatment and poWer gener ating machinery and equipment. [0009] Because of the virulence factors it triggers, the bac terial quorum-sensing system offers a target for use in modu lating the virulence of pathogenic bacteria. All acyl-ho moserine lactone quorum-sensing systems described to date, except that of V harveyi, utiliZe AI synthases encoded by a gene homologous to luxI of V?scheri. The response to the autoinducer is mediated by a transcriptional activator protein encoded by a gene homologous to luxR of V?scheri (Bassler and Silverman, in TWo Component Signal Transduction, Hoch et al., eds., Am. Soc. Microbiol. Washington DC, pp. 431-435,1995). [0010] Gram-negative bacteria represent numerous rel evant pathogens using quorum-sensing pathWays. Besides R aeruginosa, other gram-negative quorum sensing bacteria include1 Aeromonas hydrophila, A. salmonicida, Agrobacle rium Zumefaciens, Burkholderia cepacia, Chromobaclerium violaceum, Enlerobacler agglomeran, Erwinia carolovora,

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E. chrysanlhemi, Escherichia coli, Nilrosomas europaea, Obesumbaclerium proleus, Panloea slewarlii, Pseudomonas aureofaciens, P syringae, Ralslonia solanacearum, Rhiso bium elli, R. leguminosarum, Rhodobacler sphaeroides, Ser ralia liguefaciens, S. marcescens, [?brio anguillarum, V?s cheri, V cholerae, Xenorhabdus nemalophilus, Yersinia enlerocolilica, I’. peslis, I’. pseudoluberculosis, I’. medievalis, and I’. ruckeri. [0011] In addition to their pathogenic costs, quorum sens ing bacteria also have signi?cant economic impact in indus tries other than health care. For example, in agriculture, vari ous species of the genera Rhizobium, Bradyrhizobium and Sinorhizobium are important plant symbionts helping legumes to ?x nitrogen, While, species of the genera Erwinia, Xanlhomonas and Pseudomonas are responsible for signi? cant food-spoilage. Other industries, such as poWer genera tion, paper making and Water treatment are subject to biofoul ing by many types of slime forming bacteria, such as Deinococcus geolhermalis. [0012] Tannins are Widespread throughout the angiosperms (Okuda et al., Phytochem., 32:507-521, 1993), conferring structural bene?ts to the plant While providing protection through antioxidant and anti-feedant activity. Often classi?ed as “Waste” in natural products chemistry due to their abun dance and lack of protein speci?city (Zhu et al., 1997), tan nins and other polyphenolics have been previously ignored by the pharmaceutical industry. [0013] Many polyphenolics possess antimicrobial activity potentially explained by inhibition of microbial enZymes, substrate or iron deprivation, or inhibition of oxidative phos phorylation (Scalbert, Phytochem., 30:3875-3883, 1991). HoWever, the same study shoWs most bacteria are not suscep tible to ellagitannins, i.e. these compounds do not seem to have groWth inhibition or cidal effects. Furthermore, it has been shoWn that ellagic acid (a component of ellagitannins) can interfere With bacterial quorum sensing (Huber et al., Biosciences, 58:879-884, 2004). [0014] Thus, there is a need to identify and develop com pounds that are useful as inhibitors of bacterial quorum sens mg.

SUMMARY OF THE INVENTION

[0015] The present application is based on the discovery that ellagitannins, components in some medicinal plants, are capable of inhibiting quorum sensing (OS) in pathogenic bacteria. Thus, in one aspect, the invention provides methods of inhibiting OS in pathogenic bacteria in a mammalian sub ject contacting the bacteria With an ellagatannin in an amount effective to inhibit OS in the bacteria. In one embodiment, the bacteria is contacted With the ellagitannin in vivo. In such embodiments, the contacting comprises administering the ellagitannin to the mammalian subject. In one embodiment, the mammalian subject is af?icted With a bacterial infection associated With bacterial QS and the ellagitannin is adminis tered in an amount effective to treat the bacterial infection. In another embodiment, the mammalian subject is a?llicted With a disorder associated With bio?lm formation and the ellagi tannin is administered in an amount effective to treat the disorder. In one embodiment, the mammalian subject is human. In another embodiment, the human is immunocom promised (e.g., having, for example and Without limitation, cancer or AIDS). [0016] Another aspect of the invention provides a method of treating a bacterial infection associated with OS in a mam

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malian subject, the method comprising administering to the subject an ellagitannin in an amount effective to treat the infection. In one embodiment, the infection an infection caused by a bacterium is selected from the group consisting of: Aeromonas hydrophila, Aeromonas salmonicida, Agro baclerium Zumefaciens, Burkholderia cepacia, Chromobac Zerium violaceum, Enlerobacler agglomeran, Erwinia caro Zovora, Erwinia chrysanlhemi, Escherichia coli, Nilrosomas europaea, Obesumbaclerium proleus, Panloea slewarlii, Pseudomonas aureofaciens, Pseudomonas aeruginosa, Pseudomonas syringae, Ralslonia solanacearum, Rhisobium elli, Rhisobium leguminosarum, Rhodobacler sphaeroides, Serralia liguefaciens, Serralia marcescens, Staphylococcus aureus, Slaphyllococcus epidermidis, [?brio anguillarum, lflbrio?scheri, [?brio cholerae, Xenorhabdus nemalophilus, Yersinia enlerocolilica, Yersiniapeslis, Yersiniapseudoluber culosis, Yersinia medievalis, and Yersinia ruckeri. In some embodiments, exemplary bacterial infections include, but are not limited to, bacteremia, septicemia, endo- and pericarditis, sinusitis, upper respiratory tract infection, chronic bronchitis, pneumonia, cerebral and pulmonary lesions, meningitis, der matitis or folliculitis, necrotiZing fascitis, cellulitis, urinary tract infections, osteomylitis, enterocolitis, contact lens-asso ciated kerititis and conjunctivitis. In some embodiments, the mammalian subject to be treated is an immunocompromised individual, such as a human subject, for example and Without limitation, having cancer or AIDS.

[0017] In another aspect, the present invention provides a method of treating a disorder associated With bio?lm forma tion in a mammalian subject, the method comprising admin istering an ellagitannin to the subject in an amount effective to disrupt bio?lm formation in the subject. In various embodi ments, the disorder associated With bio?lm formation in the subject is selected from the group consisting of cystic ?brosis, dental caries, periodonitis, otitis media, muscular skeletal infections, necrotiZing fasciitis, biliary tract infection, osteo myelitis, bacterial prostatitis, endocarditis, native valve endocarditis, cystic ?brosis pneumonia, meloidosis, or skin lesions associated With bullous impetigo, atopic dermatitis and pemphigus foliaceus or implanted device-related infec tions. In some embodiments, the condition is a nosocomial infection, including but not limited to, pneumonia or an infec tion associated With sutures, exit sites, arteriovenous sites, scleral buckles, contact lenses, urinary catheter cystitis, peri toneal dialysis (CAPD) peritonitis, IUDs, endotracheal tubes, Hickman catheters, central venous catheters, mechanical heart valves, vascular grafts, biliary stent blockage, and orthopedic devices.

[0018] Also provided is a method of modulating bio?lm formation on a surface, the method comprising contacting the surface With an ellagitannin in an amount effective for disrupt or inhibit bio?lm formation on the surface. In one embodi ment, the surface is an inanimate surface. Exemplary inani mate surfaces include, but are not limited to, metal, glass, plastic, Wood and stone surfaces. In another embodiment, the surface is an animate surface. Exemplary animate surfaces include, but are not limited to, mammalian tissues, mamma lian membranes, mammalian skin.

[0019] As used herein, the term “pathogenic bacterium” or “pathogenic bacteria” refers to both gram-negative and gram positive bacterial cells capable of infecting and causing dis ease in a mammalian host, as Well as producing infection related symptoms in the infected host, such as fever or other

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signs of in?ammation, intestinal symptoms, respiratory symptoms, dehydration, and the like. [0020] In some embodiments, and Without limitation, the bacteria is of a genus selected from the group consisting of Aeromonas, Agrobaclerium, Burkholderia, Chromobacle rium, Enlerobacler, Erwinia, Escherichia, Nilrosomas, Obe sumbaclerium, Panloea, Pseudomonas, Ralslonia, Rhiso bium, Rhodobacler, Serralia, Slaphyllococcus, Vibl’iO, Xenorhabdus, and Yersinia. For example, in some embodi ments and Without limitation, the bacteria is of a species selected from the group consisting of Aeromonas hydrophila, Aeromonas salmonicida, Agrobaclerium Zumefaciens, Burkholderia cepacia, Chromobaclerium violaceum, Emera bacler agglomeran, Erwinia carolovora, Erwinia chrysan Zhemi, Escherichia coli, Nilrosomas europaea, Obesumbac Zerium proleus, Panloea slewarlii, Pseudomonas aureofaciens, Pseudomonas aeruginosa, Pseudomonas syringae, Ralslonia solanacearum, Rhisobium elli, Rhiso bium leguminosarum, Rhodobacler sphaeroides, Serralia liguefaciens, Serralia marcescens, Staphylococcus aureus, Slaphyllococcus epidermidis, [?brio anguillarum, [?brio?s cheri, [?brio cholerae, Xenorhabdus nemalophilus, Yersinia enlerocolilica, Yersinia peslis, Yersinia pseudoluberculosis, Yersinia medievalis, and Yersinia ruckeri. [0021] Also provided is a method of treating a disorder associated With QS in a mammalian subject resistant to treat ment With a standard of care anti-bacterial therapeutic com prising administering to the subject an ellagitannin in an amount effective to inhibit QS in the bacteria causing the infection.

