THETWO TONTTITULUUS 20170326160A1 MATULUNUL DINTRE ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2017/ 0326160 A1 DELAVENNE et al . (43 ) Pub . Date : Nov . 16 , 2017 ( 54 ) ANTIBACTERIAL USE OF HALOGENATED ( 30 ) Foreign Application Priority Data SALICYLANILIDES Sep. 12 , 2014 (SE ) ...... 1451054 - 9 (71 ) Applicant: ANTIBIOTX APS , Horsholm (DK ) Publication Classification (72 ) Inventors : Emilie Flora Aurore DELAVENNE , Le Mans (FR ) ; Daniel Jean Jacques (51 ) Int. CI. SIMON , Søborg (DK ) ; Morten Otto A61K 31 /609 (2006 . 01) Alexander SOMMER , Virum (DK ) ; (52 ) U .S . CI. Rasmus Vendler TOFT- KEHLER , CPC ...... A61K 31/ 609 (2013 . 01 ) Kobenhavn Ø (DK ) (57 ) ABSTRACT The invention relates to a halogenated salicylanilide selected (21 ) Appl. No. : 15 /662 , 691 from closantel, rafoxanide , oxyclozanide and niclosamide and derivatives thereof including salts , hydrates, esters and (22 ) Filed : Jul. 28 , 2017 the like for use in the topical treatment or prevention of infections caused by Gram - positive bacteria such as Staphy lococcus, in particular Staphylococcus aureus, and Strepto Related U .S . Application Data coccus, in particular Streptococcus pyogenes . Gram positive (63 ) Continuation of application No. 15/ 510 , 304 , filed on bacteria treated with the halogenated salicylanilides exhibit Mar . 10 , 2017 , filed as application No . PCT/ EP2015 / a very low frequency of appearance of resistant mutants 070495 on Sep . 8 , 2015 . compared to commonly used topical antibiotics. Patent Application Publication Nov . 16 , 2017 Sheet 1 of 5 US 2017 /0326160 A1

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ANTIBACTERIAL USE OF HALOGENATED More controversial is the use of topical fusidic acid in the SALICYLANILIDES treatment of cutaneous and soft tissue infections . [0007 ] Increasing fusidic acid resistance in Staphylococ CROSS -REFERENCES TO RELATED cus aureus might be important for three reasons . First, it APPLICATIONS might mean that systemic fusidic acid can no longer be used [0001 ] This application is a continuation of U . S . applica in situations where it is clinically indicated . Second , failure tion Ser. No. 15 /510 ,304 , filed Mar. 10 , 2017, the National of topical treatmentmay occur, especially in primary care Stage Entry of PCT Application No . PCT /EP2015 / 070495 , settings where treatment is often empiric . Third , resistance filed Sep . 8 , 2015 , which claims foreign priority to Sweden to fusidic acid might be linked to other antibiotic resistances , Application No . 1451054 - 9 , filed Sep . 12, 2014 , each of therefore favoring spread of multiply antibiotic resistant, which is incorporated by reference herein in its entirety for Staphylococcus aureus such as MRSA (methicillin -resistant all purposes . S aureus ) . 10008 ] Mupirocin is a topical antibiotic used to treat superficial skin infections and to control the spread of FIELD OF INVENTION methicillin -resistant Staphylococcus aureus (MRSA ) .Mupi [0002 ] The invention relates to the halogenated salicy rocin resistance was observed shortly after it became avail lanilides closantel, rafoxanide , oxyclozanide or niclosamide able . Prevalence of mupirocin resistance among MRSA and derivatives thereof including salts , hydrates, esters for isolates has been described mostly in hospitalized adult and use in the topical treatment or prevention of conditions elderly patients with wide variability, ranging from 0 to 65 % caused by Gram positive bacteria such as Staphylococcus , in of isolates . Rates of resistance have been shown to correlate particular Staphylococcus aureus , and Streptococcus, in with increased use in closed inpatient settings . Very restric particular Streptococcus pyogenes . The halogenated salicy tive mupirocin prescriptions for local treatment are now lanilides have a surprisingly low frequency of appearance of recommended . resistant mutants in said strains compared to commonly used [0009 ] Impetigo is a highly contagious bacterial infection topical antibiotics . Also provided are topical formulations of the superficial layers of the epidermis . Impetigo is one of comprising a halogenated salicylanilide the most common skin diseases among children , accounting for about 10 % of skin diseases treated in US paediatric BACKGROUND OF THE INVENTION clinics. The bacteria typically considered to be responsible [0003 ] Today, there are many different classes of antibi are Staphylococcus aureus and Streptococcus pyogenes , and otics available to medical practitioners . However, many of often a combination of the two . Impetigo is usually trans the bacteria that these antibiotics are designed to treat are mitted by direct contact but fomites also play an important quickly developing resistance to drugs in all classes. Unfor role . Methicillin - resistant Staphylococcus aureus (MRSA ) is tunately the large pharmaceutical companies have limited being found with increasing frequency as a causative bac there research in finding new antibiotics . Several bacteria teria of impetigo . Impetigo has three common clinical vari have become particularly prominent in recent years and have eties : impetigo contagiosa ( common impetigo ) , bullous garnered substantial media attention , especially methicillin impetigo , and ecthyma. Features of all three types of impe resistant Staphylococcus aureus (“ MRSA ” ) and Streptococ tigo , however , may coexist in any individual patient. cus pyogenes. MRSA infections on the skin and in the [0010 ] A number of topical compositions containing phar airways are common in hospital settings and the broad maceutically active ingredients are known for the treatment spectrum resistance of the bacteria to almost all classes of of impetigo . Topical mupirocin 2 % (Bactroban ointment and antibiotics ( e . g . methicillin , fusidic acid and mupirocin ) cream ) is a treatment option , as are older treatments , such as make it particularly difficult to combat. MRSA infections in topical gentian violet and vioform . For many patients , patients who have not recently been in hospital ( so called mupirocin is a viable treatment option for MRSA , however, community acquired MRSA infections) are becoming resistance of bacteria to mupirocin has been widely reported . increasingly frequent. [0011 ] Topical fusidic acid 2 % (Fucidin cream ) is used for [0004 ] The problem of antibiotic resistance is an increas treatment of impetigo , and is thought to be equally as ing problem , but a truly comprehensive solution to the effective as mupirocin . However, the utility of fusidic acid problem has yet to emerge . A major problem in the battle for treatment of impetigo is limited by the problem of against antibiotic resistance is the lack of novel antibiotics to resistance development, as discussed above . replace those that no longer effectively treat organisms due [0012 ] Fusidic acid -resistant Staphylococcus aureus to resistance development. New treatments are urgently ( FRSA ) has been identified as a causative bacteria in out needed to address this issue of drug resistance . breaks of impetigo and its emergence has been associated 10005 ) Fusidic acid is an antibiotic derived from Fusidium with increased use of topical fusidic acid . Accordingly , coccineum that has been used for over 35 years to treat utility of fusidic acid as first - line agent for the treatment of infections with Staphylococcus aureus . in particular , fusidic impetigo is questionable due to current resistance levels in acid is prescribed for skin infections caused by Staphylo the target bacteria . Retapamulin 1 % ( Altabax ointment ) , coccus aureus . Such infections include impetigo , angular recently approved by the FDA , is a drug in the new class of cheilitis ( an infection around the mouth ) , and infected der pleuromutilin antibiotics for the topical treatment of impe matitis . It works by stopping the growth of the bacteria tigo due to Staphylococcus aureus (methicillin - susceptible causing the infection . only ) or Streptococcus pyogene . 