USOO9675592B2

(12) United States Patent (10) Patent No.: US 9,675,592 B2 Daum et al. (45) Date of Patent: Jun. 13, 2017

(54) METHODS AND COMPOSITIONS FOR (52) U.S. Cl. BACTERIA INFECTIONS CPC ...... A61K 31/431 (2013.01); A61K 9/0019 (71) Applicants: THE UNIVERSITY OF CHICAGO, (2013.01); ) A6IK3I/05 (2013.01):( ) Chicago, IL (US): THE BOARD OF (Continued) REGENTS OF THE UNIVERSITY (58) Field of Classification Search OF ILLINOIS, Urbana, IL (US) CPC ... A61K 3 1/519; A61K 31/427; A61K 31/428; A61K 31/446 (72) Inventors: Robert S. Daum, Chicago, IL (US); Continued Susan Boyle-Vavra, Chicago, IL (US); (Continued) Michael E. Johnson, Urbana, IL (US) (56) References Cited (73) Assignees: The University of Chicago, Chicago, U.S. PATENT DOCUMENTS IL (US); The Board of Regents of the University of Illinois, Urbana, IL (US) 5,776,919 A 7, 1998 Sukigara et al...... 514, 161 2010.0029597 A1 2/2010 Cottarel et al...... 514,154 (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 FOREIGN PATENT DOCUMENTS U.S.C. 154(b) by 0 days. WO WO 2012151474 11 2012 WO WO 2014O71198 5, 2014 (21) Appl. No.: 14/776,965 (22) PCT Filed: Mar. 14, 2014 OTHER PUBLICATIONS (86). PCT No.: PCT/US2014/029237 Berge, et al., J Pharmaceutical Sci. 66(1):1-19, 1977.

S 371 (c)(1)C s (Continued) (2) Date: Sep. 15, 2015 Primary Examiner — Shengjun Wang (87) PCT Pub. No.: WO2014/144710 Storney Agent, or Firm — Norton Rose Fulbright PCT Pub. Date: Sep. 18, 2014 (57) ABSTRACT (65) Prior Publication Data Compositions and methods are provided for treating or US 2016/0120852 A1 May 5, 2016 inhibiting a bacterial infection involving at least one anti Related U.S. Application Data biotic and a compound that potentiates the antibiotic activity of the antibiotic. In certain embodiments the antibiotic is a (60) Provisional application No. 61/789,716, filed on Mar. beta lactam. In further embodiments, the antibiotic is oxa 15, 2013. cillin. In additional embodiments, the potentiating com pound is an inhibitor of VraSR operon expression. In specific (51) Int. Cl. embodiments, the bacterial infection involves an antibiotic A6 IK 3/44 3.08: resistant bacteria. A 6LX 3/59 2006.O1 (Continued) 20 Claims, 6 Drawing Sheets

0.5 (ciacin)2 4mg. 5 US 9,675,592 B2 Page 2

(51) Int. Cl. 31/519 (2013.01); A61K3I/5377 (2013.01); A6 IK3I/53 (2006.01) A61K 31/7036 (2013.01); A61K 45/06 A6 IK 3/425 (2006.01) (2013.01) A6 IK 3L/45 (2006.01) (58) Field of Classification Search A6 IK3I/05 (2006.01) USPC ...... 514/736, 342, 260.1, 262.1, 367, 371, A6 IK 3/43 (2006.01) 514/245, 394, 259.31, 403,364 A6 IK 3/43 (2006.01) See application file for complete search history. A6 IK 45/06 (2006.01) (56) References Cited A6 IK 9/00 (2006.01) A6 IK 3/46 (2006.01) A6 IK 3/42.45 (2006.01) OTHER PUBLICATIONS A6 IK 3/428 (2006.01) Boyle-Vavra, et al., Antimicrob Agents Chemother: 57(1):83-95, A6 IK 3/4439 (2006.01) 2013. A6 IK 3L/454 (2006.01) Boyle-Vavra, et al., FEMS Microbiol Lett. 262(2):163-171, 2006. A 6LX 3L/24709 (2006.01) EllisO, et al., el, Antimicro C, teis Cleppother:ES' SS: Z 2011 A6 IK 3/4725 (2006.01) Livak & Schmittgen, St. 25(4):402-408, 2001. A6 IK 3/496 (2006.01) Montgomery, et al., J Infect Dis. 198(4):561-570, 2008. A 6LX 3/5.377 (2006.01) Rohner et al. “Synergistic Effect of Quinolones and Oxacillin on A6 IK3I/7036 (2006.01) Methicillin-Resistant Staphylococcus Species' Antimicrobial (52) U.S. Cl. Agents and Chemotherapy, 33(12): 2037-2041, 1989. CPC ...... A61K 31/416 (2013.01); A61K 31/428 Yin, et al., Antimicrobial Agents Chemother: 50(1):336-343, 2006. International Search Report and Written Opinion issued in PCT/ (2013.01); A61K 31/4245 (2013.01); A61 K US2014/029237, mailed on Aug. 11, 2014. 31/4439 (2013.01); A61K 31/454 (2013.01); Extended European Search Report for EP 14764384.5, mailed Oct. A6 IK3I/4709 (2013.01); A61K 31/4725 12, 2016. (2013.01); A61 K3I/496 (2013.01); A61 K Reynolds et al., Tuberculosis 92 (2012) 72-83. U.S. Patent Jun. 13, 2017 Sheet 1 of 6 US 9,675,592 B2

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------O O.5 1 2 3 4. 5 6 6 18 Oxacillin) mg/L. time (hours

* Ill 1 Oxacillin2 3 4mg/L. 5 6 F.G. 1 A-1C U.S. Patent Jun. 13, 2017 Sheet 2 of 6 US 9,675,592 B2

A 5 | vasr VraSR wraSR active 20 inactive uncertain average

15 average 10

as re-- """ g 20 3. uninhibited ' 15 og average s - 2 SD aWeaCe g 10 5 5 - 2 SD O ------T- DDDD • F- s -Y N) () a cu C N CO CO - a -a - a -a -a -a -a -a -a -a N) N. N. N. N. N. d - a Nad co -na cu o N Co co o -a n) ca) a U. ODs bin number

FIG. 2A-2B U.S. Patent Jun. 13, 2017 Sheet 3 of 6 US 9,675,592 B2

1 2 3 4 5 6 7

His-VraS-C VraS-C U.S. Patent Jun. 13, 2017 Sheet 4 of 6 US 9,675,592 B2

3OO 2 250

200

150

O 10 20 30 40 (F2645-0188 (uM) FIG. 4 U.S. Patent Jun. 13, 2017 Sheet 5 of 6 US 9,675,592 B2

U.S. Patent Jun. 13, 2017 Sheet 6 of 6 US 9,675,592 B2

Y a . 3 3 3. ful 3. O D.5 2 4 8 6. 32 f 3 2 4 B 16 32 F2645-0.188 F5882-3050 V 2 4. -

8 -

16 - 32 64

O 0,5 1 2 4 8 16 32 FO020-1560 F1813-0712 O 2 4 8 16 32 64

O 0.5 1 2 4 8 16 32 F2703-0396 O F2619-0556 O 2 0.25 4 0.5 8 1

16 32 64 Growth in MBC (3-log Min conc to broth after 24 hr reduction in E reduce Ox MIC CFU) by 32x FIG. 6 US 9,675,592 B2 1. 2 METHODS AND COMPOSITIONS FOR 2,5-dichlorophenyl, 2,5-dichlorothiophen-3-yl, 2,6-difluo BACTERIA INFECTIONS rophenyl, 2-bromothiophen-5-yl, 2-chlorothiophen-5-yl, 2-naphthyl 2-phenoxyphenyl, 2-phenylduinolin-4-yl, 3-(2- CROSS-REFERENCE TO RELATED chlorophenyl)-5-methylisoxazol-4-yl, 3,4,5-trimethoxyphe APPLICATIONS nyl, 3,5-dichlorophenyl, 3,5-dimethoxyphenyl, 3-bro mophenyl, 3-chlorobenzobthiophen-2-yl, 3-chlorophenyl, This application is a national phase application under 35 3-fluorophenyl, 3-methoxyphenyl, 3-n-butoxyphenyl, U.S.C. 371 of International Application No. PCT/US2014/ 3-phenoxyphenyl, 3-tolyl, 3-trifluoromethylphenyl, 4-(2,6- 09237, filed on Mar. 14, 2014, which claims the benefit of dimethylmorpholinosulfonyl)phenyl, 4-(2-ethylpiperidin-1- priority of U.S. Provisional Patent Application No. 61/789, 10 ylsulfonyl)phenyl, 4-(2-methylpiperidin-1-ylsulfonyl)phe 716, filed on Mar. 15, 2013. The entire contents of each of nyl, 4-(2-phenylduinoline), 4-(3,4-dihydroisoquinolin-2 the above-referenced disclosures are specifically incorpo (1H)-ylsulfonyl)phenyl, 4-(3,4-(dihydroquinolin-1 (2H)- rated herein by reference without disclaimer. ylsulfonyl)phenyl, 4-(3,5-dimethylpiperidin-1-ylsulfonyl) phenyl, 4-(4,4-dimethyloxazolidin-3-ylsulfonyl)phenyl, BACKGROUND OF THE INVENTION 15 4-(morpholinosulfonyl)phenyl, 4-(N,N-diallylsulfamoyl) 1. Field of the Invention phenyl, 4-(N,N-diethylsulfamoyl)phenyl, 4-(N,N-diisobu The present invention relates generally to the field of tylsulfamoyl)phenyl, 4-(N,N-dimethylsulfamoyl)phenyl, medicine. More particularly, it concerns the use of chemical 4-(N-ethyl-N-benzylsulfamoyl)phenyl, 4-(N-ethyl-N-n-bu compounds that inhibit the VraSR operon and potentiate the tyl-sulfamoyl)phenyl, 4-(N-ethyl-N-phenylsulfamoyl)phe action of antibiotics. nyl, 4-(N-isopropyl-N-benzylsulfamoyl)phenyl 4-(N- 2. Description of Related Art methyl-N-benzylsulfamoyl)phenyl, 4-(N-methyl-N- Staphylococcus aureus is a well-adapted human parasite cyclohexylsulfamoyl)phenyl, 4-(N-methyl-N-n- that is both a commensal and an important pathogen. It is butylsulfamoyl)phenyl, 4-(N-methyl-N-phenylsulfamoyl) responsible for a wide variety of infectious diseases that 25 phenyl, 4-(piperidin-1-ylsulfonyl)phenyl, 4-(pyrrolidin-1- range from minor skin abscesses to severe infections and ylsulfonyl)phenyl, 4-benzoylphenyl, 4-benzylphenyl, toxinoses requiring hospitalization. Staphylococcus aureus 4-biphenyl, 4-chlorophenyl, 4-diphenyl, 4-ethoxyphenyl, strains resistant to nearly all beta-lactams, so-called methi 4-fluorophenyl, 4-phenoxyphenyl, 4-tert-butylphenyl, cillin-resistant Staphylococcus aureus (MRSA), are a lead 30 7-methoxybenzofuran-2-yl, benzodthiazol-2-yl, benzod ing cause of healthcare associated and, since the 1990s, thiazol-4-yl, benzodthiazol-6-yl, benzofuran-2-yl, diphe community-associated infection. An epidemic of MRSA nylmethyl, methyl-4-benzoate, phenyl, or thiophen-2-yl, and infections has enhanced the urgency to identify alternative R may be 1,2,3,4-tetrahydronaphthalen-6-yl, 2-(methyl antibacterial agents for Successful treatment. Unfortunately, thio)phenyl, 2,3-dihydro-1,4-dioxin-2-yl, 2,3-dihydrobenzo this need comes at a time when the industry-driven pipeline 35 b1,4-dioxin-2-yl, 2,3-dihydrobenzob 1,4-dioxin-6-yl, for antibacterial development has slowed. 2,4-dichlorophenyl, 2,4-dimethoxyphenyl, 2,4-dimethyl phenyl, 2,5-dichlorophenyl, 2,5-dichlorothiophen-3-yl 2.5- SUMMARY OF THE INVENTION dimethylphenyl, 2-bromothiophen-2-yl, 2-chlorophenyl, 2-chlorothiophen-2-yl, 2-chlorothiophen-5-yl, 2-furanyl, Methods and compositions are provided for inhibiting, 40 2-methoxyphenyl, 2-pyridinyl, 3-(methylthio)phenyl, 3,4,5- preventing, or treating a bacterial infection, particularly trimethoxyphenyl, 3,4-dimethoxyphenyl, 3,4-dimethylphe bacteria that rely on a VraSR operon for signal transduction. nyl, 3,5-dimethoxyphenyl, 3-methoxyphenyl, 3-pyridinyl, This operon is conserved among Staphylococcus aureus 4-chlorophenyl, 4-(methylthio)phenyl, 4-bromophenyl, strains and methods and embodiments concern staphylococ 45 4-ethoxyphenyl, 4-fluorophenyl, 4-isopropylbenzyl, cal bacterial infections. In particular, methods and compo 4-methoxybenzyl, 4-methoxyphenyl, 4-pyridinyl, 4-trifluo sitions involve an antibiotic and a compound that can be romethoxyphenyl, 7-ethoxybenzofuran-2-yl, 7-methoxy qualified as potentiating the activity of the antibiotic and/or benzofuran-2-yl, benzofuran-2-yl, benzyl, phenyl, or thio an inhibitor of VraSR operon expression or activity. phen-2-yl. In some embodiments, there are methods for inhibiting a 50 In some embodiments, a composition comprises an anti staphylococcus infection comprising administering to a Sub biotic potentiator having the structure: ject having a staphylococcus infection or at risk of a staphy lococcus infection: (a) an antibiotic, and (b) an antibiotic potentiator, wherein the antibiotic potentiator is a compound (II) of the formula: 55 OH

(I) O Br. R2 -->-l R, 60 wherein R' may be -(4-chlorophenoxy)methyl, 1-(thiophen CO 2-yl)cyclopentyl, 1.2.3,4-tetrahydronaphthalen-6-yl, 1-iso 65 propyl-1H-pyrazol-5-yl, 2-(thiophen-2-yl)guinolin-4-yl, In further embodiments a composition comprises an anti 2,3-dihydrobenzob.14 dioxin-6-y1, 2,4-dichlorophenyl, biotic potentiator having the formula: US 9,675,592 B2 3 4

S O (III)III (VII)VII

| N- %all N R, 5 N S-NH leN -Ns/ || S wherein R may be 2-phenoxyphenyl, 2-bromothiophen-5-yl, MN or 2,5-dichlorophenyl. 10 Other embodiments concern a composition comprising an antibiotic potentiator having the formula: In some embodiments, a composition comprises an anti biotic potentiator having the structure:

(IV) 15 (VIII)

CO 25 In further embodiments a composition comprises an anti biotic potentiator having the structure: wherein R may be fluoro, or chloro. In further embodiments a composition comprises an anti- 30 (IX) biotic potentiator having the structure: O

(V) N F 35 F 1.

C N - Nals N H H 40 In some embodiments there are compositions comprising an antibiotic potentiator having the structure: (X) 45 N 7 S O

In yet further embodiments, a composition comprises an H / antibiotic potentiator having the structure: 50 Other compositions concern an antibiotic potentiator hav ing the structure: (VI) (XI) 55

HN

Nch 60 HO N C.