[0022] In some embodiments, the methods described herein further comprise the step of administering a standard of care anti-bacterial therapeutic to the subject in need of treatment. In the context of methods of the invention, “stan dard of care” refers to a treatment that is generally accepted by clinicians for a certain type of patient diagnosed With a type of illness. For cardiac disorders, for example, an aspect of the invention is to improve standard of care therapy With co-therapy With one or more ellagitannins described herein. Exemplary standard of care anti-bacterial therapeutics include, but are not limited to, colloidal silver, penicillin, penicillin G, erythromycin, polymyxin B, viomycin, chloro mycetin, streptomycins, cefaZolin, ampicillin, methicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin aZactam, tobra mycin, cephalosporins (including cephalothin, cefaZolin, cephalexin, cephradine, cefamandole, cefoxitin, and 3rd-gen eration cephalosporins), Carbapenems (including imipenem, meropenem, Biapenem), bacitracin, tetracycline, doxycy cline, gentamycin, quinolines, neomycin, clindamycin, kana mycin, metronidaZole, treptogramins (including Quinupris tin/dalfopristun (SynercidTM)), Streptomycin, Ceftriaxone, Cefotaxime, Rifampin, Glycopeptides (including vancomy cin, teicoplanin, LY-333328 (Ortivancin)), Macrolides (in cluding erythromycin, clarithromycin, aZithromycin, linco mycin, and clindamycun), Ketolides (including Telithromycin, ABT-773), Tetracyclines, Glycylcyclines (in cluding Terbutyl-minocycline (GAR-936)), Aminoglyco sides, Chloramphenicol, Imipenem-cilastatin, Glycopeptides (including oritavancin, LY-333328, dalbavancin), Fluoroqui nolones (including o?oxacin, spar?oxacin, gemi?oxacin, cina?oxacun (DU-6859a)) and other topoisomerase inhibi tors, Trimethoprim-sulfamethoxaZole (TMP-SMX), Ciprof loxacin, topical mupirocin, OxaZolidinones (including AZD 2563, LineZolid (ZyvoxTM)), Lipopeptides (including

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Daptomycin, Ramoplanin), ARBELIC (TD-6424) (Thera vance), TD-6424 (Theravance), isoniaZid (INN), rifampin (RIF), pyraZinamide (PZA), Ethambutol (EMB), Capreomy cin, cycloserine, ethionamide (ETH), kanamycun, andp-ami nosalicylic acid (PAS). [0023] Combination therapy comprising an ellagitannin and a standard of care anti-bacterial therapeutic described herein for the treatment of a bacterial infection associated With QS is speci?cally contemplated. For example, in one embodiment, the invention provides a method of treating a bacterial infection associated With bacterial QS in a mamma lian subject in need of treatment comprising administering to the subject a therapeutically-effective amount of a combina tion therapy comprising (a) an ellagitannin and (b) a standard of care anti -bacterial therapeutic. In another embodiment, the invention provides a method of treating a disorder associated With bio?lm formation in a mammalian subject comprising administering to the subject a therapeutically-effective amount of a combination therapy comprising (a) an ellagitan nin and (b) a standard of care anti-bacterial therapeutic. In another embodiment, the invention provides a method of treating a disorder associated With bacterial QS (or bio?lm formation) in a mammalian subject comprising administering to the subject a therapeutically-effective amount of a combi nation therapy comprising (a) an ellagitannin and (b) a stan dard of care additional/ second agent as described herein.

[0024] Such combination therapy Would be provided in a combined amount effective to inhibit QS in the bacteria and/ or treat the bacterial infection and/or treat the disorder asso ciated With bio?lm formation. This process involves admin istering to a subject in need thereof an ellagitannin and a standard of care anti-bacterial therapeutic at the same time, Which may be achieved by administering a single composi tion or pharmacological formulation that includes both an ellagitannin and a standard of care therapeutic, or by admin istering tWo distinct compositions or formulations, at the same time, Wherein one composition includes an ellagitannin and the other includes a standard of care anti-bacterial thera peutic. In another embodiment, the combination therapy involves administering to a subject in need thereof an ellagi tannin and a standard of care anti-bacterial therapeutic at different times, Which may be achieved by administering tWo distinct compositions or formulations, at different time inter vals, Wherein one composition includes an ellagitannin and the other includes a standard of care anti-bacterial therapeu tic.

[0025] In some embodiments, the ellagitannin is selected from the group consisting of vescalagin, castalagin, punica lin, rhoipteleanin H, rhoipteleanin I, rhoipteleanin J, tellima grandin I, tellimagrandin II (eugeniin), pterocaryanin C, san guin H-4, sanguin H-5, casuarictin, potentillin, hemicetal congener pedunculagin, davidiin, corilagin, geraniin, carpi nusin, chebulinic acid, chebulagic acid, elaeocarpusin, repandusinic acid A, repandusinin, stachyurin, casuarinin, pedunculagin, 5-desgalloyl-stachyurin, casuariin, roburin A, roburin D, cercidininA, cercidinin B, cuspinin, platycaryanin D, nupharin A, sanguiin H-6, grandinin, coriariin, agrimo niin, rugosin D, oenothein B, Woodfordin C, strictinin and trapanin B. In one embodiment, the ellagitannin is vescalagin. In another embodiment, the ellagitannin is castalagin.

[0026] Compositions comprising the ellagitannin and a pharmaceutically-acceptable carrier, diluent or excipient are also contemplated.

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[0027] Also provided is the use of an ellagitannin in the manufacture of a medicament for the treatment of a disorder associated With bacterial OS (or for treatment of a disorder associated With bio?lm formation). [0028] The foregoing summary is not intended to de?ne every aspect of the invention, and additional aspects are described in other sections, such as the Detailed Description. The entire document is intended to be related as a uni?ed disclosure, and it should be understood that all combinations of features described herein may be contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. [0029] In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narroWer in scope in any Way than the variations de?ned by speci?c paragraphs herein. For example, certain aspects of the invention that are described as a genus, and it should be understood that every member of a genus is, individually, an aspect of the invention. Also, aspects described as a genus or selecting a member of a genus, should be understood to embrace combinations of tWo or more members of the genus. [0030] It should be understood that While various embodi ments in the speci?cation are presented using “comprising” language, under various circumstances, a related embodi 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 C. ereclus crude extract and the visualization of phenolic anti-QS activity. [0032] FIG. 2 is a schematic of fractionation of C. ereclus crude extract.

[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, and gonorrhea (Melendez, 1982, the disclosure of Which is incorporated herein by reference in their entireties). The

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activity of this plant on the bacterial QS system may explain its traditional use for these diseases. The data presented herein identi?ed tWo hydrolyZable tannins, vescalagin and castalagin, to be responsible for anti-QS activity in C. ereclus. Thus, the use of ellagitannins as an inhibitor of OS activity is speci?cally contemplated.

[0038] [0039] The term “ellagitannin” as used herein means a compound having a polyol core that is esteri?ed With at least tWo galloyl moieties, Wherein at least tWo of the galloyl moieties are oxidatively carbon-carbon coupled to each other. In one embodiment, the polyol core is a carbohydrate. In another embodiment, the polyol core is glucose. In another embodiment, the polyol core is D-glucose. In yet another embodiment, the polyol core is an open-chain D-glucose. When the ellagitannin comprises a carbohydrate polyol core, the anomeric carbon can form a C- or O-glycosidic bond With a galloyl moiety. In one embodiment, the ellagitannin forms a C-glycosidic bond With the galloyl moiety.

[0040] In some embodiments, the carbon-carbon coupled galloyl moieties are 4,6-hexahydroxybiphenoyl (HHBP or castalagin) and/or 2,3,5-nonahydroxyterphenoyl (NHTP or vescalagin).

I. Ellagitannins

HO OH

OH

galloyl moiety

NHTP Unit

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[0041] In one embodiment, the ellagitannin comprises a C-glycosidic, open-chain D-glucose core coupled to HHBP and NHTP (e.g. castalagin and vescalagin, respectively). [0042] Castalagin and vescalagin (Mayer et al., 1967; Mayer et al., 1970) belong to a sub-class of hydrolyZable tannins knoWn as C-glycosidic ellagitannins derived from gallic acid metabolism (Quideau & Feldman, 1996). Casta lagin and vescalagin are highly Water-soluble compounds featuring an open-chain glucose core esteri?ed to numerous oxidatively coupled galloyl moieties (speci?cally a 4,6 hexahydroxybiphenoyl (HHBP) unit and a 2,3,5-nonahy droxyterphenoyl (NHTP) unit) (Khanbabaee & van Ree, 2001). These complex structural units confer stereochemical rigidity to the molecule, and in fact, C-glycosidic ellagitan nins Would seem to have a higher tendency for selective protein interaction than other classes of polyphenolics (Haslam, 1996; Zhu et al., 1997). [0043] Ellagitannins knoWn in the art, other than castalagin and vescalagin, are also contemplated for use in the methods described herein. Such ellagitannins include, but are not lim ited to, punicalin [4,6-(SiS)-gallagyl-D-glucopyranose], Rhoipteleanin H, Rhoipteleanin I, Rhoipteleanin J, tellima grandin I, tellimagrandin II (eugeniin), pterocaryanin C, san guine H-4, sanguine H-5, casuarictin, potentillin, hemicetal congener pedunculagin, davidiin, corilagin, geraniin, carpi nusin, chebulinic acid, chebulagic acid, elaeocarpusin, repandusinic acid A, repandusinin, stachyurin, casuarinin, pedunculagin, 5-desgalloyl-stachyurin, casuariin, roburin A, roburin D, cercidininA, cercidinin B, cuspinin, platycaryanin D, nupharin A, sanguiin H-6, grandinin, coriariin, agrimo niin, rugosin D, oenothein B, Woodfordin C, strictinin and trapanin B. For a revieW of ellagitannins, see Quideau et al., Chem. Rev., 96:475-504, 1996 and Khanbabaee et al., Nat. Prod. Rep., 18:641-649, 2001). [0044] [0045] The invention provides in one aspect a method of inhibiting bacterial QS comprising contacting the bacteria With an ellagitannin in an amount effective to inhibit QS in the bacteria. In one embodiment, the bacteria is contacted With the ellagitannin in vivo. In such embodiments, the contacting comprises administering the ellagitannin to the mammalian subject. In one embodiment, the mammalian subject is a?licted With a bacterial infection associated With bacterial QS and the ellagitannin is administered in an amount effec tive to treat the bacterial infection. In another embodiment, the mammalian subject is a?licted With a disorder associated With bio?lm formation and the ellagitannin is administered in an amount effective to treat the disorder. In one embodiment, the mammalian subject is human. In another embodiment, the human is immunocompromised (e.g., having, for example and Without limitation, cancer or AIDS). Practice of methods of the invention in other mammalian subjects, especially mammals that are conventionally used as models for demon strating therapeutic e?icacy in humans (e.g., primate, por cine, canine, or rabbit animals), is also contemplated. [0046] In another embodiment, the bacteria is contacted With the ellagitannin ex vivo. In such an embodiment, for example, the contacting comprises administering the ellagi tannin to a surface in an amount effective to inhibit bio?lm formation associated With bacterial quorum sensing on sur face (including Without limitation, a medical device).

