|HAO WANATHI MOVIEUS009943500B2 PLENA MATUMA WA MT (12 ) United States Patent ( 10 ) Patent No. : US 9 ,943 , 500 B2 Page (45 ) Date of Patent: Apr . 17 , 2018 ( 54 ) METHODS OF TREATING TOPICAL A61K 9 /0046 ; A61K 9 / 06 ; A61K 47 /10 ; MICROBIAL INFECTIONS A61K 47 / 14 ; A61K 47 / 44 ; A61K 9 /0048 ; A61K 47/ 06 ; A61L 15 / 46 ; A61L ( 71 ) Applicant: LUODA PHARMA PTY LIMITED , 2300 /404 ; A61L 26 /0066 Caringbah ( AU ) See application file for complete search history . (72 ) Inventor : Stephen Page , Newtown ( AU ) ( 56 ) References Cited (73 ) Assignee : Luoda Pharma Pty Ltd , Caringbah U . S . PATENT DOCUMENTS (AU ) 3 ,873 , 693 A 3 / 1975 Meyers et al . 3 , 920 ,847 A * 11/ 1975 Chalaust .. .. A61K 9 /0014 Subject to any disclaimer, the term of this 514 / 512 ( * ) Notice : 4 ,772 , 470 A * 9 / 1988 Inoue .. .. .. .. .. A61K 9 / 006 patent is extended or adjusted under 35 424 / 435 U . S . C . 154 ( b ) by 0 days . 2005/ 0187199 Al * 8 /2005 Peyman .. .. .. A61K 8 / 36 514 / 154 ( 21) Appl. No .: 14 / 766 , 232 FOREIGN PATENT DOCUMENTS ( 22 ) PCT Filed : FebD . 10 , 2014 EP 0294538 A2 12 / 1988 WO WO - 2003/ 088965 A1 10 / 2003 ( 86 ) PCT No . : PCT/ AU2014 /000101 WO WO - 2006 /081327 A2 8 /2006 $ 371 ( c ) ( 1 ) , WO WO - 2008 /075207 A2 6 / 2008 ( 2 ) Date : Aug. 6 , 2015 OTHER PUBLICATIONS (87 ) PCT Pub . No .: W02014 / 121342 Weese et. al. , Veterinary Microbiology , 2010 , Elsevier, vol. 140 , pp . PCT Pub . Date : Aug . 14 , 2014 418 - 429 . * Brindle , Encyclopedia of Chemical Technology . Polyether antibi otics, Nov . 2013 , Wiley , Abstract and pp . 1 - 30 . * (65 ) Prior Publication Data Huczynski et. al ., Journal of Molecular Structure , 2008 , Elsevier , US 2016 / 0000748 A1 Jan . 7 , 2016 vol. 891 , pp . 481 -490 . * Chen et . al. , Journal of Medicinal Chemistry , 2001, American Foreign Application Priority Data Chemical Society , vol. 44 , pp . 2374 - 2377 . * ( 30 ) Dykhuizen , Antonie van Leeuwenhoek , 1998 , Kluwer Academic Publishers , vol. 73 , pp . 25 - 33. * Feb. 8 , 2013 (AU ) .. .. 2013900412 Foroumadi et . al ., European Journal ofMedicinal Chemistry , 2003 , Elsevier, vol. 38 , pp . 851 - 854 . * (51 ) Int. Ci. Lu -Lu et . al ., Infection and Immunity , 1992, American Society for A61K 31/ 352 (2006 .01 ) Microbiology , vol. 60 ( 9 ) , pp . 3807 - 3813 . * A61K 31 / 35 ( 2006 . 01 ) Akiyama et al ., Staphylococcus aureus infection on experimental A61K 31 /351 ( 2006 .01 ) croton oil - inflamed skin in mice . Staphylococcus aureus infection A61K 31 / 7048 ( 2006 . 01 ) on experimental croton oil - inflamed skin in mice . J . Dermatol . Sci . 8 ( 1 ) : 1 - 10 ( 1994 ) . A61K 9 / 06 ( 2006 . 01) Gilbert et al ., The 10x ' 20 Initiative : pursuing a global commitment A61K 9 / 00 ( 2006 . 01 ) to develop 10 new antibacterial drugs by 2020 . Clin . Infect . Dis. A61K 45 /06 ( 2006 .01 ) 50 ( 8 ) : 1081 - 3 (2010 ) . A61K 47 /06 ( 2006 .01 ) Guo et al. , In vivo bioluminescence imaging to evaluate systemic A61K 47 / 10 ( 2017 .01 ) and topical antibiotics against community -acquired methicillin resistant Staphylococcus aureus - infected skin wounds in mice . A61K 47 / 14 ( 2017 .01 ) Antimicrob . Agents Chemother . 