65 Further compositions and methods concern an antibiotic Cl potentiator having the structure: US 9,675,592 B2 5 6 Further compositions and methods concern an antibiotic In some embodiments, a composition or method concerns potentiator having the formula: an antibiotic potentiator that is depicted herein or as a prodrug or comparable salt thereof. In additional embodiments, there are methods for inhib (XII) 5 iting a staphylococcus infection comprising administering to R2 a Subject: (a) an antibiotic, and (b) an antibiotic potentiator of that is depicted herein, or a prodrug or salt thereof. In still N21 further embodiments, there are methods for inhibiting a staphylococcus infection comprising administering to a Sub 2 10 ject: (a) an antibiotic, and (b) an antibiotic potentiator that is depicted herein, or a prodrug or salt thereof. ere It is specifically contemplated that one or more antibiotic potentiators or a prodrug or salt thereof, may be excluded from methods and compositions discussed herein. In still wherein R' may be N-(4-(phenylamino)-N'-(phenyl)acet 15 further embodiments, methods and compositions include an amide)amine, N-3-(N',N'-dimethylamine)propylamine, antibiotic as the antibiotic potentiator. N-(4-N'-phenylacetamide)amine, or hydroxyl, and R may Other methods involve treating a patient with a staphy be tert-butyl, isopropyl, or phenyl, and R may be phenyl, or lococcus infection comprising administering to the patient 4-chlorophenyl. an antibiotic and administering to the patient a compound In further embodiments a composition comprises an anti selected from the group consisting of I-XV above, or pro biotic potentiator having the structure: drug or a salt thereof. Further embodiments concern methods for treating a patient with a staphylococcus infection comprising admin (XIII) istering to the patient an effective amount of an antibiotic 25 and an effective amount of a compound selected from the group consisting of I-XV above, or prodrug or a salt thereof. leN In some embodiments, methods and compositions con cern an antibiotic that is a beta-lactam antibiotic. In certain embodiments, the antibiotic is a penicillinase-resistant beta 30 lactam antibiotic. In further embodiments, the penicillinase resistant beta-lactam antibiotic is oxacillin. Certain embodi CCJ) ments concern a penicillinase-resistant beta-lactam antibiotic that is methicillin, nafcillin, cloxacillin, dicloxa cillin or flucloxacillin. Other antibiotics for use in methods 35 and compositions are discussed below and may include Other compositions concern an antibiotic potentiator hav Vancomycin, teichoplanin, and/or daptomycin. An antibiotic ing the formula: may be given before, with, or after an antibiotic potentiator is administered to the patient. The antibiotic and the anti (XIV) biotic potentiator may or may not be formulated similarly. In C 40 Some embodiments, they are co-formulated or are in the same composition or solution. In one example, one or both of them may be formulated for oral administration. In C c n N lsO N1 R, another example, they may both be formulated for i.v. H H administration. It is also contemplated that compounds may 45 be administered to a subject by the same or different routes wherein R may be 1H-indazol-6-yl, or 3-(4-methoxyphe of administration. noxy)methyloxadiazol-5-yl-methyl. Embodiments can be used with a Subject that Staphyllo coccus can infect, Such as mammals, and particularly In some embodiments, a composition comprises an anti humans, monkeys, primates, apes, dogs, cats, cows, horses, biotic potentiator having the formula: 50 pigs, goats, mice, or rats. In certain embodiments, the Subject is a human patient. In particular methods, the Subject has been tested for a staphylococcus infection. In other (XV) embodiments, the Subject is or has been diagnosed with a staphylococcus infection. Methods may further involve test 55 ing or evaluating a patient for staphylococcus infection. In R2 additional embodiments, methods also include diagnosing a patient for Staphylococcus infection. In some cases, a subject 4' is at risk of acquiring a staphylococcus infection. This % NH O N includes, but is not limited to, patient undergoing an invasive 60 hospital procedure (such as one requiring anesthesia) or Surgery, patients having undergone an invasive hospital procedure or Surgery, patients placed on an i.v., or patients on a ventilator. In certain embodiments, a Subject has one or more symptoms of a staphylococcal infection. 65 In some methods, the Subject has or is at risk for native wherein, R' and R may each be separately hydrogen, valve endocarditis or prosthetic valve endocarditis. In other methyl, chloro or methylthio. embodiments, the Subject has or is at risk for joint infection, US 9,675,592 B2 7 8 meningitis, osteomyelitis, pneumonia, septicemia, sinusitis, system or an injection under the skin. It is also contemplated or skin or soft tissue infection. In further embodiments, the that compounds may be administered to a Subject by the subject is administered about 2-3 g of oxacillin intrave same or different routes of administration. nously every 4 to 6 hours. In other methods, the subject is In some embodiments the antibiotic and the antibiotic administered about 1-2 g of oxacillin intravenously or potentiator are in the same composition. In other embodi intramuscularly every 4 to 6 hours or about 500 mg to about ments the antibiotic and the antibiotic potentiator are admin 1 g of oxacillin orally every 4 to 6 hours. In some methods, istered simultaneously in the same or different compositions. before being administered a potentiator, the Subject has A subject is administered an antibiotic up to 24 hours prior previously been treated with oxacillin or another antibiotic to administration of the antibiotic potentiator in some cases. that induces cell wall stress. In some embodiments, the prior 10 In others, the antibiotic potentiator is administered up to 24 treatment with an antibiotic has been ineffective to treat the hours prior to administration of the antibiotic. In some infection or the subject or infection has been found to be embodiments, the antibiotic and antibiotic potentiator are resistant to the antibiotic. In certain embodiments, the pre administered within 24 hours of each other. vious treatment was started within 1, 2, 3, 4, 5 weeks and/or Pharmaceutical compositions are also provided. In some 1, 2, 3, 4, 5, or 6 months (or any range derivable therein) of 15 embodiments, a pharmaceutical composition includes an being treated with a potentiator. antibiotic and a compound selected from the group consist In some cases, treating a Staphylococcus infection com ing of I-XV above, or prodrug or a salt thereof. In additional prises reducing abscess formation or incidence or reducing embodiments, a pharmaceutical composition includes a bacterial load in the Subject. In other embodiments, treating single unit dose of a selected antibiotic and a compound a Staphylococcus infection comprises reducing symptoms of selected from the group consisting of I-XV above. any infection including but not limited to reducing fever, In some cases, a pharmaceutical composition comprises at reducing Swelling at the infection site, and/or reducing pain least an additional antibacterial agent. The additional anti at the infection site. bacterial agent may be a further antibiotic, a staphylococcal With Some methods and compositions, an infection is vaccine composition or a polypeptide that specifically binds from a staphylococcus that belongs to the species Staphy 25 to a second staphylococcal protein. It is contemplated that lococcus aureus. In certain embodiments, the staphylococ the pharmaceutical composition may be a pill, capsule, cus infection is methicillin resistant Staphylococcus aureus tablet, lozenge, troche, Solution, cream, gel, paste, liquid or (MRSA). In alternative embodiments, the staphylococcus solid. In embodiments where antibiotic and an antibiotic infection is methicillin sensitive Staphylococcus aureus potentiator are in different compositions, each may be pro (MSSA), and the composition is provided to treat the 30 vided in one of these forms. infection while reducing the likelihood of acquisition of Additional embodiments concern a system for treating a methicillin resistance. bacterial infection comprising a pharmaceutically accept Certain methods and compositions may involve a second able composition comprising an antibiotic and a pharma antibiotic. In some embodiments, methods involve admin ceutically acceptable composition comprising an antibiotic istering the second antibiotic. In particular cases, the second 35 potentiator that is a compound selected from the group antibiotic is gentamicin or rifampin. Other second antibiot consisting of I-XV above. In some system embodiments, the ics are discussed below. Methods or compositions may also antibiotic is in an aqueous formulation. Further embodi include a staphylococcal vaccine. Methods include, in par ments involve a system in which the antibiotic is in an ticular embodiments, administering a staphylococcal vac aqueous formulation that is an intravenous solution or an cine. This may be administered before, after, or with an 40 aqueous formulation that is injectable into the patient or into antibiotic and/or an antibiotic potentiator. an intravenous Solution. In other embodiments, an antibiotic In some embodiments, the antibiotic is administered at a potentiator is in an aqueous formulation. In certain cases, an dose of about 0.1 mg/kg to about 50 mg/kg. In particular antibiotic potentiator is in an aqueous formulation that is an embodiments, the Subject is a pediatric patient, which means intravenous solution or an aqueous formulation that is under 18 years of age for a human patient. For a pediatric 45 injectable into the patient or into an intravenous solution. patient, in Some embodiments an antibiotic is administered Embodiments discussed in the context of compositions about 25 mg/kg to about 50 mg/kg intravenously or intra may also be applied as methods, and vice versa. muscularly every 6 to 12 hours or about 12.5 mg/kg orally As used herein the specification, “a” or “an may mean every 6 hours. In certain embodiments, the antibiotic is one or more. As used herein in the claim(s), when used in oxacillin. 50 conjunction with the word “comprising, the words “a” or Methods may also involve an antibiotic potentiator 'an' may mean one or more than one. administered in a dose of 0.1 mg/kg to about 100 mg/kg. The use of the term 'or' in the claims is used to mean In some methods administration of a compound to a “and/or unless explicitly indicated to refer to alternatives Subject is oral, Sublingual, Sublabial, gastrointestinal, rectal, only or the alternatives are mutually exclusive, although the epicutaneous (topical), intradermal, Subcutaneous, nasal, 55 disclosure Supports a definition that refers to only alterna intravenous, intraarterial, intramuscular, intracardiac, tives and “and/or.” As used herein “another may mean at intraosseous, intrathecal, intraperitoneal, intravesical, intra least a second or more. vitreal, intracavernous, intravaginal, intrauterine, epidural, Throughout this application, the term “about is used to intracerebral and/or intracerebroventricular. In specific indicate that a value includes the inherent variation of error embodiments, administration is topical, enteral, or parent 60 for the device, the method being employed to determine the eral. In some instances, administration is by application onto value, or the variation that exists among the study Subjects. the skin, inhalation, an enema, eye drops, ear drops, absorp Other objects, features and advantages of the present tion across mucosal membranes, the mouth, a gastric feeding invention will become apparent from the following detailed tube, a duodenal feeding tube, a Suppository, an injection description. It should be understood, however, that the into a vein, an injection into an artery, an injection into the 65 detailed description and the specific examples, while indi bone marrow, an injection into muscle tissue, an injection cating preferred embodiments of the invention, are given by into the brain, an injection into the cerebral ventricular way of illustration only, since various changes and modifi US 9,675,592 B2 9 10 cations within the spirit and scope of the invention will clinically important antibacterials that work by interfering become apparent to those skilled in the art from this detailed with bacterial cell wall synthesis. This VraSR operon is description. responsible for activation of a set of genes that presumably coordinately act together to adapt to cell wall stress. Com BRIEF DESCRIPTION OF THE DRAWINGS pounds that inhibit expression of the VraSR operon are shown below to enhance the ability of a beta-lactam anti The following drawings form part of the present specifi biotic to kill MRSA strains and treat infections caused by cation and are included to further demonstrate certain them (potentiation). aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in 10 II. Antibiotics combination with the detailed description of specific embodiments presented herein. Embodiments concern methods and compositions for FIG. 1A-C: Optimal conditions for measuring growth and treating bacterial infections, including infections involving luminescence of the test strain, 923 pPvra-lux. (A) Absor antibiotic-resistant bacteria. Substances are provided that bance at 18 hours as a function of oxacillin concentration. 15 can be used in conjunction with antibiotics that are effective (B) Luminescence (LU) was monitored over 18 hours with or potentially effective for treating or inhibiting a bacterial oxacillin at 0 g/mL (blue Solid line), 1 ug/mL (green line) infection. The antibiotics specifically contemplated for use and 2 g/mL (red solid line). (C) Luminescence at two hours in embodiments include beta-lactam antibiotics. Some post inoculation as a function of oxacillin concentration. embodiments concern the use of one or more antibiotics in FIG. 2A-B: Distribution of OD and LU. Criteria for combination with one or more compounds that inhibit the classification of growth and luminescence inhibition are VraSR operon. Further embodiments specifically contem based on reference data of strain 923 pPvra-lux grown in the plate the use of compounds that inhibit the VraSR operon presence or absence of oxacillin (2 Lig/mL) (+/-OX). (A) together with one or more beta-lactam antibiotics. Yet fur luminescence (B) absorbance. Solid bars-oxacillin added ther embodiments contemplate the use of compounds that (+Ox), empty bars—no oxacillin (-Ox). 25 inhibit the VraSR operon together with one or more antibi FIG. 3: VraS Cytoplasmic domain (VraS-C) expression otics known to act via disruption of bacterial cell wall and purification gel documentation and analysis. VraS-C processes. was expressed and purified as described in Example 3. The In some embodiments, Staphylococcus aureus is involved gel is loaded as follows: Lane 1: Supernatant; Lane 2: in the bacterial infection to be treated. Other embodiments flow-through from the first HisTrap column; Lane 3: wash 30 contemplate the use of compounds that inhibit the VraSR with 68 mM imidazole; Lane: 4 elution fraction with 200 operon against the homologous LiaRS system present in mM imidazole; Lane 5 elution fraction with 350 mM Bacillus subtilis, Streptococcus pneumoniae, and Strepto imidazole Lane 6: after digestion with 3C HRV protease: coccus mutansi. Further embodiments contemplate the use and Lane 7: final His-tag cleaved VraS-C. of said compounds to target the homologous CessR system FIG. 4: SPR (Surface Plasmon Resonance) binding assay 35 in several Lactococcus species. Yet further embodiments results for F2645-0188. Depicted is the fitting curve of contemplate the use of these compounds against other compound F2645-0188 with a single rectangular hyperbolic homologous two-component systems of cell-wall-mediated equation using Sigmaplot 11.0. The determined K, was antibiotic resistance. 13.2+3.2 M. All data were normalized for immobilization Certain embodiments contemplate the use of beta-lactam levels of VraS-C protein. 40 antibiotics. Beta-lactam antibiotics are a broad class of FIG. 5: Structure-activity relationship (SAR) analysis of antibiotics, consisting of antibiotic agents that contain a primary and secondary assay data for activity of oxadiazole beta-lactam nucleus in their molecular structures. This carboxaminde analogs. Based on the SAR analysis, Substitu includes penicillin derivatives (penams), cephalosporins ents were identified that may act to increase activity, (cephems), monobactams, and carbapenems. Most beta decrease activity, or may be neutral to activity. Those that 45 lactam antibiotics work by inhibiting cell wall biosynthesis may increase activity are shown in section A (top). Those in bacteria and are the most widely used group of antibiotics. that may decrease activity are shown in section C (bottom). It is contemplated that compounds that inhibit the VraSR Those that may produce no apparent increase or decrease in operon potentiate the action of any beta-lactam antibiotic. activity are shown in section B (middle) producing no This group comprises any of Amoxicillin, Ampicillin, Piv apparent increase or decrease in activity 50 ampicillin, Hetacillin, Bacampicillin, Metampicillin, Talam FIG. 6: Checkerboard MICs (boxes with horizontal lines) picillin, Epicillin, Carbenicillin Carindacillin, Ticarcillin, and MBCs (minimal bactericidal concentrations; boxes with Temocillin Azlocillin, Piperacillin, Mezlocillin Mecillinam black borders) of OX and inhibitors. Black-bordered boxes Pivmecillinam, Sulbenicillin Clometocillin, BenZathine mark the concentration of inhibitor that decreased the Oxa benzylpenicillin, Procaine benzylpenicillin, AZidocillin. cillin MIC to 0.5 to 1 (=susceptible). 55 Penamecillin, Phenoxymethylpenicillin, Propicillin, Benza thine phenoxymethylpenicillin, Pheneticillin, Cloxacillin, DESCRIPTION OF ILLUSTRATIVE Dicloxacillin, Flucloxacillin, oxacillin, Meticillin, Nafcillin, EMBODIMENTS Faropenem Biapenem, Ertapenem, Doripenem, Imipenem, Meropenem, Panipenem, Cefazolin, Cefacetrile, Cefadroxil, I. Bacterial Growth Inhibition 60 Cefalexin, Cefaloglycin, Cefalonium, Cefaloridine, Cefalo tin, Cefapirin, Cefatrizine, Cefazedone, Cefazaflur, Cefra VraS and VraR constitute a two-component signal trans dine, Cefroxadine, Ceftezole, Cefaclor, Cefamandole, Cef duction system that requires a recently renamed third com minox, Cefonicid, Ceforanide, Cefotiam, Cefprozil, ponent, VraT (previously called YvdF) that is encoded on the CefbuperaZone, Cefuroxime, CefuZonam, cephamycin, same operon (Boyle-Vavra 2006: Yin 2006; Francis 2000). 65 Cefoxitin, Cefotetan, Cefnetazole, carbacephem, Loracar The VraSR operon, conserved among Staphylococcus aureus bef, Cefixime, Ceftriaxone, Ceftazidime, CefoperaZone, strains, senses and responds to cell-wall stress elicited by Cefcapene, Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, US 9,675,592 B2 11 12 Cefnmenoxime, Cefodizime, Cefotaxime, Ce?pimizole, Cef potentiate Vancomycin in vitro was tested by performing piramide. Cefpodoxime, Cefsulodin, Cefteram, Ceftibuten, checkerboard assays with a Vancomycin intermediate resis Ceftiolene, Ceftizoxime, oxacephem Flomoxef. Latamoxef, tant Staphylococcus aureus strain, VISA 2283 (MIC Vanco Cefepime, CefoZopran, Ce?pirome, Cefauinome, Ceftobi mycin 16 mg/L); a hetero VISA isolate, hVISA 2275 (MIC prole, Ceftaroline fosamil, Ceftiofur, Cefauinome, Cefove of Vancomycin of 4 mg/L), and two van A containing VRSA cin, Aztreonam, Tigemonam, Carumonam or Nocardicin A. strains, VRS1 (MIC of Vancomycin 1024 mg/L) and VRS2 In embodiments using beta-lactamase sensitive antibiotics (MIC of Vancomycin 16 mg/L). In the checkerboard assay, Such as Amoxicillin, Ampicillin, pivampicillin, hetacillin, increasing concentrations of Vancomycin are arrayed with bacampicillin, metampicillin, talampicillin, epicillin, car increasing concentrations of the indicated potentiator. The benicillin carindacillin, ticarcillin, temocillin azlocillin, pip 10 eracillin, mezlocillin mecillinam pivmecillinam, Sulbenicil FICi for each combination was calculated according to the lin clometocillin, benzathine benzylpenicillin, procaine following formula: benzylpenicillin, azidocillin, penamecillin, phenoxymethyl penicillin, propicillin, benzathine phenoxymethyl-penicillin, FIC = Ox|MIC in combination compound in combination or pheneticillin, addition of a beta-lactamase inhibitor is 15 further contemplated. MIC of Wan alone MIC of compound alone Further embodiments contemplate the use of other anti biotics that induce the cell wall stress stimulon (CWSS) in An FIC, of s.0.5 defines a synergistic interaction. An Staphylococcus aureus and thus involve VraSR Such as fosfomycin, D-cycloserine, tunicamycin, bacitracin, bam FIC-0.5 and <0.7 is possibly synergistic. As shown in the bermycin, Vancomycin, moenomycin, teicoplanin, lyso analysis of the checkerboard assays (Table 1), gossypol fully staphin, teichoplanin, and daptomycin. In still further potentiated Vancomycin (FIC i-0.5) in hVISA, VISA, VRS2 embodiments, the antibiotic is a cationic peptide such as and VRS1. Pyrvinium partially potentiated Vancomycin cecropins, andropin, moricin, ceratotoxin, melittin, magai (FIC id-0.5 and <1.0) in hVISA, and fully potentiated in both nin, dermaseptin, bombinin, brevinin-1, esculentin and 25 of the VanA containing VRSA strains but not in the VISA buforin II, CAP 18, or LL37. strain. Clomiphene partially potentiated Vancomycin in Experimental data provide Support for using compounds hVISA, VISA, and VRS1 and it fully potentiated in strain that inhibit the VraSR operon as potentiators of Vancomycin VRS2. For gossypol, the degree of Vancomycin potentiation and related compounds, including those that induce expres was not dependent on the Vancomycin MIC of the strain sion of VraSR. The following data from the co-pending 30 since pyrvinium was a stronger potentiator in VRS1 (which application PCT/US2013/068085, which is hereby incorpo has a Vancomycin MIC of 1024 mg/L without compound) rated by reference in its entirety, demonstrates that the than it was for the VISA strain (which has a vancomycin compounds clomiphene, pyrvinium, and gossypol, which MIC of 8 in the absence of pyrvinium). In the VRSA strain, were found to inhibit the VraSR operon and to potentiate the MIC of Vancomycin decreased by 64 fold whereas in the oxacillin, were also effective potentiators of Vancomycin. 35 VISA strain, pyrvinium decreased the Vancomycin MIC by The ability of clomiphene, pyrvinium, and gossypol to 2 to 4 fold. TABLE 1. Clomiphene (Clo), Vancomycin (Van or V), Pyrvinium (Pyr), Gossypol, (Gos). Van+ indicates Vancomycin MIC when used in combination with compound, Clo-, Pyr- or Gps+ signifies MIC of the compound when used in combination with the indicated Van concentration). MIC MIC (in fold decrease Synergy Strain (mg/L) combination)* FIC FIC Van MIC category Clo Van Clo Van+ Clo- Van FIC CO FIC FIC V - Co