[0047] In another aspect, the invention provides a method of treating a bacterial infection associated With QS in a mam

II. Therapeutic Uses of Ellagitannins

Nov. 28, 2013

malian subject comprising administering to the subject one or more ellagitannins in an amount effective to inhibit QS in the bacteria. [0048] In yet another aspect, the invention provides a method of treating a disorder associated With bio?lm forma tion in a mammalian subject are also provided. Such methods comprise administering one or more ellagitannins to the sub ject in an amount effective to disrupt bio?lm formation in the subject. [0049] In one embodiment, the disorder associated With bio?lm formation in the subject is selected from the group consisting of cystic ?brosis, dental caries, periodonitis, otitis media, muscular skeletal infections, necrotiZing fasciitis, bil iary tract infection, osteomyelitis, bacterial prostatitis, endocarditis, native valve endocarditis, cystic ?brosis pneu monia, meloidosis, or skin lesions associated With bullous impetigo, atopic dermatitis and pemphigus foliaceus or implanted device-related inventions. In another embodiment, the condition is a nosocomial infection, including but not limited to, pneumonia or an infection associated With sutures, exit sites, arteriovenous sites, scleral buckles, contact lenses, urinary catheter cystitis, peritoneal dialysis (CAPD) perito nitis, IUDs, endotracheal tubes, Hickman catheters, central venous catheters, mechanical heart valves, vascular grafts, biliary stent blockage, and orthopedic devices. [0050] In some embodiments and Without limitation, the bacteria is of a genus selected from the group consisting of Aeromonas, Agrobaclerium, Burkholderia, Chromobacle rium, Enlerobacler, Erwinia, Escherichia, Nilrosomas, Obe sumbaclerium, Panloea, Pseudomonas, Ralslonia, Rhiso bium, Rhodobacler, Serralia, Staphylococcus, Vibl’iO, Xenorhabdus, and Yersinia. For example, in some embodi ments and Without limitation, the bacteria is of a species selected from the group consisting of Aeromonas hydrophila, Aeromonas salmonicida, Agrobaclerium Zumefaciens, Burkholderia cepacia, Chromobaclerium violaceum, Enlero bacler agglomeran, Erwinia carolovora, Erwinia chrysan Zhemi, Escherichia coli, Nilrosomas europaea, Obesumbac Zerium proleus, Panloea slewarlii, Pseudomonas aureofaciens, Pseudomonas aeruginosa, Pseudomonas syringae, Ralslonia solanacearum, Rhisobium elli, Rhiso bium leguminosarum, Rhodobacler sphaeroides, Serralia liguefaciens, Serralia marcescens, Slaphyllococcus aureus, [?brio anguillarum, [?brio ?scheri, Vibrio cholerae, Xenorhabdus nemalophilus, Yersinia enlerocolilica, Yersinia peslis, Yersinia pseudoluberculosis, Yersinia medievalis, and Yersinia ruckeri.

[0051] In various embodiments, an ellagitannin for use in the methods described is selected from the group consisting of vescalagin, castalagin, punicalin, Rhoipteleanin H, Rhoipteleanin I, Rhoipteleanin J, tellimagrandin I, tellima grandin II (eugeniin), pterocaryanin C, sanguine H-4, san guine H-5, casuarictin, potentillin, hemicetal congener pedunculagin, davidiin, corilagin, geraniin, carpinusin, che bulinic acid, chebulagic acid, elaeocarpusin, repandusinic acid A, repandusinin, stachyurin, casuarinin, pedunculagin, 5-desgalloyl-stachyurin, casuariin, roburin A, roburin D, cer cidinin A, cercidinin B, cuspinin, platycaryanin D, nupharin A, sanguiin H-6, grandinin, coriariin, agrimoniin, rugosin D, oenothein B, Woodfordin C, trapanin B and any combination thereof. In one embodiment, the ellagitannin is castalagin. In another embodiment, the ellagitannin is vescalagin. [0052] In one embodiment, the methods described herein further comprise the step of administering a standard of care

US 2013/0317094 A1

anti-bacterial therapeutic to the subject in need of treatment. In the context of methods of the invention, “standard of care” refers to a treatment that is generally accepted by clinicians for a certain type of patient diagnosed With a type of illness. For bacterial infections associated With bacterial QS, for example, an aspect of the invention is to improve standard of care therapy With co -therapy With one or more ellagitannins. Exemplary standard of care anti-bacterial therapeutics include, but are not limited to, colloidal silver, penicillin, penicillin G, erythromycin, polymyxin B, viomycin, chloro mycetin, streptomycins, cefaZolin, ampicillin, methicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin aZactam, tobra mycin, cephalosporins (including cephalothin, cefaZolin, cephalexin, cephradine, cefamandole, cefoxitin, and 3rd-gen eration cephalosporins), carbapenems (including imipenem, meropenem, Biapenem), bacitracin, tetracycline, doxycy cline, gentamycin, quinolines, neomycin, clindamycin, kana mycin, metronidaZole, treptogramins (including Quinupris tin/dalfopristun (SynercidTM)), Streptomycin, Ceftriaxone, Cefotaxime, Rifampin, glycopeptides (including vancomy cin, teicoplanin, LY-333328 (Ortivancin), dalbavancin), mac rolides (including erythromycin, clarithromycin, aZithromy cin, lincomycin, and clindamycun), ketolides (including Telithromycin, ABT-773), tetracyclines, glycylcyclines (in cluding Terbutyl-minocycline (GAR-936)), aminoglyco sides, chloramphenicol, Imipenem-cilastatin, ?uoroquinolo nes (including o?oxacin, spar?oxacin, gemi?oxacin, cina?oxacun (DU-6859a)) and other topoisomerase inhibi tors, Trimethoprim-sulfamethoxaZole (TMP-SMX), Ciprof loxacin, topical mupirocin, OxaZolidinones (including AZD 2563, LineZolid (ZyvoxTM)), Lipopeptides (including Daptomycin, Ramoplanin), ARBELIC (TD-6424) (Thera vance), TD-6424 (Theravance), isoniaZid (INN), rifampin (RIF), pyraZinamide (PZA), Ethambutol (EMB), Capreomy cin, cycloserine, ethionamide (ETH), kanamycun, and p-ami nosalicylic acid (PAS). [0053] Also provided is a method of modulating bio?lm formation on a surface, the method comprising contacting the surface With an ellagitannin in an amount effective for affect ing bio?lm formation on the surface. In one embodiment, the surface is an inanimate surface. Exemplary inanimate sur faces include, but are not limited to, metal, glass, plastic, Wood and stone surfaces. In another embodiment, the surface is an animate surface. Exemplary animate surfaces include mammalian tissues, mammalian membranes, mammalian skin.

[0054] A. Combination Therapy [0055] Combination therapy comprising one or more ella gitannins and a standard of care anti-bacterial therapeutic described herein for the treatment of a bacterial infection associated With QS is speci?cally contemplated. For example, in one aspect, the invention provides a method of treating a bacterial infection associated With bacterial QS in a mammalian subject in need of treatment comprising admin istering to the subject a therapeutically-effective amount of a combination therapy comprising (a) one or more ellagitan nins and (b) a standard of care anti-bacterial therapeutic.

[0056] In other embodiments, the combination of an ella gitannin With one or more additional therapeutics/second agents in methods of the invention may reduce the amount of either agent needed as a therapeutically effective dosage, and thereby reduce any negative side effects the agents may induce in vivo. Exemplary additional therapeutic/second agents include, but are not limited to, dornase alfa (Pul

Nov. 28, 2013

moZyme®), CTFR-correcting drugs (including but not lim ited to, gentamicin), anti-in?ammatory agents, NSAIDS, aldosterone antagonists, anti-bacterial agents, a COX-2 inhibitors, an ot-adrenergic antagonist, an [3-adrenergic antagonist, an anti-allergic compound, an anti-diabetic com pounds, an anti-hyperlipidemic compound, an anti-tussive compound, an angiotensin II antagonist, an angiotensin con verting enZyme (ACE) inhibitor, a bronchodilator, an anti sense nucleotide, anti-thrombotic and vasodilator compound, an antithrombogenic agent, a phosphodiesterase inhibitor, a tissue plasminogen activator, a thrombolytic agent, a ?brin olytic agent, a vasospasm inhibitor, an endothelin antagonist, an expectorant, an H2 receptor antagonist, a neural endopep tidase inhibitor, a calcium channel blocker, a potassium chan nel blocker, a nitrate, a nitric oxide promoter, a vasodilator, an antimicrobial agent, an antibiotic, a platelet reducing agent, a proton pump inhibitor, a rennin inhibitor, a steroid, an anti mitotic, a microtubule inhibitor, an actin inhibitor, a remod eling inhibitor, an agent for molecular genetic intervention, a cell cycle inhibitor, an inhibitor of the surface glycoprotein receptor, an anti-metabolite, an anti-proliferative agent, a chemotherapeutic agent, an anti-in?ammatory steroid, an immunosuppressive agent, an antibiotic, a radiotherapeutic agent, iodine-containing compounds, barium-containing compounds, a heavy metal functioning as a radiopaque agent, an extracellular matrix component, a cellular component, a biologic agent, ascorbic acid, a free radical scavenger, an iron chelator, an antioxidant, a radiolabelled form or other radio labelled forrn of any of the foregoing, or a mixture of any of these.

[0057] Exemplary antifungal agents including nystatin, liposomal nystatin, amorol?ne, butena?na, nafti?ne, terbin a?ne, ?ucytosine, ?uconaZole, itraconasole, ketoconaZole, posaconaZole, ravuconaZole, voriconaZole, clotrimaZole, econasole, miconaZole, oxiconaZole, sulconaZole, tercona Zole, ticonaZole, nikkomycin Z, caspofungin, micafungin, amphotericin B (AmB), AmB lipid complex, AmB colloidal dispersion, pimaricin, griseofulvin, ciclopirox olamine, halo progin, tolnaftate, undecylrnate. [0058] Exemplary antiviral agents include, but are not lim ited to, acyclovir, docosanol, ribarivin, interferons, cellulose acetate, carbopol, carrageenan (CAS No. 9000-07-1), plecon aril, amantidine, rimantidine, fomivirsen, Zidovudine, lami vudine, Zanamivir, oseltamivir, brivudine, abacavir, adefovir, amprenavir, arbidol, ataZanavir, atripla, cidofovir, combivir, edoxudine, efavirenZ, emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, gardasil, ibacitabine, immunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitor, lamivu dine, lopinavir, loviride, mk-0518, maraviroc, moroxydine, nel?navir, nevirapine, nexavir, nucleoside analogues, oselta mivir, penciclovir, peramivir, pleconaril, podophyllotoxin, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, teno fovir, tenofovir disoproxil, tipranavir, tri?uridine, triZivir, tro mantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, Zalcitabine, Zanamivir and Zidovu dine; [0059] Exemplary anti-microbial agents, include, but are not limited to, acediasulfone, aceturate, acetyl sulfameto ssipiraZine, acetyl sulfamethoxypyraZine, acranil, albenda Zole, alexidine, amatadine, ambaZone, amdinocillin, amika cin, p-aminosalicylic acid, p-aminosalicylic acid hydraZine, amoxicillin, ampicillin, anisomycin, apalcillin, apicyclin, apramycin, arbekacin, argininsa, aspoxicillin, aZidamfenicol,