0006 ] Fusidic acid resistance in S . aureus can be readily [ 0013 ] A wound is an injury to the body (as from violence , selected for by in vitro exposure to the antibiotic , leading to accident, or surgery ) that typically involves laceration or the recommendation that for systemic therapy fusidic acid breaking of a membrane (as the skin ) and usually damage to should only be given in combination with another agent. underlying tissues (Merriam Webster Dictionary ). Burns are US 2017 /0326160 A1 Nov . 16 , 2017 injuries to tissues caused by heat, friction , electricity , radia quickly cleared via the urinary tract or by enzymatic tion , or chemicals . Wounds and burns are often colonized by metabolism in the liver . Therapeutically it is useful against microbiologic pathogens , including Gram - positive bacteria , cestoda in humans . such as Staphylococcus aureus and / or Streptococcus pyo [0025 ] Niclosamide has also been shown to prevent the genes ; and Gram -negative bacteria , e . g . Pseudomonas penetration of Schistosoma mansoni through the human aeruginosa. skin . As well as used as an anticancer drug , pesticide and as [ 0014 ] Despite the very common occurrence of skin infec an anti - trypanosoma drug . Virtually all applications and tions, only a limited number of topical antibiotics are proposed applications of niclosamide target eukaryotic approved for the treatment of wounds and particularly organisms. infected wounds. Mupirocin (Bactroban ) is an antibiotic , (0026 ] Niclosamide has also been shown to inhibit viral developed by GSK . Emerging resistance to mupirocin is replication in human cells . However , the mechanism is becoming a concern . In coagulase - negative staphylococci believed to be through targeting human host cells to provide isolates, mupirocin resistance rates are higher, ranging from conditions that prevent the viral life rather than specifically 12 . 7 % in Europe to 38 . 8 % in the United States . Retapamulin targeting the virus . Accordingly , the viral application of ( Altabax , GSK ) is another topical antibiotic used for wound niclosamide result from its ability to target an eukaryotic treatment. Fucidin (LEO Pharma ) is also effective in primary process . and secondary skin infections caused by sensitive strains of [0027 ] Niclosamide is commercially available in a number Staphylococcus aureus, Streptococcus species and Coryne of formulations including, but not limited to Bayer73® , bacterium minutissimum . Bayer2353® , Bayer25648® , Bayluscid® , Baylucide® , Ces [0015 ] Bacterial infections are a leading cause of death tocid® , Clonitralid , Dichlosale® , Fenasal® , HL 2447® , worldwide , and bacterial resistance is greatly reducing avail lomesan® , Iomezan® , Manosil® , Nasemo® , Niclosamid® , able treatment options. There is therefore a need for new Phenasal® , Tredemine® , Sulqui® , Vermitid® , Vermitid® , antibiotics , for which development of resistance is not and Yomesan® . widespread in the target bacteria , for the prevention and 10028 ] Other halogenated salicylanilide compounds are treatment of topical infections caused or contributed to [0029 ] Closantel , ( N - [ 5 - chloro - 4 - [ ( 4 - chlorophenyl) cya Gram - positive bacteria such as Staphylococcus aureus and nomethyl ) - 2 -methylphenyl ) - 2 - hydroxy - 3 , 5 - diiodobenz Streptococcus pyogenes is strongly warranted . amide ) is used as a veterinary anthelmintic : [ 0016 ] The halogenated salicylanilides are a series of compounds generally used as anthelmintic agents . One such compound is niclosamide ( 5 - chloro - N - ( 2 - chloro - 4 - nitrophe nyl) - 2 -hydroxybenz - amide ; also known as 2 ' , 5 - dichloro4 ' 3- nitrosalicylandide , 2 -hydroxy - 5 - chloro - N - ( 2 - chloro - 4 - nitro - C ( 0 ) NH phenyl) - benzamide , 5 - chloro - 2 -chloro - 4 ' 5 nitrosalicylanilide or 5 - chloro -N -( 2 -chloro - 4 -nitrophenyl ) salicylamide ) : OH H3C Closantel [0030 ] Acute toxicity of closantel : [0031 ] LD50 , rats , p . o ., 262 - 342 mg/ kg (depending on _ CO _ NH _ -NO2 the study ) , median 302 mg/kg [ 0032 ] LD50, rats, s . c ., 67 mg/ kg OH [0033 ] LD50 , mice , p . o ., 331 mg/ kg [0034 ] LD50 , mice, i. m ., 57 mg/ kg Niclosamide [0035 ] Rafoxanide (3 '- chloro -4 '- ( p - chlorophenoxy )- 3 ,5 diiodosalicylanilide) is known for veterinary use as a fas [0017 ] Acute toxicity of niclosamide : ciolicide and anthelmintic [0018 ] LD50 , mice , p .o ., > 5000 mg/ kg [0019 ] LD50 , rats , p . o. , 5000 mg/ kg [0020 ] LDso , rats , dermal , 2000 mg/ kg [ 0021 ] LD50 , rabbits , p .o ., 5000 mg/ kg [0022 ] LD50 , cats , p . o ., > 1000 mg/kg - C ( O ) NH [0023 ] Niclosamide is a known taenicide ( tapeworm killer ) effective against several parasitic tapeworms of live OH stock and pets ( e . g . Taenia spp , Moniezia spp ) and also against rumen flukes ( Paramphistomum spp ) and blood rafoxanide flukes ( Schistosoma spp ) . This is in contrast with most other salicylanilides, which generally exhibit activity as flukicides [0036 ] Acute toxicity of rafoxanide: but not as taenicides . [0037 ] LD50 , rats , p .o . , 980 -> 2000 mg /kg (depending [0024 ] Niclosamide is currently used in humans as an on the study ) , median > 1490 mg/ kg anthelmintic drug to treat intestinal infections and displays [ 0038 ] LD50 ,mice , p . o . , 232 - 300 mg/ kg ( depending on overall low toxicity , it is poorly soluble in water, shows low the study) , median 266 mg/ kg intestinal absorption , and once in the bloodstream , it is [0039 ] LD50 , rabbits , p . o ., 3200 mg/kg US 2017 /0326160 A1 Nov . 16 , 2017

[0040 ] Oxyclozanide (3 ,3 ', 5 , 5' , 6 - pentachloro - 2 -hydrox (Bioorg . Med . Chem . Letters , 8 ( 14 ), 1923 -28 (1998 )) , but ysalicylanilide ) , is known for veterinary use as an anthel nowhere mentioned the problem with resistance develop mintic , primarily against trematodes . ment. [0051 ] R . Rajamuthiah et al . identified closantel as a hit in a high throughput liquid screening assay and found anti HO staphylococcal activity of closantel against vancomycin HQ resistant S . aureus isolates and other Gram -positive bacteria . There is no mention of the problem of resistance develop - C ( O ) NH – ment ( PloS One, 2014 , 9 ( 2 ) : e89189 ) . [ 0052 ] WO 2008/ 155535 describes the use ofhalogenated OH salicylanilides for the treatment of acne , wherein propioni bacteria is the bacteria causing the acne . There is no mention oxyclosanide of the problem with resistance development. [0041 ] LDso , rats, p .o ., 980 -3519 mg/ kg (depending on SUMMARY OF THE INVENTION the study ), median 2250 mg/ kg [0053 ] Many bacteria are resistant to , or rapidly develop , [0042 ] LD50 , mice, p .o ., 300 mg/ kg resistance against antibiotic agents and thus it is undesirable [0043 ] LD50 , rabbits , p . o ., 3200 mg/ kg or ineffective to treat a mammal suffering from a topical bacterial