VISA 2275 4 12 2 1.5 O.S O.13 O.63 2 partial VISA 2275 4 12 1 6 O.25 O.S 0.75 4 partial VISA 2283 8 12 4 3 O.S O.25 0.75 2 partial VRS2 16 12 1 6 O.O6 O.S O.S6 16 partial VRS2 16 12 2 3 O.13 O.25 O.38 8 synergistic VRS2 16 12 8 1.5 OSO O.13 O.63 2 partial VRS1 1024 12 512 3 OSO O.25 0.75 2 partial 1024 12 2S6 6 O.25 O.S 0.75 4 partial Pyr Van Pyr Van- Pyr+ Van FIC Pyr FIC FICi V+ Pyr VISA 2275 4 16 2 O.S6 OSO O.04 O.S4 2 partial VRS2 16 112 1 O.28 O.O6 O.25 O.31 16 synergistic VRS2 16 112 2 O.14 O.13 O.13 O.25 8 synergistic VRS1 1024 2.24 1 1.1 O.OO O.49 O.49 1024 synergistic VRS1 1024 2.24 64 O.S6 O.O6 O.25 O.31 16 synergistic VRS1 1024 2.24 512 O.14 OSO O.O6 O.S6 2 partial VISA non syn

Gos Van Gos Van- Gos- Van FIC Gos FIC FICW - G

VISA 2275 4 10.4 1 1.3 O.25 O.12S O.38 4 synergistic VISA 2283 8, 10.4 4 1.3 OSO O.12S O.63 2 partial VISA 2283 8, 10.4 2 5.2 O.25 O.S 0.75 4 partial US 9,675,592 B2 13 14 TABLE 1-continued Clomiphene (Clo), Vancomycin (Van or V), Pyrvinium (Pyr), Gossypol, (Gos). Van+ indicates Vancomycin MIC when used in combination with compound, Clo-, Pyr- or Gps+ signifies MIC of the compound when used in combination with the indicated Van concentration). MIC MIC (in fold decrease Synergy Strain (mg/L) combination)* FIC FIC Van MIC category VRS2 16 S.2 1 1.3 O.O6 O.25 O.31 16 synergistic VRS1 1024 20.8 16 1.3 O.O2 O.0625 O.08 64 synergistic VRS1 1024 20.8 1 2.6 O.OO1 O.12S O.13 1024 synergistic

In additional embodiments compounds listed in Table 5 very severe infections. In conventional administration, it is are envisioned to be used as antibiotics, alone or in combi 15 effective largely against gram-positive organisms. In chil nation with the antibiotics or potentiators discussed herein. dren, a dose in additional embodiments is 20 to 50 mg/kg per Penicillin G is administered in some embodiments to day, in 2 equally divided doses every 12 hours. The oral dose adults in doses ranging from 600,000 to >1,000,000 units. for nafcillin in Some embodiments ranges from 1 gram per Penicillin G is administered in doses of 20-24 million units day to 6 grams per day in 4 to 6 divided doses. daily, in divided doses every 4-6 hours. For children, a dose Oxacillin is administered in certain embodiments to adults of penicillin G is about 50,000 units/kg/dose. One unit of in doses of 2 to 12 grams per day, in 4 to 6 equally divided penicillin G contains 0.6 ug of pure sodium penicillin G (i.e., doses. In conventional administration, it is effective largely 1 mg is 1667 units). against gram-positive organisms. In children, oxacillin is Amoxicillin may be administered to adults in doses administered in doses of 100 to 300 mg/kg per day. ranging from 750 mg to 1.5 grams per day, in 3 divided 25 doses. For children, doses of amoxicillin range from 20 to 40 Piperacillin is administered to adults in doses ranging mg/kg per day in 3 equally divided doses. Amoxicillin is from 100 mg/kg, or 6 grams per day, in 2 to 4 equally also available in combination with clavulanic acid, a beta divided doses, up to a maximum of 24 grams per day, in 4 lactamase inhibitor. A 250 mg dose of the combination drug to 6 equally divided doses. Higher doses have been used amoxicillin/clavulanate will contain 250 mg of amoxicillin 30 without serious adverse effects. and either 125 or 62.5 mg of clavulanic acid. The combi Ticarcillin is administered to adults in doses ranging from nation is preferably administered to adults orally in doses of 4 grams per day to 18 grams per day administered in 4 to 6 750 mg per day divided into 3 equal doses every 8 hours, equally divided doses. The usual dose is 200 to 300 mg/kg with a dose of 1.5 grams per day for severe infections, given per day. For children, a typical dose of ticarcillin ranges in 3 equally divided doses. In children, the oral dose is 20 to 35 from 50 mg/kg per day to 300 mg/kg per day, given in 3, 4 40 mg/kg per day in 3 equally divided doses. or 6 equally divided doses. The combination ticarcillin/ In some embodiments amplicillin is administered to adults clavulanate is administered to adults in doses of 200 to 300 in doses of 6 to 12 grams per day for severe infections, in 3 mg/kg per day (based on ticarcillin content), in 4 to 6 equally to 4 equally divided doses. In children, the dose of amplicillin divided doses. For adults, the usual dose is 3.1 grams (which is 50 to 200 mg/kg per day in 3 to 4 equally divided doses. 40 contains 3 grams of ticarcillin and 100 mg of clavulanic Larger doses of up to 400 mg/kg per day, for children, or 12 acid) every 4 to 6 hours. The combination is also available grams per day, for adults, may be administered. Ampicillin in a dose of 3.2 grams, which contains 3 grams of ticarcillin is also available in combination with Sulbactam, a beta and 200 mg of clavulanic acid. lactamase inhibitor. Each 1.5 gram dose of amplicillin/ In additional embodiments, methods and compositions Sulbactam contains 1 gram of amplicillin and 0.5 grams of 45 concern cefamandole that is administered to adults in doses sulbactam. The combination is preferably administered to ranging from 1.5 grams per day, given in 3 equally divided adults in doses of 6 to 12 grams per day divided into 4 equal doses every 8 hours, to 12 grams per day for life-threatening doses every 6 hours, not to exceed a total of 12 grams per infections, given in 6 equally divided doses every 4 hours. day. In children, cefamandole is typically administered in doses In certain embodiments, azlocillin is typically adminis 50 ranging from 50 to 150 mg/kg per day, in 3 to 6 equally tered to adults in doses of 8 to 18 grams per day, given in 4 divided doses, not to exceed a total of 12 grams per day. to 6 equally divided doses. Cefazolin is administered in some methods to adults in In further embodiments, carbenicillin is administered to doses of 750 mg per day, given in 3 equally divided doses adults in doses of 30 to 40 grams per day, given by every 8 hours. In severe, life-threatening infections, it may continuous infusion or in 4 to 6 equally divided doses. Daily 55 be administered at doses of 6 grams per day divided into 4 doses of up to 600 mg/kg have been used to treat children equal doses every 6 hours; in rare instances, up to 12 grams with life-threatening infections. per day have been used. In children, the dose of cefazolin is Mezlocillin is administered in some embodiments to 20 to 50 mg/kg per day, divided into 3 or 4 equal doses, with adults in doses of 100 to 300 mg/kg per day, given in 4 to 100 mg/kg per day administered for severe infections. 6 equally divided doses. The usual dose is 16 to 18 grams per 60 In further embodiments, methods and compositions con day; for life threatening infections, 350 mg/kg per day may cern cefonicid that is administered to adults in doses ranging be administered, but in doses not to exceed 24 grams per day from 500 mg once daily, to 2 grams once daily for life given in 6 equally divided doses every 4 hours. For children, threatening infections. For intramuscular administration, a 2 the dose of mezlocillin is 150 to 300 mg/kg per day. gram dose should be divided into two 1-gram injections. Nafcillin is in some embodiments intravenously admin 65 In some methods, cefoperaZone is administered to adults istered to adults in doses of 3 grams per day, given in 6 in doses ranging from 2 grams per day, given in 2 equally equally divided doses every 4 hours, with doubled doses for divided doses every 12 hours, to 12 grams per day for severe US 9,675,592 B2 15 16 infections, given in 2, 3 or 4 equally divided doses. Doses up D-cycloserine is administered orally in children and to 16 grams per day have been administered without com adults in doses ranging from 10 to 15 milligrams per plications. kilogram daily, usually 500-750 milligrams for adults. In certain embodiments, methods and compositions con Bacitracin is administered topically to adults every 3-4 cern cefotetan that is administered to adults in doses of 1 to hours for 5-7 days for treatment of superficial infections. It 4 grams per day, in 2 equally divided doses every 12 hours. may also be administered to children and infants via intra Cefotetan may be administered in higher doses for life muscular injection of 900-1000 international units per kilo threatening infections, not to exceed a total dose of 6 grams gram per day, usually in divided doses. per day. Vancomycin is typically administered to adults via intra Cefotaxime is administered in certain embodiments to 10 vascular injection in doses ranging from 30-45 milligrams adults in doses ranging from 1 to 12 grams per day, not to per kilogram per day. This may be divided over 2-4 admin exceed 12 grams per day (2 grams every 4 hours) for istrations daily. It may also be dosed in larger loading doses life-threatening infections. In children, the parenteral dose of of 25-30 milligrams per kilogram. It may further be admin cefotaxime is 50 to 180 mg/kg, divided into 4 to 6 equal istered orally in doses ranging from 500 milligrams up to 2 doses. 15 grams daily. In children it may be administered intravascu In other embodiments, cefoxitin is administered to adults larly in doses ranging from 15-45 milligrams per kilogram in doses ranging from 3 to 12 grams per day, given in 3, 4, per day and orally in doses of 40 milligrams per kilogram or 6 equally divided doses. In children, cefoxitin is admin per day, often with a daily maximum of 2 grams. istered in doses of 80 to 160 mg/kg per day, given in 4 or 6 Daptomycin is administered to adults intravascularly in equally divided doses, not to exceed a total dose of 12 grams doses ranging from 6-8 milligrams per kilogram per day. per day. The overall composition includes each of the antibiotics In additional embodiments, methods and compositions in a therapeutically effective amount, along with a second concern ceftazidime, which is administered to adults in compound that may be referred to as an antibiotic potentia doses ranging from 500 mg per day, given in 2 to 3 equally tor. The specific amount(s) is dependent on the antibiotic and divided doses (every 8 or 12 hours), up to a maximum of 6 25 other compound that is used, the disease or infection to be grams per day. In children, ceftazidime is administered treated, and the number of times of day that the composition intravenously in doses of 30 to 50 mg/kg, to a maximum of is to be administered. 6 grams per day. An antibiotic potentiator refers to a compound or Sub In some cases, ceftizoxime is administered in certain stance that is used in conjunction with an antibiotic to treat embodiments to adults in doses ranging from 1 gram per 30 or inhibit a bacterial infection, Such as a staphylococcal day, given in 2 equally divided doses every 12 hours, to 12 infection. These compounds combine with an antibiotic to grams per day for life-threatening infections, given in 3 increase the inhibitory effect of the antibiotic on the bacteria. equally divided doses every 8 hours. The usual adult dose is Embodiments include an antibiotic potentiator or other 1 to 2 grams every 8 or 12 hours. For children, a parenteral compound that increases the ability of the antibiotic to dose is 50 mg/kg every 6 or 8 hours, for a total daily dose 35 inhibit the growth of bacteria in an in vitro assay, Such as an of 200 mg/kg in Some embodiments. assay disclosed in Example 2 herein, by about, at least about, Ceftriaxone is administered in additional embodiments or at most about the following amounts compared to the parentally to adults in doses ranging from 1 to 2 grams per antibiotic in the absence of the other compound: 0.1, 0.2, day, given in 2 equally divided doses every 12 hours. It may 0.3, 0.4,0.5,0.6,0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, be given in higher doses, not to exceed a total of 4 grams per 40 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, day. In children, the dose of ceftriaxone is 50 to 75 mg/kg 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, per day, not to exceed 2 grams per day. Ceftriaxone may be 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, administered in doses of 100 mg/kg per day, not to exceed 5.9, 6.0, 6.1, 6.2, 6.3, 6.4., 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 4 grams per day. 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, In further embodiments, cefuroxime is administered to 45 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, adults in doses ranging from 2.25 to 4.5 grams per day, in 3 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, equally divided doses every 8 hours. For life-threatening 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 1, infections, 6 grams per day may be administered in 4 equally 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, divided doses every 6 hours, and for meningitis, 9 grams per 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, day may be administered in 3 equally divided doses every 8 50 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, hours. For children, the dose of cefuroxime is 50 to 150 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, mg/kg per day in 3 to 4 equally divided doses, or 240 mg/kg 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, per day. 84, 85, 86, 87, 88, 89,90,91, 92,93, 94, 95, 96, 97,98, 99, Cephalexin is formulated for oral administration, and is 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, Sometimes administered orally to adults in doses ranging 55 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, from 1 to 4 grams per day in 2 to 4 equally divided doses. 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270,275, For children, doses may be 20 to 50 mg/kg per day in 280, 285,290, 295, 300, 305,310,315, 320, 325, 330, 335, divided doses, with doses being doubled for severe infec 340,345, 350, 355, 360, 365,370,375, 380,385, 390,395, tions. 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, Cephalothin is usually administered to adults in doses of 60 490, 500,510,520, 525, 530, 540, 550,560,570, 575,580, 8 to 12 grams per day. 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, Fosfomycin is administered either orally or parenterally. 690, 700, 710, 720, 725,730, 740, 750, 760, 770, 775, 780, In an oral formulation it is given as a single 3-gram dose 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875,880, mixed in 3-4 ounces of water and may be given daily. It may 890, 900, 910, 920, 925,930, 940, 950, 960, 970,975, 980, also be administered intravenously or intramuscularly in 65 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, doses ranging from 2 to 4 grams daily, or up to 16 grams 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, daily in certain cases. 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, US 9,675,592 B2 17 18 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, -continued 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, N- N 10000 times, or any range derivable therein. W O