US 2013/0317094 A1

aZidocillin, aZithromycin, aZlocillin, aZtreonam, bacampicil lin, benZoylpas, benZyl penicillin acid, benZyl sulfamide, bicoZamycin, bipenam, brodimoprim, capreomycin, carbeni cillin, carbomycin, cafaZedone, carindacillin, carumonam, cefcapene piVoxil, cefaclor, cefadroxil, cefafroxil, cefaman dole, cefatamet, cefatriZine, cefaZedone, cefaZolin, ceihu peraZone, cefclidin, cefdinir, cefditoren, ce?xime, ce?nenoxime, ce?netaZole, cefminox, cefodiZime, cefonicid, cefoperaZone, ceforanide, cefotaxime, cefotetan, cefotiam, cefoxitin, cefoZopran, cefpimiZole, cefpiramide, cefpirome, cefpodoxime proxetil, cefproZil, cefroxadine, cefsulodin, ceftaZidime, cefteram, cefteZole, ceftibuten, ceftiofur, cefti Zoxime, ceftriaxone, cefuroxime, cefuZonam, cephacetrile sodium, cephadrine, cephalexin, cephaloglycin, cephalori dine, cephalosporin C, cephalothin, cephapirin sodium, ceph radine, chloramphenicol, chlorotetracycline, cinoxacin, cipro?oxacin, claritromycin, clavulanic acid, clina?oxacin, clindamycin, clofaZimine, clofoctal, clometocillin, clomocy cline, cloxacillin, cloxyquin, cyclacilline, cycloserine, dano?axcin, dapsone, deoxycycline, deoxydihydrostrepto mycin, dibekacin, dicloxacillin, di?oxacin, dihydrostrepto mycin, dimetridaZole, diminaZene, dirirtomycin, doripenam, e?ornithine, enoxacin, enro?oxacin, enviomycin, epicillin, erythromycin, etacillin, ethambutol, ethionamide, famciclo Vir, fenbecillin, ?eroxacin, ?omoxef, ?oxacillin, ?umequine, furonaZide, fortimycin, furaZolium chloride, gentamycin, glyconiaZide, grepa?oxacin, guamecycline, halofuginone, hetacillin, homidium, hydroxyl-stilbamidine, ibostamycin, imidocarb, imipenam, ipronidazole, isoniaZide, iosamycin, inosine, lauroguadine, lenampicillin, levo?oxin, lincomycin, lome?oxacin, loracarbef, lymecyclin, mafenide, mebenda Zole, meclocyclin, meropenem, metampicillin, metacicline, methacycline, methicillin sodium, metronidaZole, 4'-(meth ylsulfamoyl) sulfanilanilide, meZlocillin, meZiocillin, micronomycin, midecamycinAl, minocycline, miocamycin, miokamycin, morfaZinamide, moxalactam, mupirocin, myxin, nadi?oxacin, nalidixic acid, negamycin, neomycin, netlimycin, nifurfoline, nifurpirinol, nifurpraZine, nimora Zole, nitroxoline, nor?oxacin, novobiocin, o?oxacin, olean domycin, opiniaZide, oxacillin, oxophenarsine, oxolinic acid, oxytetracycline, panipenam, paromycin, paZu?oxacin, pe?oxacin, penicillin G potassium salt, penicillin N, penicil lin O, penicillin V, penethamate hydroiodide, pentamidine, phenamidine, phenethicillin potassium salt, phenyl ami nosalicyclate, pipacycline, pipemidic acid, piperacillin, pirli mycin, piromidic acid, pivampicillin, pivcefalexin, pro?ro mycin, propamidine, propicillin, protionamide, puraltadone, puromycin, pyraZinamide, pyrimethamine, quinacillin, quinacrine, quinapyramine, quintine, ribostamycin, rifabu tine, rifamide, rifampin, rifamycin, rifanpin, rifapentine, rif axymine, ritipenem, rokitamycin, rolitetracycline, rosamy cin, ru?oxacin, salaZosulfadimidine, salinaZid, sancycline, sara?oxacin, sedacamycin, secnidaZole, sisomycin, spar ?oxacin, spectinomycin, spiramycin, spiramycin I, spiramy cin ll, spiramycin Ill, stilbamidine, streptomycin, streptoni ciZid, sulbactam, sulbenicillin, succisulfone, sulfanilamide, sulfabenZamide, sulfacetamide, sulfachloropyridaZine, sul fachrysoidine, sulfacytine, sulfadiaZine, sulfadicramide, sul fadimethoxine, sulfadoxine, sulfadraZine, sulfaetidol, sul fafenaZol, sulfaguanidine, sulfaguanole, sulfalene, sulfameraZine, sulfameter, sulfamethaZine, sulfamethiZole, sulfamethomidine, sulfamethoxaZole, sulfamethoxypy ridaZine, sulfamethylthiaZol, sulfamethylthiaZole, sulfame trole, sulfamidochrysoidine, sulfamoxole, sulfanilamide,

Nov. 28, 2013

4-sulfanilamido salicylic acid, 4-4'-sulfanilylbenZylamine, p-sulfanilylbenZylamine, 2-p-sul?nylanilinoethanol, sulfa nilylurea, sulfoniaZide, sulfaperine, sulfaphenaZole, sul faproxyline, sulfapyraZine, sulfapyridine, sulfathiaZole, sul faethidole, sulfathiourea, sul?somidine, sulfasomiZole, sulfasymaZine, sul?soxaZole, 4,4'-sul?nyldianiline, N4-sul fanilylsulfanilamide, N-sulfanilyl-3,4-xylamide, sultamicil lin, talampicillin, tambutol, taurolidine, teiclplanin, temocil lin, tetracycline, tetroxoprim, thiabendaZole, thiaZolsulfone, tibeZonium iodide, ticarcillin, tigemonam, tinidaZole, tobra mycin, tosu?oxacin, trimethoprim, troleandromycin, tros pectomycin, trova?oxacin, tubercidine, miokamycin, olean domycin, troleandromycin, Vancomycin, VeraZide, Viomycin, Virginiamycin and Zalcitabine. [0060] Exemplary aldosterone antagonists include, but are not limited to, canrenone, potassium canrenoate, dro spirenone, spironolactone, eplerenone (INSPRA®), epoxymexrenone, fadroZole, pregn-4-ene-7,21-dicarboxylic acid, 9,1 1-epoxy-17-hydroxy-3-oxo, .gamma.-lactone, methyl ester, (70.,1 10., 1 7B .)-; pregn-4-ene-7,21-dicarboxylic acid, 9,11-epoxy-17-hydroxy-3-oxo-dimethyl ester, (7a, 11a,17[3)-; 3'H-cyclopropa(6,7)pregna-4,6-diene-21-car boxylic acid, 9,1 1-epoxy-6,7-dihydro-17-hydroxy-3 -oxo-, .gamma.-lactone, (70.,1 10., l7[3.)-; pregn-4-ene-7,21-dicar boxylic acid, 9,1 1-epoxy-17-hydroxy-3-oxo-, 7-(1-methyl ethyl) ester, monopotassium salt, (7(X,ll(X,l7[3.)-, pregn-4 ene-7,21-dicarboxylic acid, 9,1 1,-epoxy-17-hydroxy-3 oxo-, 7-methyl ester, monopotassium salt, (7(X,ll(X,l7[3.)-, 3'H-cyclopropa(6,7) pregna-1,4,6-triene-21-carboxylic acid, 9,11-epoxy-6,7-dihydro-17-hydroxy-3-oxo-, y-lactone, (6B, 7[3,110t)-; 3'H-cyclopropa(6,7)pregna-4,6-diene-21-car boxylic acid, 9,1 1-epoxy-6,7-dihydro-17-hydroxy-3 -oxo-, methyl ester, (6[3,7[3,1 10,176), 3'H-cyclopropa (6,7)pregna 4,6-diene-21-carboxylic acid, 9,11-epoxy-6,7-dihydro-17 hydroxy-3-oxo-, monopotassium salt, (6[3,7[3,1 1(>t,17[3)-; 3'H-cyclopropa(6,7)pregna-1,4,6-triene-21-carboxylic acid, 9,1 1-epoxy-6,7-dihydro-17-hydroxy-3-oxo-y-lactone, (6B, 7[3,110t,17[3)-; pregn-4-ene-7,21-dicarboxylic acid, 9,11-ep oxy-17-hydroxy-3-oxo-, .gamma.-lactone, ethyl ester, (7a, 11(>t,17[3)-; pregn-4-ene-7,21-dicarboxylic acid, 9,11-epoxy 17-hydroxy-3-oxo-, y-lactone, 1-methylethyl ester, (70.,1 10., 17[3)-; RU-28318, and the like [0061] Exemplary ot-adrenergic receptor antagonists receptor antagonists, include, but are not limited to, phento lamine, tolaZoline, idaZoxan, deriglidole, RX 821002, BRL 44408, BRL 44409, BAM 1303, labetelol, ifenprodil, rau Wolscine, corynathine, raubascine, tetrahydroalstonine, apoyohimbine, akuammigine, .beta.-yohimbine, yohimbol, yohimbine, pseudoyohimbine, epi-3.alpha.-yohimbine, 10-hydroxy-yohimbine, 1 1 -hydroxy-yohimbine, tamsulosin, benoxathian, atipameZole, BE 2254, WB 4101, HU-723, tedisamil, mirtaZipine, setiptiline, reboxitine, delequamine, naftopil, saterinone, SL 89.0591, ARC 239, urapidil, 5-me thylurapidil, monatepi, haloperidol, indoramin, SB 216469, moxisylyte, traZodone, dapiproZole, efaroxan, Recordati 15/2739, SNAP 1069, SNAP 5089, SNAP 5272, RS 17053, SL 89.0591, KMD 3213, spiperone, AH 11110A, chloroeth ylclonidine, BMY 7378, niguldipine, and the like. [0062] Exemplary [3-adrenergic antagonists include, but are not limited to, acebutolol, alprenolol, amosulalol, aroti nolol, atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol, bucindolol, bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, buto?lolol, caraZolol, capsinolol, car teolol, carvedilol (COREG®), celiprolol, cetamolol, cin

US 2013/0317094 A1

dolol, cloranolol, dileValol, diprafenone, epanolol, ersentil ide, esmolol, esprolol, hydroxalol, indenolol, labetalol, landiolol, laniolol, leVobunolol, mepindolol, methylpranol, metindol, metipranolol, metriZoranolol, metoprolol, moprolol, nadolol, nadoxolol, nebiVolol, nifenalol, nipradilol, oxprenolol, penbutolol, pindolol, practolol, prone thalol, propranolol, sotalol, sotalolnadolol, sul?nalol, talip rolol, talinolol, tertatolol, tilisolol, timolol, toliprolol, tomalolol, trimepranol, xamoterol, xibenolol, 2-(3-(1,1-dim ethylethyl)-amino-2-hydroxypropoxy)-3-pyridenecarboni trilHCl-, 1-butylamino-3-(2,5 -dichlorophenoxy)-2-pro panol, 1 -isopropylamino -3 -(4-(2 -cyclopropylmethoxyethyl) phenoxy)-2-propanol, 3 -isopropylamino-1 -(7 -methylindan 4-yloxy)-2-butanol, 2-(3-t-butylamino-2-hydroxy-propy lthio)-4-(5-carbamoyl-2-thienyl)thiaZol, 7-(2-hydroxy-3-t butylaminpropoxy)phthalide, Acc 9369, AMO-140, BIB 165, CP-331684, Fr-172516, lSV-208, L-653328, LM-2616, SB-226552, SR-58894A, SR-59230A, TZC-5665, UK-1745, YM-430, and the like.