disease or infection caused by bacteria that are [0044 ] Low safety margin after oral administration resistant to the antibiotics. Described herein is the topical [0045 ] The molecular mode of action of salicylanilides, use of a halogenated salicylanilide for the treatment of an including niclosamide , is not completely elucidated . They infection caused by Gram positive bacteria . The topical use all are uncouplers of the oxidative phosphorylation in the of the halogenated salicylanilides according to the invention cell mitochondria , which disturbs the production of ATP . have a substantially reduced frequency of spontaneous resis This impairs the parasites motility and probably other pro tant mutants compared to commonly used topical antibiot cesses as well . Niclosamide acts on the tapeworms also ics . through inhibition of glucose absorption [0054 ] In a first aspect of the invention there is provided [0046 ] Niclosamide has been proposed as a possible sys a halogenated salicylanilide selected from the group con temic treatment for chronic lung infections caused by the sisting of closantel , rafoxanide, oxyclozanide and niclos proteobacterium Pseudo -monas aeruginosa and the actino amide and derivatives thereof including salts , hydrates and bacterium Mycoplasmum tuberculosis . Niclosamide has esters for use in topical prevention or treatment of an been shown to reduce the quorum sensing response as well infection or disease caused by Gram positive bacteria , as the production of quorum sensing metabolites in P . wherein the Gram positive bacteria is not a propionibacteria . aeruginosa . Since quorum sensing is considered an impor The invention thus provides a method of treating a disease tant process for the pathogenicity during chronic lung infec or infection caused by Gram positive bacteria in a subject, tions caused by this bacterium , it led to proposal that themethod comprising topically administering to the subject niclosamide could be used as an adjuvant therapy for these an effective amount of a halogenated salicylanilide selected infections . Niclosamide does not affect the growth of P . from the group consisting of closantel, rafoxanide , oxyclo aeruginosa and accordingly does not have any direct anti zanide and niclosamide and derivatives thereof including bacterial activity . The concentration required for optimal salts , hydrates and esters ; wherein the Gram positive bac activity was 20 uM , however, some inhibition was detected teria is not a propionibacteria . at 1 uM . ( F . Imperi et al. , Antimicrobial, Agents and Che [0055 ]. In a second aspect of the invention there is pro motherapy, 557 ( 2 ), 996 - 1005 ( 2013 ) ) . vided a halogenated salicylanilide selected from the group [0047 ] Ghazi et al. ( Zentralbl. Mikrobiol. 141 ( 1986 ), consisting of closantel, rafoxanide , oxyclozanide and niclos 225 - 232 ) tested the antibacterial effect and toxicity of syn amide and derivatives thereof including salts , hydrates and thesized salicylanilide derivatives against Escherichia coli, esters for use in the topical prevention or treatment of an Bacillus subtilis , Pseudomonas aeruginosa and Staphylo infection or disease by Gram -positive bacteria which is coccus aureus but nothing is mentioned about the rate of resistant to a drug selected from fusidic acid , mupirocin and resistance development. retapamulin . The invention thus provides a method of treat [0048 ] J . Vinsova et al. describe the antibacterial activity ing or preventing a disease or infection caused by Gram of salicylanilides (Molecules , vol. 12, no . 1 , pp . 1 - 12 , 2007 ; positive bacteria in a subject , the method comprising topi Bioorganic and Medicinal Chemistry Letters , vol. 19 , no . 2 , cally administering to the subject an effective amount of a pp . 348 - 351, 2009 ; European Journal of Medicinal Chem halogenated salicylanilide selected from the group consist istry , vol. 45 , no . 12 , pp . 6106 -6113 , 2010 ) , but nowhere ing of closantel, rafoxanide , oxyclozanide and niclosamide mentioned the problem with resistance development. and derivatives thereof including salts , hydrates and esters ; [0049 ] M . J . Macielag et al. tested the antibacterial activity wherein the Gram - positive bacteria which is resistant to a of closantel and related derivatives against the drug - resistant drug selected from fusidic acid , mupirocin and retapamulin . organisms, methicillin - resistant Staphylococcus aureus [0056 ] In a third aspect of the invention there is provided (MRSA ) and vancomycin - resistant Enterococcus faecium a halogenated salicylanilide selected from the group con (VREF ) ( J . Med . Chem . , 41 ( 16 ) , 2939 -45 ( 1998 ) ) but sisting of closantel, rafoxanide, oxyclozanide and niclos nowhere mentioned the problem with resistance develop - amide and derivatives thereof including salts, hydrates and ment. esters for use in topical prevention or treatment of an [0050 ] D . J . Hlasta et al found that closantel had antibac infection or disease for which fusidic acid , mupirocin or terial activity against drug resistant S . aureus and E . faecium retapamulin was an approved treatment in Europe on 12 Sep . US 2017 /0326160 A1 Nov . 16 , 2017

2014 ; wherein the disease or infection is caused by Gram known to those of skill in the art (see , e . g . WO 2004 / positive bacteria . Similarly , the invention may provide A 006906 ), which is herein incorporated by reference for all method of treating or preventing a disease or infection purposes . caused by Gram positive bacteria in a subject, the method [0061 ] It may be that the halogenated salicylanilide is comprising topically administering to the subject an effec selected from closantel , rafoxanide , oxyclozanide , niclos tive amount a halogenated salicylanilide selected from the amide , clioxanide , 4 ' -chloro -5 - nitrosalicylanilide , 2 - chloro group consisting of closantel, rafoxanide , oxyclozanide and 5 '- methoxy - 3 -nitrosalicyl - anilide , 2 '- methoxy - 3 , 4 ' - di- nitro niclosamide and derivatives thereof including salts , hydrates salicyl -anilide , 2 !, 4 ' - dimethyl- 3 -nitrosalicylanilide , and esters ; wherein the infection or disease is an infection or 2 ' - chloro - 3 , 4 -dinitrosalicylanilide , 2 - ethyl- 3 - nitrosalicy disease for which fusidic acid , mupirocin or retapamulin lanilide and 2 ' bromo- 3 -rosalicyl - anilide a pharmaceutically acceptable salt or solvate thereof. was an approved treatment in the US on 12 Sep . 