Embodiments concern a number of compounds that can 5 be used in combination with an antibiotic Such as oxacillin. o-s, O The compounds listed below may be used individually or A. collectively with one or more antibiotics in methods and N compositions provided herein. In other embodiments, the O compounds listed below, or a prodrug or salt thereof, may be 10 used individually or collectively with one or more antibiot ics in methods and compositions provided herein. FO559-O106

15 O OH o-s, H / { O Br OA. ^1 O

25 FOO20-1560 FO559-0107 Nu-( 30 ( | N als N O leN H OMe o-s, ? n1 FO447-O264 / N 35

Y-B 40 FO559-O127

F0447-O273 O

45 Nu-( OMe o-s, / | N als N C leN H in 1-1 50 C F0447-O277

N FO559-0129 55 / l O W NN O or n O O Cl S/ u OMe A. A NN 60 C S O

65 FO559-0036 FO559-0148 US 9,675,592 B2 19 20 -continued -continued MeO NN MeO NNN O

ClO N 5 - H O H O 4. MeO 4. M nN.1 e M n1 O 10

FO559-O158 FO559-O246 15

MeO NN N O MeO N SullO N (lO N 2O H / H O MeO , S N An/ N ~ 25

FO559-0171 FO559-0264

MeO N 30 N O MeO N-- 7 ls MeO O N ClO N O H O MeO A. ( 35 e in 14 ANN O O

FO559-0178 40 FO559-O279

MeO N N O 7 l O ls 45 MeO O N MeO O N O O MeO M 4. e 4. nN.14 O nN.1 50

FO559-0189 FO559-0307 55 MeO NN N O WNNN O ls MeO (lO N MeO O N 60 O O ( MeO (M troM N FO559-0311

US 9,675,592 B2 23 24 -continued -continued NNN O yN O

O CulO N 5 O)-(lO O N 21 S- AO N-N O n / N 10 FO608-0448 FO608-0071

NN NN N O 7 O (l is Cy-(lO O O N O NH H S O 2O C FO608-01.23

FO608-0491 NNN O 25 ls O N /N O 30 ---, Br H O M FO608-0146 S OAYNE, 35 N-N- Y-( O FO608-0543 ls C Qu, O NH N 40 / NN O

C -l N e FO608-0258 S / 45 NS FO608-0554 Nu-( N O O)-(l NNN O O N 50 Cul O N H O

O A 4.n FO608-0410 55 / O NNN O O

O)-(lO FO608-0560NN O N / O MO 60 ---, 2N O2N tyS 65 FO608-0447 FO608-0570 US 9,675,592 B2 25 -continued -continued N N NN O 7 O

(l N 5 O ls N S H i C CF 10 FO608-0665 FO608-0575 N W O NNN ls 15 NH byN C S

co FO608-0668 FO608-0601 2O O O N NN O o-s, O H O A. Sl N H 25 N O n

FO608-0614 FO608-0715 N NN O 30 O O

O )-l H O

A. (a. 35 O loC OO FO608-0659 FO608-0728 N O C 40 O -c.W l N O O O 4. c C 45 M. nNN O FO608-0661 N ( O FO608-0741 N o-s, e 50 | S. W -l O F / leN C FO608-0662 55 F FO608-0895 N NN O N S. / O S.--- (l -s, C leN H

C C FO608-0664 65 FO608-0928 US 9,675,592 B2 27 28 -continued -continued NN fy-g N O O N2 N

Br 10 FO608-0950

NN N O F1142-4597 15 N NN | N-( O C W O N2 N -- Br C C F1374-0014 FO608-1062 NN N O 25 W ls O N e NN N O H C / N N-(i.O le N C 30 F1374-0036 N / -s,NN O FO608-1080 35 H C

CF F1374-0037 40 N NN O C

(l N 21 H 45 C S

F1374-0043

FO608-116.1 NNN 50 W ls O O N H C O 55 ON F1374-0055 N 60 / l O O by C C S 65 F1142-4245 F1374-0058

US 9,675,592 B2 32 MeO -continued -continued e N 7 O WNNN O ls CF -l N MeO O N H OMe O C 5 F1374-0320 F1374-0562 N MeO N NN O / O 10 7 ls O ls N CF H MeO O by OMe 15 C S

F1374-0352 F1374-0583 MeO N W O MeO 7. N O C

O ls N 2O -l N H H

MeO C F1374-0651 25 MeO NN N O F1374-0379 W --- MeO N / O 30 MeO

O ls N O H F1374-0666 N 35 NN O F1374-0396 C (l N e N W l O H MeO O N 40 H F1374-0757 N O 7 NN O

45 C - X

F1374-0836 F1374-0443 /N O 50 Cl MeO O N e / 55 Cell Br Br F1374-0875 F1374-0455 NN NN W N O N-( ls 60 MeO O N H

CF 65 Colo F1374-0495 F1374-0982

US 9,675,592 B2 39 40 -continued -continued MeO N C NNN O ( ls C MeO 5 (l H N C MeO O or F2517-0007 F2518-OOO2 10

MeO N / NN O

MeO 15 Cl O MeO or F2517-0014

MeO NNN F2518-OO24 MeO H C NNN 25 MeO or (l F2517-0037

EtO N NN O 30 F2518-0039 EO (l N H C

EtO 35

C 7 Br F2517-0165 F2518-0041 EtO N 40 NN O C NNN O EO (l C N 45 EtO sco Cl C F2517-0182 F2518-0049

C NNN NNN 50

EO C 55

F2518-0055 C 60

FCO

C 65 F2517-0863 F2518-0085

US 9,675,592 B2 43 44 -continued -continued N NN O NS N O (l (lO --- O or n Y-c H C S C

F2518-0360 F2518-0483 N NN O 10 NS / N O (l N H B -sulN r H 15 C F2518-0373 N NN O / F2518-0484 NN --- X W -lN O F2518-0414 Br i y el N NN O 25 F2518-0504 B Sull N NN r H W N O 30 B -l N r H F25 18-0436 N NN O F2518-0517 35 N B Sull N Br NN O r H

Br

F2518-0447 40 N NN O

Br Sl OMe 45

F2518-0534 OMe F2518-0451 /N O 50 Br - S /N O H X Br O ls N e H / 55 F2518-0558 N C F2518-0474 N C ClO 21 NN O 60 N Cl O (l C

MeO C 65 F2518-1563 F2518-0481 US 9,675,592 B2 45 46 -continued -continued

O N 5 -O- . -- 4. NN H

10 F2645-0405

F2619-0556 N NN O ---, 15 O H SMe 1. F F2645-0012 N - Nals N N H H ( O F2703-0396 -l N 25 C-SMe H N C C S O F2645-OO67 S N YNH O ? O 30 / OEt N 3. -s, H H SMe OEt 35 F27.30-0247 OEt F2645-0099

O 40

SMe / 7 n N 45 F2767-OO12 N NN

F2645-O115 (l N H 50

OMe

F2767-0017 Ry 55 W NN O F2645-0.188 O O 60 c 4.O H / NN

-Co 65 F2767-0026 F2645-0379

US 9,675,592 B2 49 50 -continued -continued N-N N WNNN O \ NH S S Br S O ls N C 1. O y-gN O N

C

F2645-0.188 F5093-0046 10 NS N1\ N O C X-N S N 7 ls Br S O N O N 4.O 15 W nN.1 F1374-0033 O F

F F

C F5093-0101 H NN N O 25 N-s O Br O)-()S O N S F1374-0037 N NX N1 N 30 F5093-0111 C X-N S N O y-Q NN N O S O N le

Br O)-()S O N 21 35 C

O F1374-2739 ci-( MeO 40 F5093-0196 S O NN /}- )-Br NN F2518-0474 leN 45 lN (1) ) X-N S O H ---,H 50 F2518-0327 F5772-8284 C O N N 55 III. Dosages. Formulations, and Modes of l N/ Administration