[0063] Exemplary anti-allergic compounds include, but are not limited to, acrivastine, allociamide, amlexanox, bromex ine, cetiriZine, clobenZepam, chromoglycate, chromolyn, deslortidine, emedastine, epinastine, fexofenadine, formot erol, hydroxyZine, ketotifen, loratadine, levocabastine, lodoxamide, mabuterol, montelukast, nedocromil, repirinast, salmeterol, seratrodast, suplatast tosylate, terfenadine, tiara mide, and the like.

[0064] Exemplary anti-diabetic compounds include, but are not limited to, acarbose, acetohexamide, buformin, carb utamide, chlorpropamide, glibomuride, gliclaZide, glime piride, glipiZide, gliquidone, glisoxepid, glyburide, gly buthiaZol(e), glybuZole, glyhexamide, glymidine, glypinamide, insulin, metformin, miglitol, nateglinide, phen butamide, phenformin, pioglitaZone, repaglinide, rosiglita Zone, tolaZamide, tolbutamide, tolcyclamide, troglitaZone, Voglibose, and the like.

[0065] Exemplary anti-hyperlipidemic compounds include, but are not limited to, statins or HMG-CoA reductase inhibitors, such as, for example, atorvastatin (LIPITOR®), bervastatin, cerivastatin (BAYCOL®), dalvastatin, ?uin dostatin (SandoZ XU-62-320), ?uvastatin, glenvastatin, lov astatin (MEVACOR®), mevastatin, pravastatin (PRAVA CHOL®), rosuvastatin (CRESTRO®.), simVastatin (ZOCOR®), Velostatin (also knoWn as synvinolin), VYTORINTM (eZetimibe/simVastatin), GR-95030, SQ 33,600, BMY 22089, BMY 22,566, Cl 980, and the like; gem?broZil, cholystyramine, colestipol, niacin, nicotinic acid, bile acid sequestrants, such as, for example, cholestyramine, colesevelam, colestipol, poly(methyl-(3-tri methylaminopropyl) imino-trimethylene dihalide) and the like; probucol; ?bric acid agents or ?brates, such as, for example, beZa?brate (BeZalipTM), beclobrate, bini?brate, cipro?brate, clino?brate, clo?brate, eto?brate, feno?brate (LipidilTM, Lipidil MicroTM), gem?broZil (LopidTM), nico? brate, piri?brate, roni?brate, sim?brate, theo?brate and the like; cholesterol ester transfer protein (CETP) inhibitors, such as for example, CGS 25159, CP-529414 (torcetrapid), JTT 705, substituted N-[3-(1,1,2,2-tetra?uoroethoxy)benZyl]-N (3-phenoxyphenyl)-tri?uoro-3-a-mino-2-propanols, N,N disubstituted tri?uoro-3-amino-2-propanols, PD 140195 (4-phenyl-5 -tridecyl-4H-1,2,4-triaZole-3 -thiol), SC-794, SC-795, SCH 58149, and the like.

Nov. 28, 2013

[0066] Exemplary antitussive compounds, include, but are not limited to, dextromethorphan, carbetapentane, carami phen, diphenylhydramine, hydrocodene, codeine and the like.

[0067] Exemplary angiotensin II antagonists include, but are not limited to, angiotensin, abitesartan, candesartan, can desartan cilexetil, elisartan, embusartan, enoltasosartan, eprosartan, fonsartan, forasartan, glycyllosartan, irbesartan, losartan, olmesartan, milfasartan, medoxomil, ripisartan, pra tosartan, saprisartan, saralasin, sarmesin, tasosartan, telmis artan, Valsartan, Zolasartan, 3-(2'(tetraZole-5-yl)-1,1'-biphen 4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imi-daZo(4,5-b) pyridine, antibodies to angiotensin II, A-81282, A-81988, BAY 106734, BlBR-363, BIBS-39, BIBS-222, BMS 180560, BMS-184698, BMS-346567, CGP-38560A, CGP 42112A, CGP-48369, CGP-49870, CGP-63170, Cl-996, CP-148130, CL-329167, CV-11194, DA-2079, DE-3489, DMP-811, DuP-167, DuP-532, DuP-753, E-1477, E-4177, E-4188, EMD-66397, EMD-666R4, EMD-73495, EMD 66684, EXP-063, EXP-929, EXP-3174, EXP-6155, EXP 6803, EXP-7711, EXP-9270, EXP-9954, FK-739, FRI 153332, GA-0050, GA-0056, HN-65021, HOE-720, HR-720, lCl-D6888, lCl-D7155, lCl-D8731, KRl-1177, KT3-671, KT-3579, KW-3433, L-158809, L-158978, L-159282, L-159689, L-159874, L-161177, L-162154, L-162234, L-162441, L-163007, L-163017, LF-70156, LRB-057, LRB-081, LRB-087, LY-235656, LY-266099, LY-285434, LY-301875, LY-302289, LY-315995, ME-3221, MK-954, PD-123177, PD-123319, PD-126055, PD-150304, RG-13647, RWJ-38970, RWJ-46458, S-8307, S-8308, SC-51757, SC-54629, SC-52458, SC-52459, SK 1080, SL-910102, SR-47436, TAK-536, UP-2696, U-96849, U-97018, UK-77778, UP-275-22, WAY-126227, WK-1260, WK-1360, WK-1492, WY 126227, YH-1498, YM-358, YM-31472, X-6803, XH-148, XR-510, ZD-6888, ZD-7155, ZD-8731, ZD 8131, the compounds of ACS registry numbers 124750-92-1, 133240-46-7, 135070-05-2, 139958-16-0, 145160-84-5, 147403-03-0, 153806-29-2, 439904-54-8P, 439904-55-9P, 439904-56-OP, 439904-57-1P, 439904-58 2P, 155918-60-8P, 155918-61-9P, 272438-16-1P, 272446 75-OP, 223926-77-OP, 169281-89-4, 439904-65-1P, 165113-01-9P, 165113-02-OP, 165113-03-1P, 165113-03 2P, 165113-05-3P, 165113-06-4P, 165113-07-5P, 165113 08-6P, 165113-09-7P, 165113-10-OP, 165113-11-1P, 165113-12-2P,165113-17-7R165113-18-8R165113-19-9P, 165113-20-2P,165113-13-3R165113-14-4R165113-15-5P, 165113-16-6P,165113-21-3R165113-22-4R165113-23-5P, 165113-24-6P,165113-25-7R165113-26-8R165113-27-9P, 165113-28-OP, 165113-29-1P, 165113-30-4P, 165113-31 5P, 165113-32-6P, 165113-33-7P, 165113-34-8P, 165113 35-9P, 165113-36-OP, 165113-37-1P, 165113-38-2P, 165113-39-3P,165113-40-6R165113-41-7R165113-42-8P, 165113-43-9P, 165113-44-OP, 165113-45-1P, 165113-46 2P, 165113-47-3P, 165113-48-4P, 165113-49-5P, 165113 50-8P, 165113-51-9P, 165113-52-OP, 165113-53-1P, 165113-54-2P,165113-55-3R165113-56-4R165113-57-5P, 165113-58-6P, 165113-59-7P, 165113-60-OP, 165113-61 1P, 165113-62-2P, 165113-63-3P, 165113-64-4P, 165113 65-5P, 165113-66-6P, 165113-67-7P, 165113-68-8P, 165113-69-9P,165113-70-2R165113-71-3R165113-72-4P, 165113-73-5P, 165113-74-6P, 114798-27-5, 114798-28-6, 114798-29-7, 124749-82-2, 114798-28-6, 124749-84-4, 124750-88-5, 124750-91-0, 124750-93-2, 161946-65-2P, 161947-47-3P, 161947-48-4P, 161947-51-9P, 161947-52

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OP, 161947-55-3P, 161947-56-4P, 161947-60-OP, 161947 61-1P, 161947-68-8P, 161947-69-9P, 161947-70-2P, 161947-71-3P, 161947-72-4P, 161947-74-6P, 161947-75-7P, 161947-81-5P, 161947-82-6P, 161947-83-7P, 161947-84-8P, 161947-85-9P, 161947-86-OP, 161947-87-1P, 161947-88 2P, 161947-89-3P, 161947-90-6P, 161947-91-7P, 161947 92-8P, 161947-93-9P, 161947-94-OP, 161947-95-1P, 161947-96-2P, 161947-97-3P, 161947-98-4P, 161947-99-5P, 161948-00-1P, 161948-01-2P, 161948-02-3P, 168686-32-6P, 167301-42-OP, 166813-82-7P, 166961-56-4P, 166961-58 6P, 158872-96-9P, 158872-97-OP, 158807-14-8P, 158807 15-9P, 158807-16-OP, 158807-17-1P, 158807-18-2P, 158807-19-3P, 158807-20-6P, 155884-08-5P, 154749-99-2, 167371-59-7P, 244126-99-6P, 177848-35-OP and 141309 82-2P, and the like. [0068] Exemplary angiotensin-converting enzyme inhibi tors (ACE inhibitors) include, but are not limited to, alacepril, benaZepril (LOTENSIN®, CIBACEN®), benaZeprilat, cap topril, ceronapril, cilaZapril, delapril, duinapril, enalapril, enalaprilat, fasidotril, fosinopril, fosinoprilat, gemopatrilat, glycopril, idrapril, imidapril, lisinopril, moexipril, movel tipril, naphthopidil, omapatrilat, pentopril, perindopril, per indoprilat, quinapril, quinaprilat, ramipril, ramiprilat, rent ipril, saralasin acetate, spirapril, temocapril, trandolapril, trandolaprilat, urapidil, Zofenopril, acylmercapto and mer captoalkalnoyl pralines, carboxyalkyl dipeptides, carboxy alkyl dipeptide, phosphinylalkanoyl pralines, registry no. 796406, AVE 7688, BP1.137, CHF 1514, E 4030, ER 3295, FPL-66564, MDL 100240, RL 6134, RL 6207, RL 6893, SA 760, S-5590, Z 13752A, and the like. [0069] Exemplary antioxidants include, but are not limited to, small-molecule antioxidants and antioxidant enZymes. Suitable small-molecule antioxidants include, but are not lim ited to, hydralaZine compounds, glutathione, vitamin C, vita min E, cysteine, N-acetyl-cysteine, .beta.-carotene, ubiquinone, ubiquinol-10, tocopherols, coenZyme Q, super oxide dismutase mimetics, such as, for example, 2,2,6,6 tetramethyl- 1 -piperidinyloxy (TEMPO), DOXYL, PROXYL nitroxide compounds; 4-hydroxy-2,2,6,6-tetramethyl-1-pip eridinyloxy (Tempol), M-40401, M-40403, M-40407, M-40419, M-40484, M-40587, M-40588, and the like. Suit able antioxidant enZymes include, but are not limited to, superoxide dismutase, catalase, glutathione peroxidase, NADPH oxidase inhibitors, such as, for example, apocynin, aminoguanidine, ONO 1714, 517834 (benZo(b)pyran-4-one derivative), and the like; xanthine oxidase inhibitors, such as, for example, allopurinol, oxypurinol, am?utiZole, dieth yldithiocarbamate, 2-styrylchromones, chrysin, luteolin, kaempferol, quercetin, myricetin, isorhamnetin, benZophe nones such as 2,2',4,4'-tetrahydroxybenZophenone, 3,4,5,2', 3',4'-hexahydroxybenZophenone and 4,4'-dihydroxyben Zophenone; benZothiaZinone analogues such as 2-amino-4H 1,3-benZothiaZine-4-one, 2-guanidino-4H-1,3-benZothiaZin 4-one and rhodanine; N-hydroxyguanidine derivative such as, PR5 (1-(3,4-dimethoxy-2-chlorobenZylideneamino-3 hydroxyguanidine); 6-formylpterin, and the like [0070] Exemplary antithrombotic and vasodilator com pounds include, but are not limited to, abciximab, acetorphan, acetylsalicylic acid, argatroban, bamethan, benfurodil, ben Ziodarone, betahistine, bisaramil, brovincamine, bufeniode, citicoline, clobenfurol, clopidogrel, cyclandelate, dalteparin, dipyridamol, droprenilamine, enoxaparin, fendiline, ifen prodil, iloprost, indobufen, isobogrel, isoxsuprine, heparin, lami?ban, midrodine, nadroparin, nicotinoyl alcohol, nylid