2014 ; and [0062 ] It may be that the halogenated salicylanilide is wherein the disease or infection is caused by Gram positive selected from closantel , rafoxanide, oxyclozanide and bacteria . niclosamide , or a pharmaceutically acceptable salt or solvate f0057 ] The inventors have found that the halogenated thereof. salicylanilides closantel , rafoxanide , oxyclozanide , or niclosamide or a pharmaceutically acceptable derivative [0063 ] It may be that the halogenated salicylanilide is thereof are highly effective against Gram - positive bacteria , selected from closantel, rafoxanide and oxyclozanide , or a such as Staphylococcus aureus and Streptococcus pyogenes pharmaceutically acceptable salt or solvate thereof. and unexpectedly are also very effective in reducing the [0064 ] It may be that the halogenated salicylanilide is development of resistance bacteria , such as those mentioned niclosamide , or a pharmaceutically acceptable salt or solvate above. The compounds are expected to be very useful thereof. It may be that the halogenated salicylanilide is topical agents for use in the prevention and / or treatment of niclosamide in the free -base form of the compound . It may diseases or infections . One specific halogenated salicy be that the halogenated salicylanilide is a pharmaceutically lanilide is niclosamide . Examples of diseases which may be acceptable salt of niclosamide . treated by topical administration of the halogenated salicy [0065 ] In a fourth aspect of the invention there is provided lanilide include impetigo , bacterial conjunctivitis , atopic a topical pharmaceutical formulation comprising a haloge dermatitis related infections, nasal eradication , sycosis bar nated salicylanilide selected from the group consisting of bae , superficial folliculitis , paronychia erythrasma , second closantel , rafoxanide , oxyclozanide and niclosamide and ary infected dermatoses, carbuncles, furunculosis (ecthyma , derivatives thereof including salts , hydrates and esters , cellulitis , erysipelas , necrotising fasciitis , secondary skin wherein the components of the formulation are selected such infections of wounds, dermatitis , scabies, diabetic ulcers and that it provides a local pH of less than 6 at the site of the like) . infection . [0066 ] The inventors have found that the antibacterial [0058 ] In one embodiment the active ingredient is niclos activity of halogenated salicylanilides , e. g . niclosamide , is amide or a derivative thereof or as a hydrate thereof such as higher at low pH than at neutral or basic pH . It may be that niclosamide monohydrate, or salt thereof such as its etha the formulation does not comprise a buffer or pH modifier . nolamine salt , or piperazine salt or other suitable salts or This can mean that the formulation does not significantly hydrates of such salts . alter the naturally low pH of the skin at the site of infection . [ 0059 ] Niclosamide is bacteriostatic for S . aureus (See [0067 ] It may be that the halogenated salicylanilide , e . g . FIG . 1 ) , i . e . it prevents the growth , but does not kill the niclosamide , is in the form of a free base . Halogenated bacterium . Surprisingly , niclosamide has been found to have salicylanilides are typically mildly acidic , comprising as equally good effect on S . aureus strains resistant to methi they do phenolic groups. Salts of halogenated salicylanilides cillin , fusidic acid and mupirocin as non -resistant strains are typically formed with amines ( e . g . ethanolamine ) which ( See FIG . 2 ) . Biological testing ( inter alia ) has also unex can provide an increase in local pH . pectedly revealed that the halogenated salicylanilides , and [0068 ] Bacterial decolonisation may be an effective strat particularly niclosamide have lower mutation rate against egy for reducing the incidence of nosocomial (hospital MRSA 01 than compounds like fusidic acid , mupirocin and acquired ) infections , particularly those associated with retapamulin ( see Table 4 ) . MRSA . Many people carry MRSA without symptoms. [0060 ] Withoutwishing to be hound by a particular theory , Decolonising such patients may be beneficial in the preven it is believed that a number of niclosamide analogs can act tion of the spread of MRSA in a hospital environment or to in a manner similar to niclosamide . Illustrative niclosamide reduce the risk of the patient developing an infection fol analogs include, but are not limited to closantel (CAS # : lowing a surgical or medical procedure in hospital. 57808 -65 - 8 ) , oxyclozanide (CAS # : 2277 - 92 - 1 ) , rafoxanide [ 0069 ] In a fifth aspect of the invention there is provided (CAS # : 22662 - 39 - 1 ), clioxanide (CAS # : 14437 -41 -3 ). a method of treating , preventing or eliminating bacterial Other suitable niclosamide analogs include brotianide (CAS colonization by Gram positive bacteria in a subject , the # : 23233 - 88 - ' 7 ) , 4 ' - chloro - 3 - nitrosalicylanilide , 4 ' - chloro - 5 method comprising topically administering to the subject an nitrosalicylanilide , 2 -chloro - 5' - methoxy - 3- nitrosalicyl- an effective amount a halogenated salicylanilide selected from ilide , 2 ' -methoxy - 3 , 4di- nitrosalicyl- anilide , 2 ', 4 '- dimethyl the group consisting of closantel, rafoxanide , oxyclozanide 3 -nitrosalicylanilide , 24 - chloro -3 ,4 '- dinitrosalicylanilide , and niclosamide and derivatives thereof including salts , 2 ' - ethyl - 3 -nitrosalicylanilide , 2 * -bromo - 3 -nitrosalicyl - an hydrates and esters. Thus , the invention may provide a ilide , and the like ( EP2049137 ) . It is noted that these halogenated salicylanilide selected from the group consist niclosamide analogs are intended to be illustrative and not ing of closantel , rafoxanide , oxyclozanide and niclosamide limiting , Methods of making niclosamide analogs are well and derivatives thereof including salts , hydrates and esters , US 2017 /0326160 A1 Nov . 16 , 2017 for use in treating, preventing or eliminating bacterial colo pH modifier . Thus, it may be that the halogenated salicy nization in a subject by Gram positive bacteria by topical lanilide , e . g . niclosamide, is in the form of a free base . The administration . components of the formulation may be selected such that it [0070 ] It may be that the halogenated salicylanilide is provides a local pH of greater than 4 .5 (e .g . greater than 5 ) topically administered to the subject prior to performing a at the site of infection . Reference to a " local pH ” is to the pH surgical procedure on the subject. It may be that the halo at the site where the formulation is applied for example the genated salicylanilide is administered to the nose of the pH on the surface of the skin after applying the formulation subject , either intranasally or to the external skin of the nose . containing the halogenated salicylanilide Thus it may be that the halogenated salicylanilide is topi [0077 ] Itmay be that halogenated salicylanilide is selected cally administered to the nose of the subject to performing from rafoxanide , oxyclozanide , closantel and niclosamide a surgical procedure on the nose or face of the subject. and derivatives thereof including salts , hydrates and esters . [0071 ] It may be that the Gram - positive bacteria develops It may be that halogenated salicylanilide is selected from spontaneous mutations which confer resistance to the halo rafoxanide , oxyclozanide and niclosamide and derivatives genated salicylanilide at a frequency of less than 10 - at the thereof including salts , hydrates and esters . Thus, it may be minimum inhibitory concentration (MIC ) of the halogenated that the halogenated salicylanilide is niclosamide or a salt or salicylanilide to the Gram positive bacteria . It may be that hydrate thereof. the Gram - positive bacteria develops spontaneousmutations [0078 ] It may be that the halogenated salicylanilide (e . g . which confer resistance to the halogenated salicylanilide at rafoxanide , oxyclozanide , closantel or niclosamide ) is in the a frequency of less than 10 - 7 at the MIC of the halogenated form of a free base . It may be that the halogenated salicy salicylanilide to the Gram positive bacteria . It may be that lanilide ( e . g . rafoxanide , oxyclozanide , closantel or niclos the Gram - positive bacteria develops spontaneous mutations amide ) is not in the form of a hydrate . Where the haloge which confer resistance