F5882-3050 A variety of methods and compositions are contemplated C for inhibiting, preventing, or treating a bacterial infection O 60 using one or more antibiotics in combination with an anti biotic potentiator, such as one that inhibits or interferes with C H-- N--2N. expression of the VraSR operon. In particular embodiments, O X V the potentiator is a small molecule compound. Compositions NN O OMe that can be used to inhibit or treat a bacterial infection such 65 as a staphylococcal infection may include one or more of the F5882-3269 following Substances: a first antibiotic, an antibiotic poten tiator, a second antibiotic, a bacterial vaccine, an adjuvant, US 9,675,592 B2 51 52 or a combination thereof. It is contemplated that a solution 850, 860, 870, 875,880, 890, 900, 910, 920, 925,930, 940, or composition may have additional components as well, 950, 960, 970,975, 980, 990, 1000, 1100, 1200, 1300, 1400, which themselves may or may not be active ingredients. In 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, Some embodiments, a composition contains about, at least 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 0.07, 0.08, 0.09, 0.1, 0.2,0.3, 0.4,0.5,0.6, 0.7, 0.8, 0.9, 1.0, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 6000, 7000, 8000, 9000, 10000 milligrams (mg) or micro 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, grams (mcg) or lug/kg or micrograms/kg/minute or mg/kg/ 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, min or micrograms/kg/hour or mg/kg/hour, or any range 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 10 derivable therein, of a compound, such as a first antibiotic, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, an antibiotic potentiator, a second antibiotic, a bacterial 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.19.2, 9.3, 9.4, vaccine, an adjuvant, or a combination thereof. Additionally, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, a Subject may be administered a solution or composition that 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, is described in the previous paragraph. 19.0, 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 A dose may be administered on an as needed basis or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 hours (or 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, any range derivable therein) or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, times per day (or any range derivable therein). A dose may 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, be first administered before or after signs of an infection are 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90,91, 92,93, 94, exhibited or felt by a patient or after a clinician evaluates the 95, 96, 97,98, 99, 100, 105, 110, 115, 120, 125, 130, 135, patient for an infection. In some embodiments, the patient is 140, 145, 150, 155, 160, 165,170, 175, 180, 185, 190, 195, administered a first dose of a regimen 1, 2, 3, 4, 5, 6, 7, 8, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 9, 10, 11, 12 hours (or any range derivable therein) or 1, 2, 260, 265, 270,275, 280, 285,290, 295,300, 305,310,315, 3, 4, or 5 days after the patient experiences or exhibits signs 320, 325, 330, 335, 340, 345,350, 355, 360, 365, 370, 375, 25 or symptoms of an infection (or any range derivable therein). 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, The patient may be treated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 460, 470, 475, 480,490, 500,510,520, 525,530, 540, 550, more days (or any range derivable therein) or until Symp 560,570, 575,580,590, 600, 610, 620,625, 630, 640, 650, toms of an infection have disappeared or been reduced or 660, 670, 675, 680, 690, 700, 710, 720, 725,730, 740, 750, after 6, 12, 18, or 24 hours or 1, 2, 3, 4, or 5 days after 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 30 symptoms of an infection have disappeared or been reduced. 860, 870, 875,880, 890, 900, 910, 920, 925,930, 940, 950, Moreover, a dose of one compound Such as the antibiotic 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, may be delivered prior to, concurrently with, and/or after an 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, antibiotic potentiator. In certain embodiments where the first 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, antibiotic, an antibiotic potentiator, a second antibiotic, a 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 35 bacterial vaccine, and/or an adjuvant are administered with 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, respect to at least one dose separately and/or at different 6000, 7000, 8000, 9000, 10000 milligrams (mg) or micro times to a subject the intervening time between is or is about grams (mcg) or mg/ml or ug/ml of a Substance identified 10, 20, 30, 40, 50, minutes (or any range derivable therein) above, or a combination of Substances or components, or and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours (or any range any range derivable therein. 40 derivable therein) or 1, 2, 3, 4, or 5 days (or any range In certain embodiments, a Subject is administered about, derivable therein). In certain embodiments, an antibiotic is at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, given with or before the antibiotic potentiator, such as about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,0.3, 0.4,0.5,0.6,0.7, 0.8, 0.9, or up to about or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 45 120, 130, 140, 150, 160, 170, or 180 minutes (or any range 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, derivable therein) before the antibiotic potentiator. 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4., 6.5, An antibiotic may be administered for example, by any 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, one of the following routes of administration: parenteral, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.19.2, 9.3, intravenously, topically, intraocular, intranasal, rectal, vagi 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 50 nal, Subcutaneous, intramuscular, arterial, Sublingual, trans 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, mucosal, transdermal, or oral administration. 18.5, 19.0, 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, The antibiotic compositions may be formulated for 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, administration by a variety of routes of administration. For 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, example, the antibiotic product may be formulated in a way 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 55 that is Suitable for topical administration; administration in 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, the eye or the ear; rectal or vaginal administration; as nose 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89,90, 91, 92,93, drops; by inhalation; as an injectable; or for oral adminis 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, tration. In an embodiment, the antibiotic product is formu 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, lated in a manner Such that it is Suitable for oral adminis 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 60 tration. 255, 260,265, 270, 275, 280, 285,290, 295,300, 305,310, For example, in formulating the antibiotic product for 315, 320, 325, 330, 335, 340,345, 350, 355, 360, 365, 370, topical administration, such as by application to the skin, the 375, 380,385, 390, 395, 400, 410, 420, 425, 430, 440, 441, at least two different dosage forms, each of which contains 450, 460, 470, 475, 480,490, 500,510,520, 525, 530, 540, an antibiotic, may be formulated for topical administration 550,560,570, 575,580,590, 600, 610, 620,625, 630, 640, 65 by including Such dosage forms in an oil-in-water emulsion, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725,730, 740, or a water-in-oil emulsion. In Such a formulation, the imme 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, diate release dosage form is in the continuous phase, and the US 9,675,592 B2 53 54 delayed release dosage form is in a discontinuous phase. The breakdown of the component after ingestion or administra formulation may also be produced in a manner for delivery tion. These ingredients can be surfactants, such as sodium of three dosage forms as hereinabove described. For lauryl Sulfate, Sodium monoglycerate, Sorbitan monooleate, example, there may be provided an oil-in-water-in-oil emul Sorbitan monooleate, polyoxyethylene Sorbitan monooleate, Sion, with oil being a continuous phase that contains the glyceryl monostearate, glyceryl monooleate, glyceryl immediate release component, water dispersed in the oil monobutyrate, one of the non-ionic Surfactants such as the containing a first delayed release dosage form, and oil Piuronic line of surfactants, or any other material with dispersed in the water containing a third delayed release Surface active properties, or any combination of the above. dosage form. These materials may be present in the rate of 0.05-15% It is also within the scope of the embodiments to provide 10 an antibiotic product in the form of a patch, which includes (W/W). antibiotic dosage forms having different release profiles, as In certain embodiments there is a delayed release com hereinabove described. ponent. The components in this composition are the same In addition, the antibiotic product may be formulated for immediate release unit, but with additional polymers inte use in the eye or ear or nose, for example, as a liquid 15 grated into the composition, or as coatings over the pellet or emulsion. For example, the dosage form may be coated with granule. a hydrophobic polymer whereby a dosage form is in the oil Materials that can be used to obtain a delay in release phase of the emulsion, and a dosage form may be coated suitable for this component of the embodiment can be, but with hydrophilic polymer, whereby a dosage form is in the are not limited to, polyethylene glycol (PEG) with molecular water phase of the emulsion. weight above 4,000 daltons (Carbowax, Polyox), waxes Furthermore, the antibiotic with different release profiles Such as white wax or bees wax, paraffin, acrylic acid may be formulated for rectal or vaginal administration, as derivatives (Eudragit), propylene glycol, and ethylcellulose. known in the art. This may take the form of a cream or Typically these materials can be present in the range of emulsion, or other dissolvable dosage form similar to those 0.5-25% (W/W) of this component. used for topical administration. 25 As an enteric release composition the components are the As a further embodiment, the antibiotic product may be same as the immediate release component, but with addi formulated for use in inhalation therapy by coating the tional polymers integrated into the composition, or as coat particles and micronizing the particles for inhalation. ings over the pellet or granule. In some embodiments, the antibiotic product is formu The kind of materials useful for this purpose can be, but lated in a manner suitable for oral administration. Thus, for 30 are not limited to, cellulose acetate phthalate, Eudragit L. example, for oral administration, each of the dosage forms and other phthalate salts of cellulose derivatives. These may be used as a pellet or a particle, with a pellet or particle materials can be present in concentrations from 4-20% then being formed into a unitary pharmaceutical product, for (W/W). example, in a capsule, or embedded in a tablet, or Suspended Pharmaceutical Compositions and Kits in a liquid for oral administration. 35 Suitable preparations, e.g., Substantially pure preparations Alternatively, informulating an oral delivery system, each of the agents described herein may be combined with of the dosage forms of the product may be formulated as a pharmaceutically acceptable carriers, diluents, solvents, tablet, with each of the tablets being put into a capsule to excipients, etc., to produce an appropriate pharmaceutical produce a unitary antibiotic product. Thus, for example, composition. The embodiment therefore provides a variety antibiotic products may include a first dosage form in the 40 of pharmaceutically acceptable compositions for adminis form of a tablet that is an immediate release tablet, and may tration to a subject comprising (i) an antibiotic potentiating also include two or more additional tablets, each of which agent; and (ii) a pharmaceutically acceptable carrier or provides for a delayed release of the antibiotic. excipient. The embodiment further provides a pharmaceu The formulation of an antibiotic product including at least tically acceptable composition comprising (i) an antibiotic three dosage forms with different release profiles for differ 45 potentiating agent; (ii) an antibiotic whose activity is poten ent routes of administration is deemed to be within the skill tiated by the compound; and (iii) a pharmaceutically accept of the art from the teachings herein. As known in the art, able carrier or excipient. The embodiment further provides with respect to delayed release, the time of release can be a pharmaceutically acceptable unit dosage form containing controlled by the concentration of antibiotics in the coating a predetermined amount of an antibiotic and a predeter and/or the thickness of the coating. 50 mined amount of an antibiotic potentiating agent, wherein The materials to be added to the antibiotics for the the predetermined amounts are selected so that the antibiotic immediate release component can be, but are not limited to, potentiating agent potentiates the antibiotic when the unit microcrystalline cellulose, corn starch, pregelatinized dosage form is administered to a subject. starch, potato starch, rice starch, Sodium carboxymethyl Further provided are pharmaceutically acceptable com starch, hydroxypropylcellulose, hydroxypropylmethylcellu 55 positions comprising a pharmaceutically acceptable deriva lose, hydroxyethylcellulose, ethylcellulose, chitosan, tive (e.g., a prodrug) of any of the potentiating agents of the hydroxychitosan, hydroxymethylatedchitosan, cross-linked embodiments, by which is meant any non-toxic salt, ester, chitosan, cross-linked hydroxymethyl chitosan, maltodex salt of an ester or other derivative of a potentiating agent, trin, mannitol, Sorbitol, dextrose, maltose, fructose, glucose, upon administration to a recipient, is capable of providing, levulose, sucrose, polyvinylpyrrolidone (PVP), acrylic acid 60 either directly or indirectly, the potentiating agent. A wide derivatives (Carbopol, Eudragit, etc.), polyethylene glycols, variety of appropriate pharmaceutically acceptable salts are such a low molecular weight PEGs (PEG2000-10000) and well known in the art. For example, S. M. Berge et al., high molecular weight PEGs (Polyox) with molecular describe pharmaceutically acceptable salts in detail in J. weights above 20,000 daltons. It may be useful to have these Pharmaceutical Sciences, 66:1, 1977, incorporated herein by materials present in the range of 1.0 to 60% (W/W). 65 reference. Pharmaceutically acceptable salts of the com In addition, it may be useful to have other ingredients in pounds of this embodiment include those derived from this system to aid in the dissolution of the drug, or the Suitable inorganic and organic acids and bases. US 9,675,592 B2 55 56 The term "pharmaceutically acceptable carrier, excipient, lacetic acid, trimethylacetic acid, and the like. Pharmaceu or vehicle' refers to a non-toxic carrier, excipient, or vehicle tically acceptable salts also include base addition salts which that does not destroy the pharmacological activity of the may be formed when acidic protons present are capable of agent with which it is formulated. Pharmaceutically accept reacting with inorganic or organic bases. Acceptable inor able carriers, excipients, or vehicles that may be used in the ganic bases include sodium hydroxide, Sodium carbonate, compositions of this embodiment include, but are not lim potassium hydroxide, aluminum hydroxide and calcium ited to, ion exchangers, alumina, aluminum Stearate, leci hydroxide. Acceptable organic bases include ethanolamine, thin, serum proteins, such as human serum albumin, buffer diethanolamine, triethanolamine, tromethamine, N methyl Substances such as phosphates, glycine, Sorbic acid, potas glucamine and the like. It should be recognized that the sium Sorbate, partial glyceride mixtures of Saturated veg 10 particular anion or cation forming a part of any salt of this etable fatty acids, water, salts or electrolytes, such as invention is not critical, so long as the salt, as a whole, is protamine Sulfate, disodium hydrogen phosphate, potassium pharmacologically acceptable. Additional examples of phar hydrogen phosphate, Sodium chloride, Zinc salts, colloidal maceutically acceptable salts and their methods of prepara silica, magnesium trisilicate, polyvinyl pyrrolidone, cellu tion and use are presented in Handbook of Pharmaceutical lose-based substances, polyethylene glycol, Sodium car 15 Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., boxymethylcellulose, polyacrylates, waxes, polyethylene Verlag Helvetica Chimica Acta, 2002). polyoxypropylene-block polymers, polyethylene glycol and “Prodrug' means a compound that is convertible in vivo wool fat. Solvents, dispersion media, coatings, antibacterial metabolically into an inhibitor according to the present and antifungal agents, isotonic and absorption delaying invention. The prodrug itself may or may not also have agents, and the like, compatible with pharmaceutical admin activity with respect to a given target protein. For example, istration may be included. Supplementary active com a compound comprising a hydroxy group may be adminis pounds, e.g., compounds independently active against the tered as an ester that is converted by hydrolysis in vivo to the disease or clinical condition to be treated, or compounds that hydroxy compound. Suitable esters that may be converted in enhance activity of a compound, can also be incorporated Vivo into hydroxy compounds include acetates, citrates, into the compositions. 25 lactates, phosphates, tartrates, malonates, oxalates, Salicy Pharmaceutically acceptable salts of the agents of this lates, propionates, Succinates, fumarates, maleates, methyl embodiment include those derived from pharmaceutically ene-bis hydroxynaphthoate, gentisates, isethionates, dip acceptable inorganic and organic acids and bases. Examples toluoyltartrates, methane-Sulfonates, ethanesulfonates, ben of Suitable acid salts include acetate, adipate, alginate, Zenesulfonates, p toluenesulfonates, cyclohexyl-Sulfamates, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, 30 quinates, esters of amino acids, and the like. Similarly, a citrate, camphorate, camphorsulfonate, cyclopentanepropi compound comprising an amine group may be administered onate, digluconate, dodecylsulfate, ethanesulfonate, for as an amide that is converted by hydrolysis in vivo to the mate, fumarate, glucoheptanoate, glycerophosphate, glyco amine compound. late, hemisulfate, heptanoate, hexanoate, hydrochloride, A pharmaceutical composition is formulated to be com hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lac 35 patible with its intended route of administration. Solutions tate, maleate, malonate, methanesulfonate, 2-naphthalene or Suspensions used for parenteral (e.g., intravenous), intra Sulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, muscular, intradermal, or subcutaneous application can persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, include the following components: a sterile diluent Such as propionate, salicylate. Succinate, Sulfate, tartrate, thiocya water for injection, Saline solution, fixed oils, polyethylene nate, tosylate and undecanoate. Other acids, Such as oxalic, 40 glycols, glycerine, propylene glycol or other synthetic Sol while not in themselves pharmaceutically acceptable, may vents; antibacterial agents such as benzyl alcohol or methyl be employed in the preparation of salts useful as interme parabens; antioxidants such as ascorbic acid or sodium diates in obtaining the compounds of the embodiment and bisulfite; chelating agents such as ethylenediaminetet their pharmaceutically acceptable acid addition salts. raacetic acid; buffers such as acetates, citrates or phosphates “Pharmaceutically acceptable salts' means salts of com 45 and agents for the adjustment of tonicity Such as Sodium pounds of the present invention which are pharmaceutically chloride or dextrose. pH can be adjusted with acids or bases, acceptable, as defined above, and which possess the desired Such as hydrochloric acid or sodium hydroxide. The paren pharmacological activity. Such salts include acid addition teral preparation can be enclosed in ampoules, disposable salts formed with inorganic acids Such as hydrochloric acid, Syringes or multiple dose vials made of glass or plastic. hydrobromic acid, Sulfuric acid, nitric acid, phosphoric acid, 50 Pharmaceutical compositions suitable for injectable use and the like; or with organic acids Such as 1.2 ethanedisul typically include sterile aqueous Solutions (where water fonic acid, 2 hydroxyethanesulfonic acid, 2 naphthalenesul soluble) or dispersions and sterile powders for the extem fonic acid, 3 phenylpropionic acid, 4.4 methylenebis(3 poraneous preparation of sterile injectable solutions or dis hydroxy 2 ene-1 carboxylic acid), 4 methylbicyclo[2.2.2]oct persion. For intravenous administration, Suitable carriers 2 ene-1 carboxylic acid, acetic acid, aliphatic mono- and 55 include physiological Saline, bacteriostatic water, Cremo dicarboxylic acids, aliphatic Sulfuric acids, aromatic Sulfuric phor ELTM (BASF, Parsippany, N.J.), phosphate buffered acids, benzenesulfonic acid, benzoic acid, camphorsulfonic saline (PBS), or Ringer's solution. acid, carbonic acid, cinnamic acid, citric acid, cyclopentane Sterile, fixed oils are conventionally employed as a sol propionic acid, ethanesulfonic acid, fumaric acid, glucohep vent or Suspending medium. For this purpose, any bland tonic acid, gluconic acid, glutamic acid, glycolic acid, 60 fixed oil may be employed including synthetic mono- or heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic di-glycerides. Fatty acids, such as oleic acid and its