Nov. 28, 2013

rin, oZagrel, perhexyline, phenylpropanolamine, preny lamine, papaveroline, reviparin sodium salt, ridogrel, suloc tidil, tinofedrine, tinZaparin, trifusal, vintoperol, xanthinal niacinate, and the like. [0071] Exemplary bronchodilators include, but are not lim ited to, ambroxol, atropine, bevonium methyl sulfate, bethanechol, chlorprenaline, cyclodrine, daiphenacine, N-desethyl-oxybutynin, dicyclomine, emepronium, ephe drine, epinephrine, etafredine, ethylnorepinephrine, ?avox ate, ?utoprium bromide, hexoprenaline, 2-hydroxy-2,2 diphenyl-N-( 1 ,2 ,3 ,6 -tetra hydro -pyridin-4-ylmethyl) acetamide, ipratropium bromide, isoetharine, NS 21, oxybutynin, oxitropium bromide, propanthelin, propiverine, rispenZepine, terbutaline, 1-teobromine actetic acid, terodiline, tiotropium bromide, tolterodine, trospium, vami camide, Zamiphenacine, and the like. [0072] Exemplary calcium channel blockers include, but are not limited to, amlodipine (NORVASC®), anipamil, aranidipine, aminone, aZelnidipine, barnidipine, bencyclane, benidipine, bepridil, cilnidipine, cinnariZine, clentiaZem, dil tiaZem, dotariZine, efonidipine, elgodipine, fantofarone, felo dipine, fendiline, ?unariZine, ?uspirilene, fumidipine, gallo pamil, ipenoxaZone, isradipine, lacidipine, lemildipine, lercanidipine, lomeriZine, manidipine, mibefradil, monatepil, nicardipine, nifedipine, niguldipine, niludipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, nival dipine, oxodipine, perhexylene, phenyloin, phenylpreny lamine, pranidipine, ranolaZine, ryosidine, semotiadil, tamo lariZine, temiverine hydrochloride, terodiline, tiapamil, vatanidipine hydrochloride, verapamil, Ziconotide, AE-0047, CAI, JTV-519, CHE-1521, L-651582, NS-7, NW-1015, RO-2933, SB-237376, SL-34.0829-08, S-312d, SD-3212, TA-993, YM-430, and the like. [0073] Exemplary endothelin antagonists include, but are not limited to, atrasentan, bosentan, darusentan, endothelin, enrasentan, sitaxsentan, sulfonamide endothelin antagonists, teZosentan, BMS 193884, BQ-123, SQ 28608, and the like. [0074] Exemplary expectorants include, but are not limited to, ambroxol, domiodol, erdosteine, guaiacol, guaifenesin, iodinated glycerol, letosteine, mensa, sobrerol, strepronine, terpin, tiopronin, and the like. [0075] Exemplary H2 receptor antagonists include, but are not limited to, burimamide, cimetidine, ebrotidin, famoti dine, niZatidine, roxatidine, rantidine, tiotidine, and the like. [0076] Exemplary neutral endopeptidase inhibitors include, but are not limited to, atrial natriuretic peptides, diaZapins, aZepinones, ecadotril, fasidotril, fasidotrilat, oma patrilat, sampatrilat, BMS 189,921, Z 13752 A, and the like. [0077] Exemplary NSAlDs include, but are not limited to, acetaminophen, acemetacin, aceclofenac, alminoprofen, amfenac, bendaZac, benoxaprofen, bromfenac, bucloxic acid, butibufen, carprofen, cinmetacin, clopirac, diclofenac, etod olac, felbinac, fencloZic acid, fenbufen, fenoprofen, fen tiaZac, ?unoxaprofen, ?urbiprofen, ibufenac, ibuprofen, indomethacin, isofeZolac, isoxepac, indoprofen, ketoprofen, lonaZolac, loxoprofen, metiaZinic acid, mofeZolac, miropro fen, naproxen, oxaproZin, piroZolac, pirprofen, pranoprofen, protiZinic acid, salicylamide, sulindac, suprofen, suxibuZone, tiaprofenic acid, tolmetin, xenbucin, ximoprofen, Zaltopro fen, Zomepirac, aspirin, acemetcin, bumadiZon, carprofenac, clidanac, di?unisal, enfenamic acid, fendosal, ?ufenamic acid, ?unixin, gentisic acid, ketorolac, meclofenamic acid, mefenamic acid, mesalamine, prodrugs thereof, and the like.

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[0078] Exemplary phosphodiesterase inhibitors, include but are not limited to, ?laminast, piclamilast, rolipram, Org 20241, MCI-154, ro?umilast, toborinone, posicar, lixaZi none, Zaprinast, sildena?l, pyraZolopyrimidinones, motapi Zone, pimobendan, Zardaverine, siguaZodan, Cl 930, EMD 53998, imaZodan, saterinone, loprinone hydrochloride, 3-py ridinecarbonitrile derivatives, acefylline, albifylline, bami fylline, denbufyllene, diphylline, doxofylline, etofylline, tor bafylline, theophylline, nanterinone, pentoxofylline, proxyphylline, cilostaZol, cilostamide, MS 857, piroximone, milrinone, aminone, tolafentrine, dipyridamole, papavero line, E4021, thienopyrimidine derivatives, tri?usal, ICOS 3 51, tetrahydropiperaZino(1,2-b)beta-carboline-1,4-dione derivatives, carboline derivatives, 2-pyraZolin-5-one deriva tives, fused pyridaZine derivatives, quinaZoline derivatives, anthranilic acid derivatives, imidaZoquinaZoline derivatives, tadala?l and vardena?l.

[0079] Exemplary potassium channel blockers include, but are not limited to, nicorandil, pinacidil, cromakalim (BRL 34915), aprikalim, bimakalim, emakalim, lemakalim, minoxidil, diaZoxide, 9-chloro-7-(2-chlorophenyl)-5H-py rimido(5,4,-d)(2)-benZaZepine, Ribi, CPG-11952, CGS 9896, ZD 6169, diaZixide, Bay X 9227, P1075, Bay X 9228, SDZ PCO 400, WAY-120,491, WAY-120,129, Ro 31-6930, SR 44869, BRL 38226, S 0121, SR 46142A, CGP 42500, SR 44994, artilide fumarate, loraZepam, temaZepam, ril maZafone, nimetaZepam, midaZolam, lormetaZepam, lopra Zolam, ibutilide fumarate, haloxaZolam, ?unitraZepam, esta Zolam, doxefaZepam, clonaZepam, cinolaZepam, brotiZolam, and the like.

[0080] Exemplary platelet reducing agents include, but are not limited to, ?brinolytic agents such as for example, ancrod, anistreplase, bisobrin lactate, brinolase, Hageman factor (i.e. factor XII) fragments, plasminogen activators such as, for example, streptokinase, tissue plasmino gen activators (TPA), urokinase, pro-urokinase, recombinant TPA, plasmin, plas minogen, and the like; anti-coagulant agents including but are not limited to, inhibitors of factor Xa, factor TFPI, factor Vlla, factor lXc, factor Va, factor Vllla, inhibitors of other coagulation factors, and the like; vitamin K antagonists, such as, for example, coumarin, coumarin derivatives (e.g., War farin sodium); glycosoaminoglycans such as, for example, heparins both in unfractionated form and in loW molecular Weight form; ardeparin sodium, bivalirudin, bromindione, coumarin, dalteparin sodium, danaparoid sodium; daZoxiben hydrochloride, desirudin, dicumarol, efegatran sulfate, enox aparin sodium, ifetroban, ifetroban sodium, lyapolate sodium, nafamo stat mesylate, phenprocoumon, sulfatide, tin Zaparin sodium, retaplase; trifenagrel, Warfarin, dextrans and the like; abciximab, acadesine, anipamil, argatroban, aspirin, clopidogrel, diadeno sine 5',5"'-P1,P4-tetraphosphate (Ap4A) analogs, di?brotide, dilaZep dihydrochloride, dipy ridamole, dopamine, 3-methoxytyramine, glucagon, glyco protein llb/llla antagonists, such as, for example, Ro-43 8857, L-700,462, iloprost, isocarbacyclin methyl ester, itaZigrel, ketanserin, BM-13.177, lami?ban, lifariZine, mol sidomine, nifedipine, oxagrelate, prostaglandins, platelet activating factor antagonists such as, for example, lexipafant, prostacyclins, pyraZines, pyridinol carbamate, ReoPro (i.e., abciximab), sul?npyraZone, synthetic compounds BN-50727, BN-52021, CV-4151, E-5510, FK-409, GU-7, KB-2796, KBT-3022, KC-404, KF-4939, OP-41483, TRK 100, TA-3090, TFC-612, ZK-36374,2,4,5,7-tetrathiaoctane, 2,4,5,7-tetrathiaoctane 2,2-dioxide, 2,4,5-trithiahexane,

Nov. 28, 2013

theophyllin pentoxifyllin, thromboxane and thromboxane synthetase inhibitors such as, for example, picotamide, sulotroban, ticlopidine, tiro?ban, trapidil, ticlopidine, trifenagrel, trilinolein, 3-substituted 5,6-bis(4-methoxyphe nyl)-1,2,4-triaZines; antibodies to glycoprotein IIb/IIIa; anti serotonin drugs, such as, for example, clopridogrel; sul?n pyraZone and the like; aspirin; dipyridamole; clo?brate; pyridinol carbamate; glucagon, caffeine; theophyllin pen toxifyllin; ticlopidine, and the like.