to the halogenated salicylanilide at nated salicylanilide is niclosamide it may be that it is not in a frequency of less than 10 - 8 at the MIC of the halogenated the form of the monohydrate . salicylanilide to the Gram positive bacteria . Thus , it may be [ 0079 ] It may be that the Gram - positive bacteria is not an that the Gram -positive bacteria develops spontaneous muta antibiotic resistant strain . Alternatively , it may be that the tions which confer resistance to the halogenated salicy Gram - positive bacteria is an antibiotic resistant strain . lanilide at a frequency of less than 4x10 -9 at the MIC of the 0080 ] It may be that the infection or disease is in a human halogenated salicylanilide to the Gram positive bacteria . or animal, for example wherein the infection is in a human . [ 0072 ] It may be that the Gram positive bacteria is not a [0081 ] In some embodiments , the populationof Gram propionibacteria . It may be that the Gram positive bacteria positive bacteria includes antibiotic - resistant Gram -positive is selected from Staphylococcus aureus or Streptococcus bacteria . pyogenes . It may be that the bacteria is resistant to a drug [0082 ] It may be that the Gram positive bacteria is not a selected from fusidic acid , mupirocin and retapamulin . It propionibacteria , e . g . that it is not Propionibacterium acnes. may be that the bacteria is methicillin - resistant Staphylo [0083 ] In some embodiments , the population of Gram coccus aureus (MRSA ). positive bacteria includes coccus gram -positive bacteria . In [0073 ] It may be that the infection or disease is selected some embodiments , the Gram -positive bacteria are from the from the group consisting of impetigo , bacterial conjuncti Streptococcus or Staphylococcus genus . vitis , atopic dermatitis and related infections, infected 10084 ] In some embodiments, the Gram -positive : bacteria eczema, rosacea , nasal eradication , sycosis barbae, superfi are from the Streptococcus genus. It may be that the Gram cial folliculitis , paronychia erythrasma , secondary infected positive bacteria are Streptococcus selected from Strepto dermatoses, carbuncles, furonculosis , ecthyma, cellulitis , coccus pneumoniae, Streptococcus pyogenes , Streptococcus erysipelas , necrotising fasciitis , secondary skin infections of suis, Streptococcus agalactiae or Streptococcus viridans. wounds , dermatitis , scabies and diabetic ulcers . Thus it may [0085 ] In sonic embodiments , the Gram -positive bacteria be that the infection or disease is selected from the group are Streptococcus pyogenes consisting of impetigo , bacterial conjunctivitis and atopic 0086 ] In some embodiments , the Gram - positive bacteria dermatitis . It may be that the infection or disease is selected are from the Staphylococcus genus. It may be that the from the group consisting of impetigo , bacterial conjuncti Gram -positive bacteria are Staphylococcus selected from vitis , atopic dermatitis related infections , nasal eradication , Staphylococcus epidermidis , Staphylococcus aureus , sycosis barbae , superficial folliculitis , paronychia erythr Staphylococcus saprophyticus or Staphylococcus lugdunen asma , secondary infected dermatoses , carbuncles and furon sis , in some embodiments , the coccus Gram -positive bacte culosis ( ecthyma , cellulitis , erysipelas , necrotising fasciitis , riaare Staphylococcus aureus ( e . g . methicillin -resistant secondary skin infections of wounds, dermatitis, scabies , Staphylococcus aureus ) . diabetic ulcers and the like ) . [0087 ] The Gram -positive bacteria described herein may [ 0074 ] It may be that the infection or disease is not acne be resistant to another antibiotic drug . For example , the vulgaris . bacteria may be resistant to another antibiotic drug other [0075 ] It may be that the treatment is administered topi than a halogenated salicylanilide selected from closantel , cally for 2 weeks or less. rafoxanide , oxyclozanide and or niclosamide , or a derivative [ 007 ] It may be that the halogenated salicylanilide is thereof. It may be that the bacteria is resistant to a drug comprised in a formulation the components of which are selected from fusidic acid , mupirocin and retapamulin . selected such that it provides a local pH of less than 6 at the [0088 ] Also provided are methods of treating a subject site of infection . The inventors have found that the antibac - having a Gram - positive bacterial infection that include topi terial activity of halogenated salicylanilides, e. g. niclos cally administering to a subject having a Gram -positive amide , is higher at low pH than at neutral or basic pH . Thus , bacterial infection one of the halogenated salicylanilides it may be that the formulation does not comprise a buffer or closantel, rafoxanide , oxyclozanide , or niclosamide or a US 2017 /0326160 A1 Nov . 16 , 2017 derivative thereof in amounts sufficient to decrease the cally administering to a subject a pharmaceutically effective population of Gram - positive bacteria in the subject. The amount of a halogenated salicylanilide selected from clos invention may be and characterized by a rate of developing antel , rafoxanide , oxyclozanide and niclosamide or a hydrate spontaneous resistance to these bacteria of less than 10 - 6 , or a salt or hydrate of such a salt thereof. One example which such as less than 10 - 7 or 10 - 8 , such as less than 4x10 - 9 . One is of special interest is niclosamide. example which is of special interest is niclosamide . [ 0094 ] The present invention may provide a pharmaceu [ 0089 ] Furthermore , the present invention provides a tical composition comprising a pharmaceutically effective method of treating a subject suffering from an infection amount of a halogenated salicylanilide selected from clos contributed to or caused by Gram - positive bacteria as here antel , rafoxanide , oxyclozanide and niclosamide or a deriva inbefore described , said method comprising the step of tive thereof to be administered for the topical prevention topically administering an effective amount of a halogenated and /or treatment of Gram - positive bacteria such as Staphy salicylanilide selected from closantel, rafoxanide, oxycloza lococcus aureus and /or Streptococcus pyogenes colonizing nide and niclosamide , or a derivative thereof. The invention or infecting skin affected with a dermatological condition may be characterized by a rate of developing spontaneous selected from the group consisting of impetigo , atopic resistance to these bacteria of less than 10 - , such as less dermatitis and infections associated with different skin con than 10 - 7 or 10 - 8 , such as less than 4x1031 9 . One example ditions such as eczema or psoriasis and characterized by a which is of special interest is niclosamide which may be rate of developing spontaneous resistance to these bacteria characterized by a rate of developing spontaneous resistance of less than 10 - º , such as less than 10 - ' or 10 , such as less to these bacteria of less than 10 - 8 , such as less than 4x10 - 9 . than 4x10 - 9 . One example which is of special interest is [0090 ] The present invention may provide the use of a niclosamide . pharmaceutically effective amount of a halogenated salicy 10095 ] The present invention may provide a pharmaceu lanilide selected from closantel, rafoxanide, oxyclozanide tical composition comprising a pharmaceutically effective and niclosamide or a derivative thereof