glycer acid, laurylsulfuric acid, maleic acid, malic acid, malonic ide derivatives are useful in the preparation of injectables, as acid, mandelic acid, methanesulfonic acid, muconic acid, o are natural pharmaceutically-acceptable oils, such as olive (4 hydroxybenzoyl)benzoic acid, oxalic acid, p-chloroben oil or castor oil, especially in their polyoxyethylated ver Zenesulfonic acid, phenyl-Substituted alkanoic acids, propi 65 sions. These oil solutions or Suspensions may also contain a onic acid, p-toluenesulfonic acid, pyruvic acid, Salicylic long-chain alcohol diluent or dispersant, such as carboxym acid, Stearic acid, Succinic acid, tartaric acid, tertiarybuty ethyl cellulose or similar dispersing agents that are com US 9,675,592 B2 57 58 monly used in the formulation of pharmaceutically accept For administration by inhalation, the inventive composi able dosage forms including emulsions and Suspensions. tions are preferably delivered in the form of an aerosol spray Other commonly used surfactants, such as Tweens, Spans from a pressured container or dispenser which contains a and other emulsifying agents or bioavailability enhancers Suitable propellant, e.g., a gas such as carbon dioxide, or a which are commonly used in the manufacture of pharma nebulizer. Liquid or dry aerosol (e.g., dry powders, large ceutically acceptable Solid, liquid, or other dosage forms porous particles, etc.) can be used. The present embodiment may also be used for the purposes of formulation. also contemplates delivery of compositions using a nasal In all cases, the composition should be sterile, if possible, Spray. and should be fluid to the extent that easy syringability For topical applications, the pharmaceutically acceptable 10 compositions may be formulated in a Suitable ointment exists. containing the active component Suspended or dissolved in Pharmaceutical formulations are stable under the condi one or more carriers. Carriers for topical administration of tions of manufacture and storage and must be preserved the compounds of this embodiment include, but are not against the contaminating action of microorganisms such as limited to, mineral oil, liquid petrolatum, white petrolatum, bacteria and fungi. In general, the relevant carrier can be a 15 propylene glycol, polyoxyethylene, polyoxypropylene com Solvent or dispersion medium containing, for example, pound, emulsifying wax and water. Alternatively, the phar water, ethanol, polyol (for example, glycerol, propylene maceutically acceptable compositions can be formulated in glycol, and liquid polyetheylene glycol, and the like), and a Suitable lotion or cream containing the active components suitable mixtures thereof. The proper fluidity can be main Suspended or dissolved in one or more pharmaceutically tained, for example, by the use of a coating Such as lecithin, acceptable carriers. Suitable carriers include, but are not by the maintenance of the required particle size in the case limited to, mineral oil, Sorbitan monostearate, polysorbate of dispersion and by the use of surfactants. Prevention of the 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, action of microorganisms can be achieved by various anti benzyl alcohol and water. bacterial and antifungal agents, for example, parabens, chlo For local delivery to the eye, the pharmaceutically accept robutanol, phenol, ascorbic acid, thimerosal, and the like. In 25 able compositions may be formulated as micronized Sus many cases, it will be preferable to include isotonic agents, pensions in isotonic, pH adjusted Sterile Saline, or, prefer for example, Sugars, polyalcohols such as manitol, Sorbitol ably, as Solutions in isotonic, pH adjusted Sterile Saline, or Sodium chloride in the composition. Prolonged absorption either with or without a preservative such as benzylalkonium of injectable compositions can be brought about by includ chloride. Alternatively, for ophthalmic uses, the pharmaceu ing in the composition an agent which delays absorption, for 30 tically acceptable compositions may be formulated in an example, aluminum monostearate and gelatin. Prolonged ointment such as petrolatum. absorption of oral compositions can be achieved by various A therapeutically effective amount of a pharmaceutical means including encapsulation. composition typically ranges from about 0.001 to 100 mg/kg Sterile injectable solutions can be prepared by incorpo body weight, preferably about 0.01 to 25 mg/kg body rating the active compound in the required amount in an 35 weight, more preferably about 0.1 to 20 mg/kg body weight, appropriate solvent with one or a combination of ingredients and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, enumerated above, as required, followed by filtered steril 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The ization. Preferably solutions for injection are free of endo pharmaceutical composition can be administered at various toxin. Generally, dispersions are prepared by incorporating intervals and over different periods of time as required, e.g., the active compound into a sterile vehicle which contains a 40 multiple times per day, daily, every other day, once a week basic dispersion medium and the required other ingredients for between about 1 to 10 weeks, between 2 to 8 weeks, from those enumerated above. In the case of sterile powders between about 3 to 7 weeks, about 4, 5, or 6 weeks, etc. The for the preparation of sterile injectable solutions, the meth skilled artisan will appreciate that certain factors can influ ods of preparation are vacuum drying and freeze-drying ence the dosage and timing required to effectively treat a which yields a powder of the active ingredient plus any 45 subject, including but not limited to the severity of the additional desired ingredient from a previously sterile-fil disease or disorder, previous treatments, the general health tered solution thereof. and/or age of the Subject, and other diseases present. Gen Oral compositions generally include an inert diluent or an erally, treatment of a Subject with an inventive composition edible carrier. For the purpose of oral therapeutic adminis can include a single treatment or, in many cases, can include tration, the active compound can be incorporated with 50 a series of treatments. It will be appreciated that a range of excipients and used in the form of tablets, troches, or different dosage combinations (i.e., doses of the antibiotic capsules, e.g., gelatin capsules. Oral compositions can also and antibiotic potentiating agent) can be used. be prepared using a fluid carrier for use as a mouthwash. Exemplary doses include milligram or microgram Pharmaceutically compatible binding agents, and/or adju amounts of the inventive compounds per kilogram of subject vant materials can be included as part of the composition. 55 or sample weight (e.g., about 1 microgram per kilogram to The tablets, pills, capsules, troches and the like can contain about 500 milligrams per kilogram, about 100 micrograms any of the following ingredients, or compounds of a similar per kilogram to about 5 milligrams per kilogram, or about 1 nature: a binder Such as microcrystalline cellulose, gum microgram per kilogram to about 50 micrograms per kilo tragacanth or gelatin; an excipient such as starch or lactose, gram.) For local administration (e.g., intranasal), doses a disintegrating agent such as alginic acid, Primogel, or corn 60 much smaller than these may be used. It is furthermore starch; a lubricant such as magnesium Stearate or Sterotes; a understood that appropriate doses depend upon the potency glidant such as colloidal silicon dioxide; a Sweetening agent of the agent, and may optionally be tailored to the particular Such as Sucrose or saccharin; or a flavoring agent Such as recipient, for example, through administration of increasing peppermint, methyl salicylate, or orange flavoring. Formu doses until a preselected desired response is achieved. It is lations for oral delivery may advantageously incorporate 65 understood that the specific dose level for any particular agents to improve stability within the gastrointestinal tract Subject may depend upon a variety of factors including the and/or to enhance absorption. activity of the specific compound employed, the age, body US 9,675,592 B2 59 60 weight, general health, gender, and diet of the Subject, the IV. Examples time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of The following examples are included to demonstrate expression or activity to be modulated. embodiments of the invention. It should be appreciated by The presented embodiments also include pharmaceutical those of skill in the art that the techniques disclosed in the packs or kits comprising one or more containers (e.g., vials, examples which follow represent techniques discovered by ampoules, test tubes, flasks, or bottles) containing one or the inventor to function well in the practice of the invention, more ingredients of the inventive pharmaceutical composi and thus can be considered to constitute preferred modes for tions, for example, allowing for the simultaneous or sequen its practice. However, those of skill in the art should, in light tial administration of the antibiotic potentiating agent and 10 of the present disclosure, appreciate that many changes can antibiotic agent(s) it potentiates. Optionally associated with be made in the specific embodiments which are disclosed Such container(s) can be a notice in the form prescribed by and still obtain a like or similar result without departing from a governmental agency regulating the manufacture, use or the spirit and scope of the invention. sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for 15 Example 1—Materials and Methods human administration. Different ingredients may be Sup plied in Solid (e.g., lyophilized) or liquid form. Each ingre Test Strain Construction— dient will generally be suitable as aliquoted in its respective The test strain (923) is a USA300 MRSA clinical isolate container or provided in a concentrated form. Kits may also for which the dependence of Mc resistance on VraSR has include media for the reconstitution of lyophilized ingredi been well characterized (1, 2). To produce a reporter for Vra ents. The individual containers of the kit are preferably operon transcription, the Vra promoter (P) was PCR maintained in close confinement for commercial sale. amplified from strain 923 using primers P-F (5'-aaa Examples of pharmaceutically acceptable, nontoxic acid gaattctgaagg tatgg tattagctattg-3'(SEQ ID NO: 1)) and (P- addition salts are salts of an amino group formed with R) (5'-aaaggatccgttgatgtcgatgatatgtttg-3'(SEQ ID NO: 2)) inorganic acids such as hydrochloric acid, hydrobromic acid, 25 containing EcoRI and BamHI restriction sites (underlined) phosphoric acid, Sulfuric acid and perchloric acid or with and inserted into pXen-1 (Caliper Life Sciences) in the organic acids such as acetic acid, oxalic acid, maleic acid, compatible restriction sites present upstream of the luxAB tartaric acid, citric acid, Succinic acid or malonic acid or by CDE operon (lux) from Photorhabdus luminescens. This using other methods used in the art such as ion exchange. gene cluster (lux) produces all the components required for Other pharmaceutically acceptable salts include adipate, 30 luminescence obviating the need for exogenous Substrate alginate, ascorbate, aspartate, benzenesulfonate, benzoate, (3). The ligation was transformed into E coli strain TOP10 bisulfate, borate, butyrate, camphorate, camphorsulfonate, (Invitrogen) and colonies were selected on ampicillin (100 citrate, cyclopentanepropionate, digluconate, dodecylsul ug/ml). The resulting plasmid was isolated and called fate, ethanesulfonate, formate, fumarate, glucoheptonate, pPvra::lux. With this construct, the P, drives expression of glycerophosphate, gluconate, hemisulfate, heptanoate, 35 the lux gene cluster. pP::lux was transformed into the hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lacto restriction negative intermediate Staphylococcus aureus bionate, lactate, laurate, lauryl Sulfate, malate, maleate, host strain, RN4220 (Kreiswirth) and was then isolated from malonate, methanesulfonate, 2-naphthalenesulfonate, nico RN4220 and transformed into MRSA strain 923 (923 pP tinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, lux). The Vra promoter and lux genes were confirmed as persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, 40 wild-type by Sanger sequence analysis. Strains containing propionate, Stearate. Succinate, Sulfate, tartrate, thiocyanate, pP-lux were cultured in the presence of chloramphenicol p-toluenesulfonate, undecanoate, Valerate salts, and the like. (Cm) to maintain the plasmid. Examples of pharmaceutically acceptable salts derived Validation of Test Strain 923 pP::lux— from appropriate bases include alkali metal, alkaline earth The optimal concentration of oxacillin for Vra induction metal, ammonium and N--(C1-4 alkyl)4 salts. 45 and luminescence was chosen by preliminary testing of a Representative pharmaceutically acceptable alkali or range of oxacillin concentrations (FIG. 1). Briefly, a satu alkaline earth metal salts include Sodium, lithium, potas rated overnight culture of the test strain (923 pP-lux) in sium, calcium, magnesium, and the like. TSB (containing 10 ug/ml chloramphenicol for plasmid Further pharmaceutically acceptable salts include, when selection) was diluted 1:100 into fresh TSB containing 0 to appropriate, nontoxic ammonium, quaternary ammonium, 50 6 g/mL of oxacillin. A 100LL cell plus oxacillin Suspension and amine cations, for example formed using counterions was applied to each well of a white plastic clear-bottomed Such as halide, hydroxide, carboxylate, Sulfate, phosphate, 96-well plate (Costar) and the plate was incubated at 37° C. nitrate, lower alkyl sulfonate and aryl sulfonate. Absorbance at 600 nm and luminescence were monitored Physiologically acceptable carrier or excipient: As used every 5 minutes for 24 hours using a microplate reader herein, the term "physiologically acceptable carrier or 55 (Fluostar Optima, BMG). excipient” refers to a carrier medium or excipient which Small-Molecule Screening does not interfere with the effectiveness of the biological The Life Chemicals Library was used for the small activity of the active ingredients and which is not exces molecule library. The Life Chemicals Library used in the sively toxic to the host at the concentrations at which it is screen contains approximately 25,000 drugs with history of administered. The term includes solvents, dispersion media, 60 use in humans, with the greatest possible degree of "drug coatings, antibacterial and antifungal agents, isotonic agents, likeliness”. It was supplied in 384 well format in 10 mM absorption delaying agents, and the like. The use of Such concentration in DMSO and was re-plated to a 96 well media and agents for the formulation of pharmaceutically daughter plates to a 1 mM concentration. This antimicrobial active Substances is well-known in the art (see, for example, focused chemical library consisting of 25,000 compounds “Remington’s Pharmaceutical Sciences'. E. W. Martin, 18th 65 with drug-like properties was built specifically by the inven Ed., 1990, Mack Publishing Co.: Easton, Pa., which is tors for high-throughput Screening against antimicrobial incorporated herein by reference in its entirety). drug targets. To this end, 18,750 compounds were selected US 9,675,592 B2 61 62 from the antibacterial and antimicrobial Life Chemicals Relative Quantification was performed using the AACT (LC) libraries. The parent LC libraries consist of 40,000 method facilitated by using the ABI Prism 7300 Sequence antibacterial (Life Chemicals Activity targeted: antibacteri Detection Software (version 1.2.3) (4) (Applied Biosys als library) and 27,000 antiviral (Life Chemicals Activity tems). Strain 923 grown in the absence of oxacillin was used targeted: antiviral library) compounds focused by 2D fin as the comparator condition unless the oxacillin induced gerprint methodology to have high similarity to known condition is indicated (5). antibacterial and antiviral compounds taken from current literature. The remaining 6,250 compounds of the screened Example 2-Assay Strategy 25,000 compounds were selected from the general LC screening collection (Life Chemicals General: diversity 10 Assay Development for Screening of Inhibitors of Growth library). The LC libraries have been pre-filtered to be Rule and VraSR Expression— of 5’ compliant (one exception tolerated). Additionally, a By coupling the VraSR promoter to the luxABCDE series of custom pre-filters, based in part on published data, operon, we were able to simultaneously monitor cell growth were applied to each of the LC libraries prior to compound as a function of optical density (OD) and PvraSR expression selection to remove compounds from consideration with 15 known reactive functionalities and/or known toxicities. as a function of luminescence (lux) (measured in relative Molecular weight filters specified a range of 150-650 Dal light units (LU)). Prior to screening the Life Chemicals tons. The higher MW cut-off of 650 was used to reflect the compound library for inhibitors of growth and/or lumines known trend of increased weight and hydrophilicity amongst cence, the optimal concentration of oxacillin and time points known antimicrobials. Finally, the most diverse compounds for collection of ODoo and LUs, and criteria for classifying from each starting library that met the filtering criteria were a compound. To this end, cells were cultured in 96 well selected based upon 2D fingerprints. microtiter plates, and growth and luminescence monitored in A saturated overnight culture of the test strain (923 a plate reader. pPvra-lux) in TSB (containing 10 g/ml chloramphenicol When using CLSI guidelines, strain 923 has a 24-h for plasmid selection) was diluted 1:100 into fresh TSB. A 25 oxacillin MIC of 16-32 g/mL (Boyle-Vavra et al). How 99 LL cell Suspension and 1 LL test compound (final ever, when cultured in the 96-well format, the MIC of concentration 10 uM dissolved in DMSO) was applied to a oxacillin of strain 923 was 8 Lug/mL, and in 923 containing 96-well plate and incubated at 37°C. The OD at 530 nm and pPvra-lux, the MIC of oxacillin further dropped to 5 g/mL luminescence were recorded at intervals of 0, 1, 2, 3, 4, and (FIG. 1A). Based on the MIC of oxacillin obtained in the 24 hours. All compounds were tested in the presence and 30 96-well format, we measured the OD600 and LU of cultures absence of oxacillin (2 ug/mL). For each 96-well plate of strain 923 Pvra-lux supplemented with oxacillin in con assayed, 80 compounds were tested. The remaining 16 wells centrations spanning 0 to 6 mg/L (FIG. 1). We found that contained either cells in TSB alone (7 replicates), cells with VraSR expression reached its maximum 2 hours after induc oxacillin (2 ug/mL, 7 replicates), or blank TSB (2 repli tion (FIG. 1B), and 2 g/mL oxacillin was the minimum cates). Due to instrument limitations, only half of the library 35 was screened each day (7 plates, 560 compounds). The full concentration sufficient for maximal VraSR expression (FIG. library was screened twice. Compounds identified in the 1C). Therefore, the library was screened using 2 ug/ml primary screen that satisfied the criteria described in oxacillin, luminescence was the recorded at 2 hours, and Example 2 were Subjected to secondary screening. ODoo recorded at 24 hrs. In the secondary screen each microtiter plate contained 40 To define the LU and ODoo cutoff values for defining either cells in TSB alone (16 replicates), cells with oxacillin inhibition of luminescence and growth, respectively, data (2 ug/mL, 31 replicates), or cells with test compound and was obtained from 49 wells seeded with cell culture supple oxacillin (2 g/mL, 49 wells). Each compound was assayed mented with 2 g/ml oxacillin and 49 control wells seeded in four separate biological replicates. with cultures lacking oxacillin. The values were partitioned Confirmation of VraR Inhibition Via qRT-PCR 45 into bins for ease of visualization. The data were normally A saturated overnight culture of strain 923 was diluted distributed for both LU (FIG. 2A) and OD600 values (FIG. 1:100 into fresh TSB and grown at 37° C. with shaking. 2B). Cutoff values were set at 2 SDs above or below the After one hour, test compound (10 uM) and oxacillin (2 Ca. ug/mL) were added and the culture was incubated for an Values were classified as either VraSR-inactive, (RLUs additional hour. Bacteria were lysed via lysostaphin incu 50 less than two SDs above the mean of the no-oxacillin bation (Ambi, 200 ug/mL) for 10 minutes at 25° C. RNA controls (FIG. 2A, white bars), VraSR-active (any value less was purified with the Qiagen RNeasy kit, including treat than two SDS below the mean LU of the oxacillin-induced ment with DNase and cDNA generated using the high wells (FIG. 2A, black filled bars), or VraSR-uncertain (any capacity cDNA Archive Kit (Applied Biosystems). qRT value which falls between the other two categories). PCR was performed using molecular beacons and primers 55 obtained from IDT. Specifically, a multiplex reaction con For judging cell growth, cultures were classified as either sisting of a vraR probe (PrimeTime Probe 75' 6-FAM/TTG “uninhibited or “inhibited. “Uninhibited was defined as CCA AAG/ZENACCC ATG AGT TGA AGC CA/3'IAB any value less than 2 SDs below the mean of the no-oxacillin kFQ?) (SEQ ID NO:3) with primers vraR-R (5'-TAG TTG cultures (white bars), while “inhibited' wells had any value GTG AAG GCGCTT CTG GTA-3) (SEQ ID NO: 4) and 60 <2 SDS below the mean of the no oxacillin cultures (black vraR-F (5-TCG TCG CTT CTA CAC CAT CCA TGT-3') bars, bins 4-8)(FIG. 2B). (SEQID NO: 5) to detect Vra operon transcription combined Primary Screen with a gyrB endogenous control probe (75Cy5/AAA TGG The Life Chemicals Library was screened twice for its GAC GTC CAG CTG TCG AAGTT/3IAbRQSp/) (SEQID effects on growth and VraSR induction of the test strain, both NO: 6) with primers (gyrBR 5'-CCG CCAAATTTACCA 65 in the presence and absence of oxacillin. CCA GCA TGT-3') (SEQ ID NO: 7) and (gyrBF 5'-AAC Relative Growth is defined by the following values and GGA CGT GGT ATC CCA GTT GAT-3') (SEQ ID NO:8). calculations:

US 9,675,592 B2 67 68 TABLE 3 TABLE 3-continued Count of Compound Count of Compound family family

Row Labels Total Row Labels Total

FOO15 F6153 1 FOO20 F9995 3 FO191 FO230 Grand Total 28O FO266 10 FO3O1 FO382 Lead hits were additionally filtered using the following FO401 FO415 filtering strategy. A REOS filter (rapid elimination of Swill) FO447 was applied. This is similar to the “rule of 5’ filter, with FO559 15 property filters for MW. hydrogen bonds, charge, etc. It also FO593 FO600 applies additional functional group filters. The result of FO608 32 applying this filter was elimination of 30 compounds. A FO700 PAINS (Pan Assay Interference) filter was applied. This FO737 filter detects hits that come up in lots of high-throughput FO743 F1009 assays and eliminated 6 compounds. A filter to eliminate F1013 possible false positives based on chemotype (e.g. OXadiaz F1059 oles are known to interfere with firefly luciferase) was F1094 applied. This filter eliminated approximately 220 com F1118 F1142 pounds. The result of filter application was a “Top 40 F1374 25 library (Table 4). F1605 F1813 TABLE 4 F1821 F1886 UIC Top 40 library F2090 F2273 30 Sample maximum F2289 # for non-GI RG RL wraSR F23O8 Plate COC Ox Ox repressing F2359 ull ID reader (g/mL) avg. avg syn rep synergist F2368 F3068-0037 15 400 -0.01 0.11 x x 1. confirme F2399 35 F2399-0063 8 400 -0.01 0.04 x x 1. confirme F2438 F2619-0SS6 11 400 0.01 0.28 x x 1. confirme F2473 F3348-OO3O 17 400 0.01 0.05 x x 1. confirme F2483 F2359-O3O8 34 400 0.01 0.01 x x 1. confirme F2517 F2703-0396 12 400 0.01 0.34 x x 1. confirme F2518 F3348-OS33 18 400 0.02 0.10 x x 1. confirme FS882-3 OSO 27 400 0.02 0.07 x x 1. confirme F2537 40 F2619 F2645-O188 36 40 0.03 0.05 x x 1. confirme F2645 F2962-0176 38 400 0.04 0.01 x x 1. confirme F2647 F1142-4597 30 40 0.08 -0.01 x x 1. confirme F2653 F273O-O247 13 400 0.23 0.15 x x 1. confirme F2703 F1142-4245 5 400 0.25 0.04 x x 1. confirme F2090-0579 7 400 0.30 0.11 x x 1. confirme F2708 45 F2730 F5772-8284 40 400 0.37 0.52 x 2 2. possible FO447-O273 28 40 0.39 0.08 x x 1. confirme F2767 F3348-0667 19 40 0.51 0.72 x 2 2. possible F2768 F1374-0082 6 40 0.67 0.08 2 x 2. possible F2813 FOO2O-1560 2 40 0.68 0.17 2 x 2. possible F2832 F1813-0710 31 400 0.69 0.20 2 x 2. possible F2962 50 F1813-0712 33 400 0.71 0.20 2 x 2. possible F3068 F1813-0711 32 400 0.85 0.43 2 x 2. possible F3225 FOO15-0758 1 400 O.92 O.25 x 3. no effec F3305 F2S17-0863 10 4 O.92 118 3. no effec F3348 FO600-1305 29 400 O.94 O15 x 3. no effec F3394 F2438-OOO2 9 40 O.95 0.62 3. no effec F5074 55 FO608-0146 3 40 O.96 1.11 3. no effec F5079 3 F3348-0373 39 40 O.99 O.80 3. no effec F5093 F5833-1551 25 40 O.99 O.S6 3. no effec FS123 F1118-0378 4 40 1.OO O.65 3. no effec F5237 FS669-0378 24 40 1.OO O.71 3. no effec FS854-2599 26 40 1.O1 O.54 3. no effec F5562 F3305-O3S4 16 40 1.O1 O.61 3. no effec F5669 60 F2813-0883 14 40 1.O3 1.28 3. no effec F5772 FSS62-0130 23 40 1.O3 O.S7 3. no effec F5773 F2708-0138 37 40 1.04 O.69 3. no effec F5833 FS123-0084 21 40 1.08 O.67 3. no effec F5854 F5237-0056 22 40 1.10 O.72 3. no effec F5882 F2S18-0213 35 4 1.14 O.33 x 3. no effec FS946 65 F3394-0078 2O 40 1.24 0.41 x 3. no effec FS948 US 9,675,592 B2 69 70 Synergy Confirmation of “Top 40'. TABLE 5-continued To determine if the compounds found in the oxacillin (Ox) synergy screen also inhibited growth on their own, com Top 40 leads after secondary screen pounds were tested alone at the “Screen” concentration, and at 1:10 and 1:100 dilutions to find the maximum concen- 5 PR Sample trat1On Whereh it did11 NOT inhibiinni it growthh (i.e.,(i Subinhibitory)Subinhibi # fromlibrary full Full ID # concentration(Lig/mL) (Table 5). Inhibition of luminescence by lead compounds was also tested to determine if it was artifactual due to 9 F2438-OOO2 4 interference with lux rather than expression of Pvra, which 10 F2S17-0663 4 would have given a false positive in the screen. To this end 10 14 F2613-0883 4 the ability- of the compound to inhibit------light - production from 1619 F33OS-O3S4F3348-0667 4 a transcriptional fusion between lux and the promoter from 2O F3394-0078 4 the constitutively expressed pyruvate dehydrogenase gene 21 FS123-OO84 4 (Ppdh-lux) in S. aureus (RN4220) was tested. 22 compounds 22 F5237-0056 4 synergized with Ox and genuinely inhibited Pvra lumines- 15 23 F5562-0130 4 cence including 2 of the 5 oxadiazoles tested (Table 6). 2524 F5833-1551FS669-0378 4 Using RT-PCR 12 compounds were confirmed that repress 26 FS854-2599 4 VraTSR expression (Table 6, marked by x). (- indicates a 28 FO447-O273 4 lack of repression by RT-PCR). 30 F1142-4597 4 2O 35 F2S18-0213 4 36 F2645-O188 4 TABLE 5 37 F2708-0138 4 Top 40 leads after secondary screen 39 F3348-0373 4 PR Sample # from full concentration 25 library Full ID # (ig/mL) TABLE 6

2 FOO2O-1560 40 conc Inh RT lit 3 FO608-0146 40 full ID (ig/mL) rep 1 4 F1118-0378 40 6 F1374-0062 40 30 F1374-0082 40 X 9 F2438-OOO2 40 F2399-0063 400 X 10 F2S17-0663 40 F2619-0SS6 400 X 14 F2613-0883 40 F2703-0396 400 X 16 F33OS-O3S4 40 F273O-O247 400 X 19 F3348-0667 40 F3348-0667 40 X 2O F3394-0078 40 35 FS882-3OSO 400 X 21 FS123-0084 40 F1813-0710 400 X 22 F5237-0056 40 F1813-0711 400 X 23 FSS62-0130 40 F1813-0712 400 X 24 FS669-0378 40 F2645-O188 40 X 25 F5833-1551 40 F2962-0176 400 X 26 FS854-2599 40 FOO2O-1560 40 28 FO447-O273 40 40 F1142-4245 400 30 F1142-4597 40 F2090-0579 400 35 F2S18-O213 40 F3068-0037 400 36 F2645-O188 40 F3348-0O3O 400 37 F27O6-O138 40 F3348-OS33 400 39 F3348-0373 40 FO447-O273 40 2 FOO2O-1560 4 45 F1142-4597 40 3 FO608-0146 4 F2359-O3O8 400 4 F1118-0378 4 F5772-8284 400 6 F1374-0062 4

TABLE 7 MICs (minimal inhibitory concentrations) vs multiple strains of MRSA Ox Ox MIC (mg/L) in presence of compound

MIC F2645-0188 FOO2O-1560 F2703-0396 FS882-3OSO F1813-0712 F2619-0SS6 Isolate ID SCCmec ST alone (0.25 mg/L) (1 mg/L) (2 mg/L) (2 mg/L) (4 mg/L) (4 mg/L)

923 IV 8 16 2 1 <0.25 <0.25 4 <0.25 1176 II 231. 32

MIC F2645-0188 FOO2O-1560 F2703-0396 FS882-3OSO F1813-0712 F2619-0SS6 Isolate ID SCCmec ST alone (0.25 mg/L) (1 mg/L) (2 mg/L) (2 mg/L) (4 mg/L) (4 mg/L) 8227-01 IV 87 2 O.S O.25 <0.06 <0.06 O.S <0.06 Q2461 VT 59 8 O.25 4 <0.13 O.S 16 1 ST, indicates the genetic background of the MRSA strain. SCCmec (staphylococcal cassette chromosome mec; coding for resistance to the antibiotic methicillin), designates the SCCmec type, OXMIC alone is the Ox MIC of 923 in the absence of inhibitor,