[0081] Exemplary proton pump inhibitors include, but are not limited to, disulpraZole, esomepraZole, lansopraZole, leminopraZole, omepraZole, pantopraZole, rabepraZole, timo praZole, tenatopraZole, 2-(2-benZimidaZolyl)-pyridine, tricy clic imidaZole, thienopydidine benZimidaZole, ?uoroalkoxy substituted benZimidaZole, dialkoxy benZimidaZole, N-sub stituted 2-(pyridylalkenesul?nyl)benZimidaZole, cyclohep tenepyridine, 5-pyrrolyl-2-pyridylmethylsul?nyl benZimida Zole, alkylsul?nyl benZimidaZole, ?uoro pyridylmethylsul?nyl benZimidaZole, imidaZo(4,5-b) pydridine, RO 18-5362, IY 81149, 4-amino-3-carbonyl quinoline, 4-amino-3-acylnaphthyride, 4-aminoquinoline, 4-amino-3-acylquinoline, 3-butyryl-4-(2-methylpheny lamino)-8-(2-hydroxyethoxy)quinoline, quinaZoline, tet rahydroisoquinolin-2-yl pyrimidine, YH 1885, 3-substituted 1,2,4-thiadiaZolo(4,5-a)benZimidaZole, 3-substituted imi daZo(1,2-d)-thiadiaZole, 2-sul?nylnicotinamide, pyridyl sul?nylbenZ imidaZole, pyridylsul?nyl thieno imidaZole, theinoimidaZole-toluidine, 4,5-dihydrooxaZole, thienoimi daZole-toluidine, Hoe-731, imidaZo(1,2-a)pyridine, pyrrolo (2,3-b)pyridine, and the like.

[0082] Exemplary renin inhibitors include, but are not lim ited to, aldosterone, aliskiren (SPP-100), ditekiren, enalkrein (A-64662), medullipin, terlkiren, tonin, Zankiren, RO 42-5892 (remikiren), A 62198, A 64662, A 65317, A 69729, A 72517 (Zankiren), A 74273, CP 80794, CGP 29287, CGP 38560A, EMD 47942, ES 305, ES 1005, ES 8891, FK 906, FK 744, H 113, H-142, KRI 1314, pepstatin A, RO 44-9375 (ciprokiren), RO 42-5892, RO 66-1132, RO 66-1168, SP 500, SP 800, SR-43845, SQ 34017, U 71038, YM-21095, YM-26365, urea derivatives of peptides, amino acids con nected by nonpeptide bonds, di- and tri-peptide derivatives (e.g., Act-A, Act-B, Act-C, ACT-D, and the like), amino acids and derivatives thereof, diol sulfonamides and sul?nyls, modi?ed peptides, peptidyl beta-aminoacyl aminodiol car bamates, monoclonal antibodies to renin.

[0083] Exemplary COX-2 inhibitors include, but are not limited to, nimesulide, celecoxib (CELEBREX®), etoricoxib (ARCOXIA®), ?osulide, lumiracoxib (PREXIG®, COX 189), parecoxib (DYNSTAT®), rofecoxib (V IOXX®), tira coxib (JTE-522), valdecoxib (BEXTRA®), ABT 963, BMS 347070, CS 502, DuP 697, GW-406381, NS-386, SC-57666, SC-58125, SC-58635, and the like, and combinations oftWo or more thereof.

[0084] Exemplary steroids include, but are not limited to, 21-acetoxypregnenolone, alcolometasone, algestone, amci nonide, beclomethasone, betamethasone, budesonide, chlor prednisone, clobetasol, clobentasone, clocortolone, clopred nol, corticosterone, cortisine, corticaZol (cortivatol), de?aZacort, desonide, desoximetasone, dexamethasone, di?orasone, di?ucortolone, di?uprednate, enoxolone, ?uZa cort, ?ucloronide, ?umethasone, ?unisolide, ?ucinolone

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acetonide, ?uocininide, ?uocortin butyl, ?uocortolone, ?uo rometholone, ?uperolone acetate, ?uprednidene acetate, ?u prednisolone, ?urandrenolide, ?uticasone propionate, ?uti casone propionate, forrnocortal, halcinonide, halobetasol propionate, halometasone, haloprednone acetate, hydrocor tamate, hydrocortisone and its derivatives (such as phosphate, 21-sodium succinate and the like), hydrocortisone terbutate, iso?upredone, loteprednol etabonate, maZipredone, medrysone, meprednisone, methylprednisolone, mometa sone furoate, paremethasone, prednicarbate, prednisolone and its derivatives (such as 21-stearoylglycolate, sodium phosphate and the like), prednisone, prednival, prednylidene and its derivatives (such as 21-diethylaminoactetate and the like), rimexolone, tixocortol, trimcinolone and its derivatives (such as acetonide, benetonide and the like), and the like.

[0085] The combination therapies described herein is pro vided in a combined amount effective to inhibit QS in the bacteria (and/ or treat a bacterial infection associated With bacterial QS and/or treat a disorder associated With bio?lm formation). This process may involve administering to a sub ject in need thereof one or more ellagitannins and a standard of care anti -bacterial therapeutic (and/ or additional therapeu tic/ second agent) at the same time, Which may be achieved by administering a single composition or pharmacological for mulation that includes both an ellagitannin and a standard of care therapeutic, or by administering tWo distinct composi tions or formulations, at the same time, Wherein one compo sition includes an ellagitannin and the other includes a stan dard of care anti-bacterial therapeutic. In another embodiment, the combination therapy involves administer ing to a subject in need thereof an ellagitannin and a standard of care anti -bacterial therapeutic (and/ or additional therapeu tic/second agent) at different times, Which may be achieved by administering tWo distinct compositions or formulations, at different time intervals, Wherein one composition includes an ellagitannin and the other includes a standard of care anti-bacterial therapeutic (and/ or additional therapeutic/sec ond agent).

[0086] Alternatively, the treatment With the ellagitannin(s) may precede or folloW the treatment With the standard of care anti-bacterial therapeutic (and/ or additional therapeutic/sec ond agent) by intervals ranging from minutes to Weeks. In embodiments Where the ellagitannin(s) and the standard of care anti-bacterial therapeutic (and/ or additional therapeutic/ second agent) are administered separately (either in separate compositions administered simultaneously or in separate compositions administered at different time intervals), one Would generally ensure that a signi?cant period of time did not expire betWeen the times of each delivery, such that the further therapeutic agent and the ellagitannin Would still be able to exert an advantageously combined effect. In such instances, it is contemplated that one Would administer both modalities Within about 1, about 2, about 3, about 4, about 5, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 36, about 48, or about 72 hours of each other. In one embodiment, both modalities are administered Within about 6-12 hours of each other. In some situations, it may be desirable to extend the time period for treatment signi?cantly. Exemplary routes of administration of the peptides or com positions described herein include, but are not limited to,

Nov. 28, 2013

intraderrnal, intramuscular, intraperitoneal, intraocular, intra venous, subcutaneous, topical, oral and intranasal adminis tration. [0087] C. Medical Devices [0088] In another embodiment, one or more ellagitannins is used to inhibit bio?lm formation associated With bacterial QS on a medical device by contacting the device With an ellagi tannin in an amount effective to inhibit bio?lm formation. Percutaneous devices (such as catheters) and implanted medical devices (including, but not limited to, pacemakers, vascular grafts, stents, and heart valves) commonly serve as foci for bacterial infection. The tendency of some microor ganisms to adhere to and coloniZe the surface of the device promotes such infections, Which increase the morbidity and mortality associated With use of the devices. [0089] For example, one or more ellagitannins is used to inhibit bio?lm formation on substrates used to manufacture medical devices associated With non-invasive and invasive medical procedures. Such substrates include, Without limita tion, tubular, sheet, rod and articles of proper shape for use in a number of medical devices such as vascular grafts, aortic grafts, arterial, venous, or vascular tubing, vascular stents, dialysis membranes, tubing or connectors, blood oxygenator tubing or membranes, surgical instruments, ultra?ltration membranes, intra-aortic balloons, stents, blood bags, cath eters, sutures, soft or hard tissue prostheses, synthetic pros theses, prosthetic heart valves, tissue adhesives, cardiac pace maker leads, arti?cial organs, endotracheal tubes, lenses for the eye such as contact or intraocular lenses, blood handling equipment, apheresis equipment, diagnostic and monitoring catheters and sensors, biosensors, dental devices, drug deliv ery systems, or bodily implants of any kind. For example, arthroscopic surgery is routinely performed With use of medi cal devices that minimiZe the invasiveness of the procedure. Such devices include, for example and Without limitation, ultrathin micro?beroptic endoscopes that offer the laryngolo gist unique access to the limited spaces of the temporal bone and skull base. In another example, a stent supplemented With one or more ellagitannins can be constructed. Stents are used to maintain an open lumen in tissues including the tracheo bronchial system, the biliary hepatic system, the esophageal boWel system, and the urinary tract system. [0090] III. Routes of Administration and Dosage [0091] Ellagitannin(s) either alone or in combination With a standard of care anti-bacterial therapeutic as described herein are administered by any route that delivers an effective do sage to the desired site of action, With acceptable (preferably mini mal) side-effects. Numerous routes of administration are knoWn, including for example, oral, rectal, vaginal, transmu cosal, buccal or intestinal administration; parenteral delivery, including intraperitoneal, intramuscular, subcutaneous, intramedullary injections, as Well as intrathecal, cutaneous or intraderrnal injections; respiratory or inhalation, nasal, pul monary and topical application, including ocular and trans dermal applications. [0092] When used in the above or other treatments, a "therapeutically-effective amount” or an “effective amount” of an ellagitannin or a composition comprising an ellagitan nin means a su?icient amount of the ellagitannin is provided to treat disorders or to achieve a desired result. It Will be

understood, hoWever, that the total daily usage of the ellagi tannin in a therapeutic method described herein Will be decided by the attending physician Within the scope of sound medical judgment. The speci?c therapeutically effective dose

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level for any particular patient Will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the speci?c compound employed; the speci?c composition employed; the age, body Weight, gen eral health, sex and diet of the patient; the time of adminis tration, route of administration, and rate of excretion of the speci?c compound employed; the duration of the treatment; drugs used in combination or coincidental With the speci?c compound employed; and like factors Well knoWn in the medical arts. For example, it is Well Within the skill of the art to start doses of the compound at levels loWer than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. [0093] The dose of ellagitannin administered to a mamma lian subject range from about 10 [lg to about 400 mg/day. In some embodiments, the dose is about 10 ug/day, about 25 ug/day, about 50 ug/day, about 75 ug/day, about 100 ug/day, about 125 ug/day, about 150 ug/day, about 175 ug/day, about 200 ug/day, about 225 ug/day, about 250 ug/day, about 275 ug/day, about 300 ug/ day, about 325 ug/day, about 350 ug/day, about 375 ug/ day, about 400 ug/day, about 425 ug/day, about 450 ug/ day, about 475 ug/day, about 500 ug/day, about 750 ug/day, about 1 mg/day, about 5 mg/day, about 10 mg/ day, about 25 mg/ day, about 30 mg/day, about 40 mg/day, about 45 mg/day, about 50 mg/ day, about 60 mg/day, about 70 mg/day, about 80 mg/day, about 90 mg/day, about 100 mg/ day, about 150 mg/ day, about 200 mg/day, about 250 mg/day, about 300 mg/day, about 350 mg/day or about 400 mg/day. In some embodiments, the maximum dosage is about 200 mg/ day. In some embodiments, the maximum dosage is about 300 mg/day. If desired, the effective daily dose is divided into multiple doses for purposes of administration; consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. The dosage regimen of an ellagitannin composition alone or in combination as described herein to be used in treatment of bacterial infections (orbio?lm formation) associated With QS Will be determined by the attending physician considering various factors Which modify the action of the ellagitannin, e.g., the patient’s age, sex, and diet, the severity of any infec tion, time of administration and other clinical factors. [0094] Oral dosage forms include tablets, capsules, caplets, solutions, suspensions and/ or syrups, and may also comprise a plurality of granules, beads, poWders or pellets that may or may not be encapsulated. Such dosage forms are prepared using conventional methods knoWn to those in the ?eld of pharmaceutical formulation and described in the pertinent texts, e.g., in Remington: The Science and Practice of Phar macy, supra). Tablets and capsules represent the most conve nient oral dosage forms, in Which case solid pharmaceutical carriers are employed.