57 ( 2 ) : 855 -63 ( 2013) . A61K 47 /44 ( 2017 .01 ) International Search Report and Written Opinion of the Interna A61L 15 / 46 ( 2006 .01 ) tional Searching Authority in connection with International Appli A61L 26 / 00 ( 2006 .01 ) cation No . PCT/ AU2014 /00101 , United States Patent Office , dated (52 ) U .S . CI. Feb . 10 , 2014 . CPC . .. .. .. .. A61K 31 /352 ( 2013 .01 ) ; A61K 9 / 0014 (Continued ) ( 2013 .01 ) ; A61K 9 /0017 ( 2013 .01 ) ; A61K 9 /0043 (2013 .01 ) ; A61K 9 /0046 (2013 .01 ) ; Primary Examiner — Sarah Pihonak A61K 9 / 0048 ( 2013 .01 ) ; A61K 9 / 06 ( 2013 .01 ) ; ( 74 ) Attorney , Agent, or Firm — Marshall , Gerstein & A61K 31/ 35 ( 2013 .01 ) ; A61K 31 /351 Borun LLP ( 2013 .01 ) ; A61K 31 / 7048 ( 2013 .01 ) ; A61K 45 / 06 ( 2013 .01 ) ; A61K 47 / 06 ( 2013 .01 ) ; A61K (57 ) ABSTRACT 47 / 10 ( 2013 .01 ) ; A61K 47 / 14 (2013 . 01 ) ; A6IK The invention is a method and composition for treating a 47 /44 (2013 .01 ) ; A61L 15 / 46 ( 2013 .01 ) ; A61L topical microbial infection in a subject . The method includes 26 /0066 ( 2013 .01 ) ; A6IL 2300/ 404 ( 2013 .01 ) the step of administering a therapeutically effective amount ( 58 ) Field of Classification Search of a polyether ionophore , or a therapeutically acceptable salt CPC .. .. A61K 31 /35 ; A61K 31 /351 ; A61K 31/ 352 ; thereof , to the subject . A61K 31 /7048 ; A61K 45 /06 ; A61K 9 / 0014 ; A61K 9 /0017 ; A61K 9 /0043 ; 12 Claims, 27 Drawing Sheets US 9 ,943 ,500 B2 Page 2 ( 56 ) References Cited OTHER PUBLICATIONS Kugelberg et al ., Establishment of a superficial skin infection model in mice by using Staphylococcus aureus and Streptococcus pyogenes . Antimicrob. Agents Chemother . 49 ( 8 ) : 3435 -41 (2005 ) . Liu , Microbial aspects of polyethers antibiotics: Activity, produc tion , and biosynthesis . Polyether Antibiotics Naturally Occurring Acid Ionophores vol. 1 : Biology . J . W . Westley , New York . Marcel Dekker Inc . 43 - 102 ( 1982 ) . McRipley et al. , Characterization and quantification of experimental surgical- wound infections used to evaluate topical antibacterial agents . Antimicrob . Agents Chemother . 10 ( 1 ) : 38 -44 ( 1976 ) . Naujokat et al . , Salinomycin as a drug for targeting human cancer stem cells . J . Biomed . Biotechnol . 2012 : 950658 ( 2012 ) . Stieritz et al. , A burned mouse model to evaluate anti -pseudomonas activity of topical agents. J . Antimicrob . Chemother . 9 ( 2 ) : 133 -40 ( 1982 ) . * cited by examiner atent Apr . 17 , 2018 Sheet 1 of 27 US 9 , 943 , 500 B2 Strain ID # Source Species Resistance Profile ATCC 49775 Human S . aureus MSS 1 Cow S . aureus MSS 2 Cat S . intermedius MSS 3 Cat S . aureus MSS 4 Unknown S . pseudintermedius MSS 5 Dog S . intermedius MSS 6 Human S . pseudintermedius MSS 7 Dog S . pseudintermedius P MSS 8 Dog Coagulase negative P , Te MSS 9 Dog S . intermedius MSS 10 Dog | S . intermedius MSS 11 Unknown Coagulase negative P , Te MSS 12 Dog S . pseudintermedius P MSS 13 Bird Coagulase negative P , Te MSS 14 Dog S . intermedius P MSS 15 Dog S . intermedius P , Te MSS 16 Pig Coagulase negative P | MSS 17 Pig S . aureus CI, E MSS 18 Dog S . intermedius P MSS 19 Dog Coagulase negative E MSS 20 Dog S . pseudintermedius CI, P , Te MRSA 1 Human S . aureus Enr , E , Gn , O , P . MRSA 2 Human S . aureus Gn , O , P , Tm MRSA 3 Human S . aureus E , Enr, O , P MRSA 4 . Human S . aureus E , Enr, O , P MRSA 5 Human | S . aureus E , Enr, Gn , O , P , Te MRSA 6 Human S . aureus E , Enr, O , P , Tm MRSA 7 Human S . aureus E , Enr, Gn , O , P , Tm MRSA 8 Human S . aureus E , Gn , O , P , Te , Tm MRSA 9 Human S . aureus E , O , P MRSA 10 Human S . aureus E , Enr, O , P MRSA 11 Human S . aureus 0 , P MRSA 12 Human S . aureus E , O , P , Te MRSA 13 Human S . aureus E , O , P MRSA 14 Human S . aureus E , O , P MRSA 15 Human S . aureus E , O , P MRSA 16 Human S . aureus O , P , Te FIGURE 1A atent Apr . 17 , 2018 Sheet 2 of 27 US 9 , 943 , 500 B2 Strain ID # Source Species | Resistance Profile MRSA 17 Human S . aureus E , Enr, Gn , O , P , Tm MRSA 18 Human S . aureus 10 , P , Tm MRSA 19 Human S . aureus E , O , P , Te , Tm MRSA 20 Human S . aureus Enr , O , P MR -CNS 1 Coagulase negative Enr, Gn , O , P , Tm Cl= clindamycin , Enr = enrofloxacin , E = erythromycin , Gn = gentamicin , O = oxacillin , P = penicillin G , Te = tetracycline , TmHLE = trimethoprim - sulfamethoxazole FIGURE 1B atent Apr . 17 , 2018 Sheet 3 of 27 US 9 , 943 , 500 B2 Negative Positive Control Control 0 , 25 871 ug ml JUIN stbinmenthow Tahan W 1 2 3 4 5 6 1 8 9 10 11 12 * * #in Simpurit" tek *WWW* tinit market *hátttt sehan * *** "EV guretat i NINY ETUSIN 4HFR* in Instrument Today! EX ini ih takich *** pulärtan 11 WHO it ta 20 1584 Nyhet! kelerinde * . 7 Herk heledag Widjeti Desting YERA *** * line * Test Cepounds * *** * *** .TUBE FD000000*** *** * Wir ora * * * *** " *** * WWWWWWW*** een Wow! ooooooooooooih * * * * * * * ** * * ** * * * * * * * * * * * * * * ** * * * * * * * * WWWWWWWWWWWWWWWWWW FIGURE 2 U. S . Patentatent Apr . 1717, , 2018 Sheet 4 of 27 US 9 , 943 , 500 B2 VC??? 128 ug /mL , Www *** arriver * *** w ti Figure 3 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ?????????????????????????????????????????????????????????????????????????? AYY Minimum Inhibitory concentrations . Meteo Sensitive (PusatiuogenudauogkjarayurXTILEIRA * sta Meto grains / / / / WW pics LA1068 124525 26315 LA9666 LTJESAntimicrobial Agent women who typiwwii w wwww Figure 4 atent Apr . 17 , 2018 Sheet 5 of 27 US 9 , 943 , 500 B2 Compound Ampicillin LP 1088 LP 1369 LP 4525 LP 6315 LP 9666 MIC50 0 0 . 5 2 MICMICO , 16 * . MC 0 . 25 - 32 0 . 5 - 4 0 . 5 - 2 0 , 25 - 1 2 - 8 2 – 128 Range Figure 5 Compound Ampicillin LP 1088 LP 1369 LP 1525 LP 6315 LP 9666 MIC50 128 1 * 05 32 NIC -> 128 1 6 MC 8 - 128 1 - 7 0 . 5 - 2 0 . 25 22 - 16 , 128 Range Figure 6 atent Apr . 17 , 2018 Sheet 6 of 27 US 9, 943, 500 B2 Minimum Bactericidal Concentration * MinimumBactericidalConcentrationwg/ml) Veiticillin -- Sensive straitis * Methicillin - - - çesistant cr. strains * LA1088 LP1369 LP4525 LP6315 LP9856 { { { { }{ Ex???à3???????? ?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Figure 7 Compound LP 1088 LP 1369 LP 4525 LP 6315 LP 9666 { BC5k > >128 64 > 128 >> 128 > 128 MBC90 >> 128 > 128 > 128 > 128 > 128 MBC Range .4 - > 128 4 - > mat128 64 - > 128 64 - > 128 128 - > 128 Figure 8 atent Apr . 17 , 2018 Sheet 7 of 27 US 9 , 943 , 500 B2 Compound LP 1369 LP 4525 LP 6315 LP 9666 MBC30 > 128 > 128 > 128 DULODOU 128128 > 128 MBC > 128 > 128 >> 128 >> 128 >> 128 MBC Range 128 – > 128 64 - > 128 4 - > 128 4 - > 128 128 – > 128 Figure 9 Microdilution Time- Kill Assay (Strain : ATCC 49775) .