Synergy Testing— 15 on last channel as another control. All compounds to be Checkerboard MIC testing. The interactions of F2645 analyzed were initially prepared as 10 mM stock solutions in 0188, F0020-1560, F2703-0396, F5882-3050, F1813-0712 100% DMSO. Compound solutions with a series of increas and F2619-0556 were assessed for synergy by checkerboard ing concentrations (0-100 uM at 1.5-fold dilution) were MIC testing using each test compound (0.25 to 16 ug/mL) applied to six parallel channels at a 30 L/min flow rate at and oxacillin (2 to 32 g/mL) in 2-fold serial dilutions room temperature. Sensorgrams were analyzed using the according to the method in CLSI MO7-A8. oxacillin and Proteon ManagerTM software, and response unit difference compounds were prepared to 4 times their final concentra (ARU) Values at each concentration were measured during tion in TSB containing 2% NaCl. 250 uL of each concen the equilibration phase. Data were double referenced with tration of oxacillin and each concentration of compound 25 both blank and interspot RU values. The ProteCon Man were applied to a 48-well plate to create a 6x8 checkerboard. A concentration series of oxacillin alone and compound agerTM software was used to fit the data to a single rectan alone were included so that the MIC of oxacillin and test gular hyperbolic curve to determine dissociation equilibrium compound alone could be determined. All wells were inocu constant (K) values. The hyperbolic function, y y, x/ lated with 500 uL of a 1x10" cfu/mL suspension of strain 30 (K+X), was used to plot response units and corresponding 923 in TSB containing 2% NaCl. Plates were incubated concentration, where y is the response, y, is the maximum overnight at 37° C. and MICs were visually determined at 24 response and X is the compound concentration. The fitting h (Table 8). curve of compound F2645-0188 is depicted in FIG. 4. 35 As shown in Table 9, compound F2645-0188 bound untagged VraS-C protein with a K, of 13.2+3.2 uM and Example 3—Binding to VraS and/or VraR His-tagged VraS-C with a K, of 56.1+16.3 uM. This indi Compound F2645-0188 Binds to the Cytoplasmic cates that F2645-0188 may potentiate oxacillin by directly Domain of VraS (“VraS-C) binding to VraS protein and inhibiting its function. F2645 The cytoplasmic domain of VraS (VraS-C) used in SPR 40 0188 has a central core structure consisting of the oxadiaz was obtained by PCR from USA300 strain 923 using ole-carboxamide, that was used to analyze the primary and primers: 5'-AAAGGATCCAAAATCAATCAG secondary screening data to evaluate the types of R and R CAAAATGA-3' (SEQ ID NO: 9) and 5'-AAAAAGCT substituents that affected synergistic activity. The results of TATCGTCATACGAATCCTCCT-3 (SEQ ID NO: 10). This 45 that analysis are shown in FIG. 5, with substituents shown clone was expressed in E. coli strain BL21 (Star, pIysS) in section A (top) acting to increase activity of the resulting (Invitrogen) (Ebook 333). VraR was cloned at BamHI and compound, Substituents shown in section C (bottom) gen HindIII sites in pet28a (Novagen) with its own stop codon. erally acting to decrease activity of the resulting compound, This is a kanamycin selection vector. VraR was PCR ampli 50 and Substituents shown in section B (middle) producing no fied from strain 275 using primers: 5'-AAAGGATCC apparent increase or decrease in activity. The full Life ATGACGATAAAGTATGTTTG-3' (SEQ ID NO: 11) and Chemicals library was then searched for analogs with sub 5'-AAAAAGCTT CGATACGAACTATTGA-3' (SEQ ID stituents that appeared likely to enhance synergistic activity. NO: 12). This clone was expressed in E. coli strain BL21 Twenty analogs were selected, as shown in Table 9, and were (DE3, pIysS) (Invitrogen) (Ebook 331). The expression and 55 purification is depicted in FIG. 3. tested for their ability to bind VraS-C or VraR. His-tagged VraS, VraR, and an unrelated protein (PurC As shown in Table 9, compounds F1374-0033, F1374 from Bacillus anthracis) were prepared in assay buffer 0037, F1374-2739, and F25 18-0474 bound to VraS-C, and containing 10 mM phosphate, pH 7.4, 2.7 mM KC1, 137 mM compounds F1374-2739 and F2518-0327 bound to VraR. NaCl, 0.5% Tween-20, and 1 mM TCEP. Each protein was 60 Like compound F2645-0188, the 1,3,4-oxadiazoles that bind immobilized on vertical channels of a HTE sensor chip VraS, VraR, or both may also potentiate oxacillin and other (Bio-Rad) using ProteCnTM XPR36 instrument (Bio-Rad). antibiotics that interfere with cell wall synthesis, including Of six vertical flow channels, VraS was immobilized on but not limited to Vancomycin, daptomycin, and teichopla channels 1 and 2, and VraR was immobilized on channels 3 65 nin. Table 9 also shows that pyrvinium, a compound shown and 4. An unrelated protein, PurG, was immobilized on the to potentiate oxacillin and Vancomycin in co-pending appli 5" channel as a control, and blank immobilization was done cation PCT/US2013/068085, also binds to VraS. US 9,675,592 B2 73 74 TABLE 9

Binding of 1,3,4-oxadiazoles, clomiphene, pyrvinium, and gossypol to VraS or VraR.

KI (M) (RUmax)

PrC Structure ID MW VraS Virar (control)

F2645 368.4 Previous run NT (Not NT O188 with untagged tested) VraS 13.23.2 (325 + 33)

New run NB NB with His (No tagged VraS Binding) 56.1 16.3 (74.1 + 10.7)

FO608 376.4 NB NB NB 0570

387.4 NB NB NB H-() O NRN O

356.8 19.6 4.4 NB NB (64.3 + 5.4) g O)-(O N C NO)-\, NN \

323.7 25.8 4.8 NB NB (45.1 + 3.2) F

C H

N-s O

391.8 NB NB

H O>

C

US 9,675,592 B2

TABLE 9-continued

Binding of 1,3,4-oxadiazoles, clomiphene, pvrvinium, and gosswpol to VraS or VraR. K (IM) RUmax

PrC Structure ID MW VraS Virar (control) Clomiphene 406.0 NB NB NB

Pyrvinium 382.5 21.3 : 36 NB NB (46.5 + 2.9)

Gossypol 518.6 NB NB NB

All of the methods disclosed and claimed herein can be REFERENCES made and executed without undue experimentation in light The following references, to the extent that they provide of the present disclosure. While the compositions and meth- 5 O exemplary procedural or other details Supplementary to ods of this invention have been described in terms of those set forth herein, are specifically incorporated herein by preferred embodiments, it will be apparent to those of skill reference. in the art that variations may be applied to the methods and Berge, et al., J Pharmaceutical Sci. 66:1, 1977. 55 Boyle-Vavra, et al., Antimicrob Agents Chemother: 57(1): in the steps or in the sequence of steps of the method 83-95, 2013. described herein without departing from the concept, spirit Boyle-Vavra, et al., FEMS Microbiol Lett. 262(2):163-171, and scope of the invention. More specifically, it will be 2006. apparent that certain agents which are both chemically and Francis, et al., Infect Inmmun. 68(6):3594-3600, 2000. physiologically related may be substituted for the agents 60 Jo, et al., Antimicrob Agents Chemother. 55:2818-23, 2011. described herein while the same or similar results would be Livak & Schmittgen, Methods. 25(4):402-408, 2001. Martin, “Remington's Pharmaceutical Sciences’, 18th Ed., achieved. All such similar substitutes and modifications Mack Publishing Co.: Pa., 1990. apparent to those skilled in the art are deemed to be within Montgomery, et al., J Infect Dis. 198(4):561-570, 2008. the spirit, scope and concept of the invention as defined by Yin, et al., Antimicrobial Agents Chemother: 50(1):336-343, the appended claims. 2006. US 9,675,592 B2 81 82

SEQUENCE LISTING

< 16 Os NUMBER OF SEO ID NOS : 12

SEQ ID NO 1 LENGTH: 32 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer SEQUENCE: 1 aaagaattct galagg tatgg tattagct at td 32

SEQ ID NO 2 LENGTH: 31 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer SEQUENCE: 2 aaaggat.ccg ttgatgtcga tigatatgttt g 31

SEQ ID NO 3 LENGTH: 26 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer FEATURE: NAME/KEY: 6-FAM LOCATION: (1) ... (1) FEATURE: NAME/KEY: ZEN LOCATION: (9) ... (9) FEATURE: NAME/KEY: 3 'IABkFO LOCATION: (26).. (26)

< 4 OOs SEQUENCE: 3 ttgccaaagc C catgagttg aagcca 26

SEQ ID NO 4 LENGTH: 24 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer

SEQUENCE: 4 tagttggtga aggcgcttct ggta 24

SEO ID NO 5 LENGTH: 24 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer SEQUENCE: 5 tcqtcqctitc tacaccatcc atgt 24

SEQ ID NO 6 LENGTH: 26 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer FEATURE: US 9,675,592 B2 83 84 - Continued <221> NAME/KEY: Cy5 <222s. LOCATION: (1) . . (1) 22 Os. FEATURE: <221> NAME/KEY: 3 IAbRQSp <222s. LOCATION: (26) ... (26) <223> OTHER INFORMATION: 3' Iowa Black RQ-Sp <4 OOs, SEQUENCE: 6 aaatgggacg tccagctgtc. gaagtt 26

<210s, SEQ ID NO 7 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer

<4 OO > SEQUENCE: 7 cc.gc.caaatt taccaccago atgt 24

<210s, SEQ ID NO 8 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer

<4 OOs, SEQUENCE: 8 aacggacgtg gitatcc cagt tat 24

<210s, SEQ ID NO 9 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer

<4 OOs, SEQUENCE: 9 aaaggat.cca aaatcaatca gcaaaatga 29

<210s, SEQ ID NO 10 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer

<4 OOs, SEQUENCE: 10 aaaaagctta t cqt catacg aatcct cot 29

<210s, SEQ ID NO 11 &211s LENGTH: 31 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer

<4 OOs, SEQUENCE: 11 aaaggat.cca tacgattaa agt attgttt g 31

<210s, SEQ ID NO 12 &211s LENGTH: 25

US 9,675,592 B2 87 88 an antibiotic potentiator having the structure: an antibiotic potentiator having the structure:

ON F 10 F 1.NN

C N N N H H 15

O an antibiotic potentiator having the structure:

O an antibiotic potentiator having the structure: N 7 S O

25 H

30 O an antibiotic potentiator having the structure:

35

O an antibiotic potentiator having the structure: 40 HO N

O Cl, 45 C O) O-

() (O. O 50 an antibiotic potentiator having the formula: O an antibiotic potentiator having the structure:

55

R3 O N N N 60 \ 1. N S V H O wherein R' may be N-(4-(phenylamino)-N'-(phenyl)acet F amide)amine, N-3-(N',N'-dimethylamine)propylamine, 65 N-(4-N'-phenylacetamide)amine, or hydroxyl, and R may be tert-butyl, isopropyl, or phenyl, and R may be hydrogen O or chloro, or US 9,675,592 B2 89 90 an antibiotic potentiator having the structure: 10. The method of claim 1, wherein treating a staphylo coccal infection comprises reducing abscess formation or incidence or reducing bacterial load in the Subject. 11. The method of claim 1, further comprising adminis f Y tering a second antibiotic or a staphylococcal vaccine; wherein the second antibiotic is gentamicin or rifampin. 12. The method of claim 1, wherein the antibiotic is administered at a dose of 0.1 mg/kg to 50 mg/kg and/or the antibiotic potentiator is administered at a dose of 0.1 mg/kg 10 to 100 mg/kg. CCJ) 13. The method of claim 12, wherein the subject is a pediatric patient. 14. The method of claim 13, wherein the pediatric patient is administered 25 mg/kg to 50 mg/kg of oxacillin intrave an antibiotic potentiator having the formula: 15 nously or intramuscularly every 6 to 12 hours or 12.5 mg/kg of oxacillin orally every 6 hours. 15. The method of claim 1, whereby administration to a C Subject is oral, Sublingual, Sublabial, gastrointestinal, rectal, epicutaneous (topical), intradermal, Subcutaneous, nasal, C c na N lO N1 R, intravenous, intraarterial, intramuscular, intracardiac, H H intraosseous, intrathecal, intraperitoneal, intravesical, intra vitreal, intracavernous, intravaginal, intrauterine, epidural, wherein R may be 1H-indazol-6-yl, or 3-(4-methoxyphe intracerebral and/or intracerebroventricular, enteral, parent noxy)methyloxadiazol-5-yl-methyl, or eral, onto the skin, inhalation, an enema, eye drops, ear 25 drops, absorption across mucosal membranes, the mouth, a an antibiotic potentiator having the formula: gastric feeding tube, a duodenal feeding tube, a Suppository, an injection into a vein, an injection into an artery, an injection into the bone marrow, an injection into muscle tissue, an injection into the brain, an injection into the 30 cerebral ventricular system or an injection under the skin. 16. The method of claim 1, wherein the antibiotic and the antibiotic potentiator are administered in the same compo sition or wherein the antibiotic and the antibiotic potentiator are administered simultaneously. 35 17. The method of claim 1, wherein the antibiotic is administered up to 24 hours prior to administration of the antibiotic potentiator; wherein the antibiotic potentiator is administered up to 24 hours prior to administration of the 40 antibiotic; or wherein the antibiotic and antibiotic potentia wherein, R' and R may each be separately hydrogen, tor are administered within 24 hours of each other. methyl, chloro or methylthio, or a prodrug or comparable 18. A pharmaceutical composition comprising an antibi salt thereof. otic and a compound selected from the group consisting of 2. The method of claim 1, wherein the antibiotic is a penicillinase-resistant beta-lactam antibiotic. 3. The method of claim 2, wherein the penicillinase 45 NS resistant beta-lactam antibiotic is oxacillin, methicillin, naf cillin, cloxacillin, dicloxacillin or flucloxacillin. R-(lO N R, 4. The method of claim 1, wherein the subject has been H tested for a staphylococcus infection, is diagnosed with a staphylococcus infection, is at risk of acquiring a staphyllo 50 coccus infection, or has one or more symptoms of a staphy wherein R' may be -(4-chlorophenoxy)methyl, 1-(thiophen lococcal infection. 2-yl)cyclopentyl, 1.2.3,4-tetrahydronaphthalen-6-yl, 1-iso 5. The method of claim 1, wherein the staphylococcus propyl-1H-pyrazol-5-yl, 2-(thiophen-2-yl)guinolin-4-yl, infection is Staphylococcus aureus or methicillin resistant 2,3-dihyrdobenzob.14 dioxin-6-y1, 2,4-dichlorophenyl, Staphylococcus aureus (MRSA). 55 2,5-dichlorophenyl, 2,5-dichlorothiophen-3-yl, 2,6-difluo 6. The method of claim 1, wherein the subject has or is at rophenyl, 2-bromothiophen-5-yl, 2-chlorothiophen-5-yl, risk for native valve endocarditis or prosthetic valve endo 2-naphthyl 2-phenoxyphenyl, 2-phenylduinolin-4-yl, carditis. 2-tolyl, 3-(2-chlorophenyl)-5-methylisoxazol-4-yl, 3,4,5- 7. The method of claim 6, wherein the subject is admin trimethoxyphenyl, 3,5-dichlorophenyl, 3,5-dimethoxyphe istered 2-3 g of oxacillin intravenously every 4 to 6 hours. 60 nyl, 3-bromophenyl, 3-chlorobenzobthiophen-2-yl, 8. The method of claim 1, wherein the subject has or is at 3-chlorophenyl, 3-fluorophenyl, 3-methoxyphenyl, 3-n-bu risk for joint infection, meningitis, osteomyelitis, pneumo toxyphenyl, 3-phenoxyphenyl, 3-tolyl, 3-trifluoromethyl nia, septicemia, sinusitis, or skin or soft tissue infection. phenyl, 4-(2,6-dimethylmorpholinosulfonyl)phenyl, 4-(2- 9. The method of claim 5, wherein the subject is admin ethylpiperidin-1-ylsulfonyl)phenyl, 4-(2-methylpiperidin-1- istered 1-2 g of oxacillin intravenously or intramuscularly 65 ylsulfonyl)phenyl, 4-(2-phenylduinoline), 4-(3,4- every 4 to 6 hours or 500 mg to 1 g of oxacillin orally every dihydroisoquinolin-2(1H)-ylsulfonyl)phenyl, 4-(3,4- 4 to 6 hours. dihydroquinolin-1 (2H)-ylsulfonyl)phenyl, 4-(3.5-

US 9,675,592 B2 95 96 -continued OH O 5 a y Br, \- --

CO 10 \, H / is O F N O | N2 leN N R, 15 N F wherein R may be 2-phenoxyphenyl, 2-bromothiophen-5-yl, F 1. or 2,5-dichlorophenyl: NN

2O C N l Nals N H H R N 7 S O O als 25 N N A N

30 N W HO N

35 Cl,

Cl 40 R2 N21 R3 45 2 N R, I eN

50 wherein R' may be N-(4-(phenylamino)-N'-(phenyl)acet amide)amine, N-3-(N',N'-dimethylamine)propylamine, N-(4-N'-phenylacetamide)amine, or hydroxyl, and R may be tert-butyl, isopropyl, or phenyl, and R may be hydrogen Na ND 55 or chloro, ls 2

O 60 leN O- C-CO Oll-IC US 9,675,592 B2 97 98 -continued C

C DO N lsO N1 5 H H wherein R may be 1H-indazol-6-yl, or 3-(4-methoxyphe noxy)methyloxadiazol-5-yl-methyl, and 10

R2 % O 15 o?2N. YNH o i

N I (R', 2O wherein, R' and R may each be separately hydrogen, methyl, chloro or methylthio, or a prodrug or comparable 25 salt thereof.