in the preparation of amount of niclosamide or a derivative thereof to be admin a medicament for use in the topical prevention and /or istered for the topical prevention and/ or treatment of Gram treatment of Gram -positive bacteria such as Staphylococcus positive bacteria such as Staphylococcus aureus and /or aureus and / or Streptococcus pyogenes colonizing or infect Streptococcus pyogenes colonizing or infecting skin affected ing skin affected with a dermatological condition selected with a dermatological condition selected from the group from the group consisting of impetigo , atopic dermatitis and consisting of impetigo , atopic dermatitis and infections infections associated with different skin conditions such as associated with different skin conditions such as eczema or eczema or psoriasis. The invention may be characterized by psoriasis and characterized by a rate of developing sponta a rate of developing spontaneous resistance to these bacteria neous mutation frequency less than 10 - , such as less than of less than 10 - , such as less than 10 - ' or 10 - 8 , such as less 10 - 7 or 10 - 8 , such as less than 4x10 - 9 than 4x10 - 9 . One example which is of special interest is [0096 ] The present invention may provide a pharmaceu niclosamide . tical composition comprising niclosamide as the active [0091 ] The present invention may provide a method for ingredient to be administered for the topical prevention the topical prevention and / or treatment of Gram - positive and / or treatment of Gram -positive bacteria such as Staphy bacteria such as Staphylococcus aureus and / or Streptococ lococcus aureus and / or Streptococcus pyogenes colonizing cus pyogenes colonizing or infecting skin affected with a or infecting skin affected with a dermatological condition dermatological condition selected from the group consisting selected from the group consisting of impetigo , atopic of impetigo , atopic dermatitis and infections associated with dermatitis and infections associated with different skin con different skin conditions such as eczema or psoriasis com ditions such as eczema or psoriasis and characterized by a prising administering to a subject a pharmaceutically effec rate of developing spontaneous mutation frequency less than tive amount of a halogenate salicylanilide selected from 10 - 6 , such as less than 10 - 7 or 10 - , such as less than closantel, rafoxanide , oxyclozanide and niclosamide or a 4x10 - 9 hydrate or a salt or a hydrate of such a salt thereof. The 10097 ] Examples of diseases which may be topically invention may be characterized by a rate of developing treated using the halogenated salicylanilide include impe spontaneous mutation frequency less than 10 - , such as less tigo , bacterial conjunctivitis , atopic dermatitis related infec than 10 - 7 or 10 - 8 , such as less than 4x10 - 9 . One example tions , nasal eradication , sycosis barbae , superficial follicu which is of special interest is niclosamide . litis, paronychia erythrasma, secondary infected dermatoses , [ 0092 ] The present invention may provide a method for carbuncles , furonculosis ( ecthyma, cellulitis , erysipelas , the topical treatment of antibiotic - resistant Gram -positive necrotising fasciitis , secondary skin infections of wounds , bacterial infections comprising administering to a subject dermatitis , scabies, diabetic ulcers and the like ). infected with antibiotic -resistant Gram -positive bacteria [0098 ] The infection or disease treated topically using the selected from the group consisting of antibiotic - resistant halogenated salicylanilide may be a skin infection , infected Staphylococcus aureus and/ or Streptococcus pyogenes, a dermatitis or infected dermatosis , for example any of the pharmaceutically effective amount of a halogenated salicy skin infections described herein . The skin infection may , for lanilide selected from closantel, rafoxanide, oxyclozanide example , be selected from impetigo ( including impetigo and niclosamide or a hydrate or a salt or a hydrate of such contagiosa , bullous impetigo , and ecthyma ) infected derma a salt thereof. One example which is of special interest is titis ( for example infected atopic dermatitis ) infected niclosamide. eczema, infected skin wounds, infected burns and infected [0093 ] The present invention may provide a method for ulcers (for example diabetic ulcers ) . the prevention and /or treatment of conjunctivitis and kera [ 0099 ] The infection or disease treated topically using the titis caused by Gram -positive bacteria such as Staphylococ halogenated salicylanilide may be a secondarily Gram posi cus aureus and /or Streptococcus pyogenes comprising topi tive infected dermatosis, for example a secondary skin US 2017 /0326160 A1 Nov . 16 , 2017

infection . Secondary Gram positive infections are common caused by Gram - positive bacteria , are highly effective in complications of primary dermatoses, primary nonbacterial inhibiting the growth of Gram -positive bacteria such as skin infections , traumatic lesions , ulcers , cutaneous infesta Staphylococcus, in particular Staphylococcus aureus , and tions , and other skin diseases . Accordingly , the halogenated Streptococcus, in particular Streptococcus pyogenes charac salicylanilide may be for use in the topical treatment of for terized by a rate of developing spontaneous mutation fre example secondary infections of a condition selected from quency less than 10 - 6 , such as less than 10 - 7 or 10 - 8 , such eczema, pediculosis , scabies, insect bites ( for example papu as less than 4x10 - 9 . One example which is of special interest lar urticaria ) , pemphigus psoriasis , skin ulcers , kerion and a is niclosamide. viral infection of the skin ( for example herpes simplex or [0104 ] A further aspect of the invention also provides a chicken pox ). method for manufacturing of a medicament for use in topical [ 0100 ] The halogenated salicylanilide may be for use to prevention or treatment of an infection caused by Gram decolonise a subject carrying a Gram positive bacteria positive bacteria , wherein the medicament is niclosamide ( including any of the Gram positive bacteria described and derivatives thereof including salts , hydrates and esters herein , for example MRSA ) , wherein the halogenated and wherein the use of the halogenated salicylanilide are salicylanilide is applied topically to the subject. Such highly effective in inhibiting the growth of Gram - positive decolonisation may be effective in preventing or reducing bacteria such as Staphylococcus , in particular Staphylococ the spread of infection to other subjects particularly in a cus aureus , and Streptococcus , in particular Streptococcus hospital environment. Decolonisation may also prevent or pyogenesl characterized by a rate of developing spontaneous reduce the risk of surgical site infections resulting from mutation frequency less than 10 - such as less than 10 - 7 or surgical or medical procedures carried out on the patient or 10 - 8 , such as less than 4x10 - 9 . at the site ofmedical devices such as catheters or IV lines or cannula . Accordingly the halogenated salicylanilide may be [0105 ] By the topical use of the halogenated salicy for use in the decolonisation of a subject prior to carrying out lanilides , such as niclosamide it is possible to treat a human a surgical procedure on the subject, wherein the halogenated suffering from an infection caused by bacteria such as salicylanilide is applied topically to the subject. Such sur Staphylococcus aureus or Streptococcus pyogenes while gical procedures include, for example elective surgical pro having a reduced rate of appearance of spontaneous resistant cedures such as hip or knee replacement . Decolonisation mutants against the agent . may be achieved by topically administering the halogenated [0106 ] Further advantages and objects with the present salicylanilide to sites on the subject which are colonised by invention will be described in more detail, inter alia with the Gram positive bacteria . It is known that a common site reference to the accompanying drawings . for bacterial colonisation such as MRSA is the nose . Accord ingly , the halogenated salicylanilide may be applied topi cally to the nose. Particularly the halogenated salicylanilide BRIEF DESCRIPTION OF THE DRAWINGS may be applied to the anterior nares (the inner surface of the AND TABLES nostrils ) . [0107 ] FIG . 