[0095] Tablets include those manufactured using standard tablet processing procedures and equipment. One method for forming tablets is by direct compression of a poWdered, crys talline or granular composition containing the active agent(s), alone or in combination With one or more carriers, additives, or the like. As an alternative to direct compression, tablets can be prepared using Wet-granulation or dry-granulation pro cesses. Tablets are also molded rather than compressed, start ing With a moist or otherWise tractable material.

[0096] In addition to the ellagitannin either alone or in combination as described herein, tablets prepared for oral administration Will in one aspect contain other materials such as binders, diluents, lubricants, disintegrants, ?llers, stabiliZ

Nov. 28, 2013

ers, surfactants, preservatives, coloring agents, ?avoring agents and the like. Binders are used to impart cohesive qualities to a tablet, and thus ensure that the tablet remains intact after compression. Suitable binder materials include, but are not limited to, starch (including corn starch and prege latiniZed starch), gelatin, sugars (including sucrose, glucose, dextrose and lactose), polyethylene glycol, propylene glycol, Waxes, and natural and synthetic gums, e.g., acacia sodium alginate, polyvinylpyrrolidone, cellulosic polymers (includ ing hydroxypropyl cellulose, hydroxypropyl methylcellu lose, methyl cellulose, ethyl cellulose, hydroxyethyl cellu lose, and the like), and Veegum. Diluents are typically necessary to increase bulk so that a practical siZe tablet is ultimately provided. Suitable diluents include dicalcium phosphate, calcium sulfate, lactose, cellulose, kaolin, manni tol, sodium chloride, dry starch and poWdered sugar. Lubri cants are used to facilitate tablet manufacture; examples of suitable lubricants include, for example, vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and oil of theobroma, glycerin, magnesium stearate, calcium stearate, and stearic acid. Disintegrants are used to facilitate disintegration of the tablet, and are generally starches, clays, celluloses, algins, gums or crosslinked polymers. Fillers include, for example, materials such as silicon dioxide, tita nium dioxide, alumina, talc, kaolin, poWdered cellulose and microcrystalline cellulose, as Well as soluble materials such as mannitol, urea, sucrose, lactose, dextrose, sodium chloride and sorbitol. Stabilizers are used to inhibit or retard drug decomposition reactions that include, by Way of example, oxidative reactions. Surfactants may be anionic, cationic, amphoteric or nonionic surface active agents.

[0097] The dosage form also includes a capsule, in Which case the ellagitannin-containing composition is in one aspect encapsulated in the form of a liquid or solid (including par ticulates such as granules, beads, poWders or pellets). Suit able capsules may be either hard or soft, and are generally made of gelatin, starch, or a cellulosic material, With gelatin capsules preferred. TWo-piece hard gelatin capsules are pref erably sealed, such as With gelatin bands or the like. (See, for e.g., Remington: The Science and Practice of Pharmacy, supra), Which describes materials and methods for preparing encapsulated pharmaceuticals. [0098] Solid dosage forms, Whether tablets, capsules, caplets, or particulates, are, if desired, coated so as to provide for delayed release. Dosage forms With delayed release coat ings are in one aspect manufactured using standard coating procedures and equipment. Such procedures are knoWn to those skilled in the art and described in the pertinent texts (See, for e.g., Remington: The Science and Practice of Phar macy, supra). In one aspect, after preparation of the solid dosage form, a delayed release coating composition is applied using a coating pan, an airless spray technique, ?uidized bed coating equipment, or the like. Delayed release coating com positions comprise in various aspects a polymeric material, e.g., cellulose butyrate phthalate, cellulose hydrogen phtha late, cellulose proprionate phthalate, polyvinyl acetate phtha late, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate, dioxypropyl methylcellulose succi nate, carboxymethyl ethylcellulose, hydroxypropyl methyl cellulose acetate succinate, polymers and copolymers formed from acrylic acid, methacrylic acid, and/ or esters thereof. [0099] Sustained release dosage forms provide for drug release over an extended time period, and optionally are

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delayed release. As Will be appreciated by those of ordinary skill in the art, sustained release dosage forms are formulated in various aspects by dispersing a drug Within a matrix of a gradually bioerodible (hydrolyZable) material such as, for example, an insoluble plastic, a hydrophilic polymer, or a fatty compound, or by coating a solid, drug-containing dos age form With such a material. Insoluble plastic matrices are in certain aspects comprised of, for example, polyvinyl chlo ride or polyethylene. Hydrophilic polymers useful for pro viding a sustained release coating or matrix cellulosic poly mers include, Without limitation: cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydrox ypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethyl cellulose phtha late, hydroxypropylcellulose phthalate, cellulose hexahydro phthalate, cellulose acetate hexahydrophthalate, and car boxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, meth acrylic acid, acrylic acid alkyl esters, methacrylic acid alkyl esters, and the like, eg and Without limitation copolymers of acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, With a ter polymer of ethyl acrylate, methyl methacrylate and trimethy lammonioethyl methacrylate chloride (sold under the trade name Eudragit RS) preferred; vinyl polymers and copolymers such as polyvinyl pyrrolidone, polyvinyl acetate, polyvinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymers; Zein; and shellac, ammoniated shellac, shellac-acetyl alcohol, and shel lac n-butyl stearate. Fatty compounds for use as a sustained release matrix material include, but are not limited to, Waxes generally (e.g., carnauba Wax) and glyceryl tristearate. [0100] Although compositions described herein are in one aspect administered orally, other modes of administration are contemplated as Well. Exemplary modes of administration include transmucosal (e.g., US. Pat. Nos. 5,288,498; 6,248, 760; 6,355,248; 6,548,490, the disclosures of Which are incorporated herein by reference in their entireties), transure thral (e.g., e.g., US. Pat. Nos. 5,919,474 and 5,925,629, the disclosures of Which are incorporated herein by reference in their entireties), vaginal or perivaginal (e.g., US. Pat. Nos. 4,211,679; 5,491,171 and 6,576,250, the disclosures ofWhich are incorporated herein by reference in their entireties) and intranasal or inhalation (e.g., US. Pat. Nos. 4,800,878; 5,112, 804; 5,179,079; 6,017,963; 6,391,318 and 6,815,424, the dis closures of Which are incorporated herein by reference in their entireties). One of skill in the art Would be able to modify a composition comprising an ellagitannin either alone or in combination With a standard of care anti -bacterial therapeutic as described herein to be used in any of the modes of admin istration described herein.

[0101] Compositions comprising an ellagitannin either alone or in combination as described herein are be used as a

topical agent. The topical agent is a solution, that is, in one aspect, a liquid formulation comprising the ellagitannin and a carrier. Other suitable forms include semi-solid or solid forms comprising a carrier indigenous to topical application and having a dynamic viscosity preferably greater than that of Water, provided that the carrier does not deleteriously react With the ellagitannin in the composition. Suitable formula tions include, but are not limited to, lip balms, suspensions, emulsions, creams, ointments, poWders, liniments, salves and the like. If desired, these compositions may be steriliZed or

Nov. 28, 2013

mixed With auxiliary agents, including but not limited to, preservatives, stabiliZers, Wetting agents, buffers or salts for in?uencing osmotic pressure and the like Well knoWn in the art. Preferred vehicles for semi-solid or solid forms topical preparations include ointment bases, conventional oph thalmic vehicles; creams; and gels. These topical prepara tions optionally contain emollients, perfumes, and/or pig ments to enhance their acceptability for various usages, provided that the additives do not deleteriously react With the ellagitannin material in the composition. [0102] Also suitable for topical application are sprayable aerosol preparations Wherein the ellagitannin, preferably in combination With a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture With a pressur iZed volatile, normally gaseous propellant, e.g., a Freon (chloro?uorocarbon) or environmentally acceptable volatile propellant. Such compositions are used for in one aspect, application to environmental surfaces, e. g., examining tables, toilet seats and the like, and/ or for application to the skin or to mucous membranes. The aerosol or spray preparations optionally contain solvents, buffers, surfactants, perfumes, and/or antioxidants in addition to the ellagitannin. [0103] The compositions are in certain aspects employed in mixture With conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances, suitable for topical application Which do not deleteriously react With the ellagitannin in the composition. The compositions of the invention optionally include diluents, ?llers, salts, buffers, stabiliZers, solubiliZers, and other materials Well knoWn in the art (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). [0104] IV. Kits and Unit Doses [0105] In related variations of the preceding embodiments, a composition comprising an ellagitannin as described herein packaged alone, e.g., in a kit or package or unit dose, or is optionally arranged to permit co-administration With one or more other therapeutic agents as described herein, but the ellagitannin and the agent are not in admixture. In an altema tive variation, the ellagitannin and the agent are in admixture. In some embodiments, the tWo components to the kit/unit dose are packaged With instructions for administering the tWo agents to a human subject for treatment of one of the above indicated disorders and diseases. The kit may comprise a composition described herein in combination With a vehicle in a cream or gel base, as a pump-spray, as an aerosol, on an impregnated bandage, or in a dropper.

EXAMPLES

Example 1

Ellagitannins Inhibited Bacterial QS

[0106] The present Example describes the isolation and veri?cation of tWo C-glycosidic ellagitannins, castalagin and vescalagin, from C. ereclus and the con?rmation of anti-QS activity of these compounds. [0107] Materials and Methods: [0108] Plant Extraction: [0109] Leaves of the medicinal plant C. ereclus (Combre taceae), Were collected and processed according to methods described previously (AdoniZio et al., 2006). Brie?y, pulver iZed plant material Was extracted into boiling Water, freeZe dried using a lyophiliZer, and stored at —200 C. until needed.