1 shows microbiological data of niclosamide [0101 ] The infections and decolonisation described in the tested against MRSA , MSSA and S . pyogenes strains. A . above paragraphs and herein may be topically treated with MIC (ug /ml ) against all targeted strains. The MIC of niclos any of the halogenated salicylanilides herein selected from amide was < 0 . 4 ug /ml against S . aureus strains , including closantel , rafoxanide , oxyclozanide or niclosamide or a the strains resistant to fusidic acid ( * ) and the ones resistant derivative thereof. Particularly the halogenated salicy to mupirocin ( i ), and < 3 . 2 ug /ml against Streptococcus lanilide may be niclosamide . pyogenes strains. Dose -response curves of yellow high [0102 ] The present invention may provide a pharmaceu tical composition comprising a pharmaceutically effective lighted strains are represented in B . amount of one of the halogenated salicylanilides closantel , [0108 ] FIG . 2 shows a time- kill curve of MRSA 01 rafoxanide , oxyclozanideor niclosamide or a derivative incubated with niclosamide (MICx10 ) . Niclosamide had a thereof to be administered for the topical prevention and/ or bacteriostatic effect against MRSA 01 ( strain used in animal treatment of Gram -positive bacteria such as Staphylococcus experiment) in the tested conditions ( initial inoculum : 7 aureus and / or Streptococcus pyogenes colonizing or infect log10 cfu /ml ; niclosamide: 4 ug/ ml MICx10 ] ) . ing skin affected with a dermatological condition selected [0109 ] FIG . 3 shows CFU in skin lesions after three days from the group consisting of impetigo , atopic dermatitis and of treatment with Samples 5 . Sample- control corresponds to infections associated with different skin conditions such as the sample without niclosamide * : P < 0 . 05 , unpaired t - test ; eczema or psoriasis characterized by a rate of developing * * : P < 0 .005 , unpaired t- test ; * * * : P < 0 .001 , unpaired t- test ; spontaneous mutation frequency less than 10 - , such as less ns: not significant. than 10 - 7 or 10 - 8 , such as less than 4x10 - 9 against MRSA 01 , MRSA 15 and MRSA 16 at a concentration of MICx1 [0110 ) FIG . 4 shows the dose - response curves of niclos when measured according to mutational frequency evalua amide , fusidic acid and mupirocin against S . aureus with tion method as described in the experimental section . methicillin - resistant strains ( A ) , fusidic acid - resistant strain Niclosamide has been found to be superior against S . aureus ( B ) and mupirocin - resistant strain ( C ) . and S . pyogenes (both in uM and ug /ml ) . [0111 ] FIG . 5 shows the growth of S . aureus at different [ 0103 ] A further aspect of the invention also provides a pH as a function of niclosamide concentration ( average of 3 halogenated salicylanilide selected from the group consist replicates ) . The growth of S . aureus without niclosamide ( 0 ing of closantel, rafoxanide, oxyclozanide , and niclosamide ug /ml ) is comparable from pH 7 .4 to pH 6 .0 . The growth and derivatives thereof including salts , hydrates and esters without niclosamide is slightly inhibited at pH 5 . 5 and for use in the topical prevention or treatment of an infection completely inhibited with pH equal or below 5 . 0 . US 2017 /0326160 A1 Nov . 16 , 2017

DETAILED DESCRIPTION OF THE cus aureus and / or Streptococcus pyogenes symptomatic INVENTION infection , ( vi ) the eradication ofGram -positive bacteria such [0112 ] Definitions as Staphylococcus aureus and / or Streptococcus pyogenes ; [0113 ] In the context of the present application and inven from an area of the body affected by another disease that tion , the following definitions apply : could enable establishment of an infection more readily , than [ 0114 The term “ Staphylococcus aureus" or " S . aureus ” in a non -disease affected area - e . g , an area of the skin as used herein , without further description , relates to any affected by eczema or atopic dermatitis , ( vii ) the suppression strain of the gram - positive bacteria classified as Staphylo of the disease caused by Gram - positive bacteria such as coccus aureus, and which have been associated with a Staphylococcus aureus and / or Streptococcus pyogenes ; from number of infections , including pneumonia , osteomyelitis , an area of the body affected by another non - infectious arthritis , endocarditis , sepsis and toxic shock syndrome, as disease that enables establishment of an infection more well as cause less severe infections of the skin and soft readily , than in a non - disease affected area - e . g . an area of tissues. the skin affected by eczema or atopic dermatitis . 01151. The term “ methicillin - resistant Staphylococcus [0120 ] Thus, in the context of the present invention , aureus ” or “MRSA ” as used herein includes strains of treatment of a condition encompasses both therapeutic and Staphylococcus aureus that are resistant to methicillin and prophylactic treatment, of either an infectious or a non can also broadly relate to Gram -positive bacteria strains (e . g . infectious condition , in a mammal such as a human or beta - lactamase -producing bacteria ) that are resistant to anti animal, but in particular a human . It may involve complete biotics falling within the general classification of penicillins . or partial eradication of the condition , removal or amelio Methicillin is the common name for (2S , 5R ,6R )- 6 -[ (2 ,6 ration of associated symptoms, arresting subsequent devel dimethoxybenzoyl ) amino ] 3 , 3 - dimethyl- 7 - oxo - 1 - thia - 1 opment of the condition , and /or prevention of, or reduction azabi - cyclo [ 3 . 2 . 0 ]heptane - 2 - carboxylic acid , which is a nar of risk of, subsequent occurrence of the condition . The row spectrum beta -lactam antibiotic that has been used to bacterial strain may be characterized by a rate of developing treat infections caused by susceptible Gram -positive bacte spontaneous mutation frequency less than 10 - , such as less ria (e . g . including Staphylococcus aureus ). than 10 - 7 or 10 - 8 , such as less than 4x10 - 9 . The treatment [ 0116 ] The term " derivative ” refer to , but shall not be will typically involve the use of the halogenated salicy limited to metabolites, pro - drugs (converted into active lanilides closantel , rafoxanide , oxyclozanide or niclosamide drugs ) , esters , hydrates and / or a pharmaceutically accept or derivatives thereof against Gram -positive bacteria such as able salt and /or hydrates of such salts of the halogenated Staphylococcus aureus and Streptococcus pyogenes. One salicylanilides of the present invention . Also within the term example which is of special interest is niclosamide . " derivatives ” are crystalline forms of the compounds and [0121 ] It may be that the infection or disease which is to co - crystals formed between the halogenated salicylanilide be treated is in an animal, e . g . a mammal . In particular, the and a suitable co - former ( s ) . A person skilled in the art is well halogenated salicylanilide can be used to treat commercial aware of various chemical methods and techniques to render animals such as livestock ( e . g . cows , sheep , chickens, pigs , a chemical substance to generate a derivate , which still geese , ducks, goats , etc . ) . Alternatively, the compounds of comprises the chemical basis , such as addition , deletion or the present invention can be used to treat companion animals substitution of a group or functional group and thus it would such as cats , dogs, horses , etc . be easy to generate a similar compound as niclosamide [0122 ] A “ topical medication ” is a medication that is which has the same effect as the original. applied to body surfaces such as the skin or mucous mem [0117 ] The term “ pharmaceutically acceptable salt” refers branes to treat ailments via a large range of classes including to salts ( e . g . ethanolamine or piperazine salts ) that retain the but not limited to creams, foams, gels , droplets , lotions , and biological effectiveness and properties of the compounds ointments . Topicalmedications differ from many other types described herein and , which are not biologically or other of drugs because mishandling them can lead to certain wise undesirable . Pharmaceutically acceptable salts are well complications in a patient or administrator of the drug . Many known to skilled persons in the art . Accordingly , it may be topical medications are epicutaneous , meaning that they are that a reference to a salt of a halogenated salicylanilide applied directly to the skin . Topical medications may also be herein may refer to a pharmaceutically acceptable salt of the inhalational, such as asthma medications , or applied to the halogenated salicylanilide . surface of tissues other than the skin , such as eye drops [0118 ] The term “ solvate ” is used herein to refer to a complex of solute , such as a compound or salt of the applied to the conjunctiva , or ear drops placed in the ear, or compound , and a solvent. if the solvent is water, the solvate medications applied to the surface of a tooth . may be termed a hydrate , for example a monohydrate , [0123 ] In topical application , a suitable pharmaceutical dihydrate , trihydrate etc ., depending on the number ofwater composition , for example a cream , lotion , gel, ointment, molecules present per molecule of substrate . paste , drops or the like , may be spread on the affected [ 0119 ] The term treatment' herein indicates (i ) the pre surface and gently rubbed in . A solution may be applied in vention of the disease caused by bacteria , such as Staphy the same way, but more typically will be applied with a lococcus aureus and / or Streptococcus pyogenes ; ( ii ) the dropper, swab , or the like, and carefully applied to the suppression of the disease caused by bacteria , such as affected areas. Staphylococcus aureus and /or Streptococcus pyogenes ; and [0124 ] Topical application of the halogenated salicy ( iii ) the relief of the disease caused by bacteria , such as lanilide according to the present nvention enables the halo Staphylococcus aureus and /or Streptococcus pyogenes ; v ) genated salicylanilide to be delivered selectively to a spe the eradication of a non - symptomatic colonization by cific site, and avoids inter - and intra -patient variations which Staphylococcus aureus from an area on the body , ( v ) the may be associated with alternative routes of drug adminis eradication of Grain -positive bacteria such as Staphylococ tration . US 2017 /0326160 A1 Nov . 16 , 2017

[0125 ] Topical pharmaceutical compositions according to between 1- 20 % , preferably 2 - 10 % , more preferably 3 -8 % the present invention may be used to treat a variety of skin and even more preferably 4 - 6 % of active ingredient ( all or membrane disorders , such as infections of the skin or numbers given by weight ). membranes ( e . g . infections of nasal membranes , axilla , [0129 ] In microbiology , minimum inhibitory concentra groin , perineum , rectum , dermatitic skin , skin ulcers , and tion (MIC ) is the lowest concentration of an antibacterial sites of insertion ofmedical equipment such as i . v . needles , that will inhibit the visible growth of a microorganism after catheters and tracheostomy or feeding tubes ) with any of the overnight incubation . bacteria described above , ( e . g . any of the Staphylococci, 10130 ] Minimum inhibitory concentrations are important Streptococci such as S. aureus ( e . g . methicillin resistant S . in diagnostic laboratories to confirm resistance ofmicroor aureus (MRSA ) ) . Particular bacterial conditions thatmay be ganisms to an antimicrobial agent and also to monitor the treated by topical pharmaceutical compositions of the pres activity of new antimicrobial agents . A MIC is generally ent invention also include the skin - and membrane - related regarded as the most basic laboratory measurement of the conditions disclosed hereinbefore , as well as : rosacea ( in activity of an antimicrobial agent against an organism . cluding erythematotelangiectatic rosacea, papulopustular [0131 ] In toxicology , the median lethal dose , LD50 ( abbre rosacea , phymatous rosacea and ocular rosacea ) ; erysipelas; viation for “ lethal dose , 50 % ” ) of a toxin , radiation , or erythrasma ; ecthyma ; ecthyma gangrenosum ; impetigo ; pathogen is the dose required to kill half the members of a paronychia ; cellulitis ; folliculitis ( including hot tub follicu tested population after a specified test duration . LD5, figures litis ); furunculosis ; carbunculosis ; staphylococcal scalded are frequently used as a general indicator of a substance 's skin syndrome ; surgical scarlet fever ; streptococcal perianal acute toxicity . disease ; streptococcal toxic shock syndrome; pitted kera [0132 ] Therapeutic index ( .therapeutic defined as the tolysis ; trichomycosis axillaris ; pyoderma ; external canal amount of a therapeutic agent causing the therapeutic effect ear infections ; green nail syndrome; spirochetes; necrotizing measured as MIC to the amount that causes death in animal fasciitis ; Mycobacterial skin infections ( such as lupus vul studies measured as LD50 . Some antibiotics require moni garis , scrofuloderma , warty tuberculosis , tuberculides, ery toring to balance efficacy with minimizing adverse effects , thema nodosum , erythema induratum , cutaneousmanifesta including : gentamicin , vancomycin , amphotericin B (nick tions of tuberculoid leprosy or lepromatous leprosy, named ' ampho -terrible ' for this very reason ) , and polymyxin erythema nodosum leprosum , cutaneous M . kansasii, M . B . Other MIC values could be used as well. malmoense , M . szulgai, M . simiae , M . gordonae, M . hae 0133 ] The rate of resistance development is quantified as mophilum , M . avium , M . intracellular, M . chelonae ( includ the frequency of spontaneous mutants in a population of ing M . abscessus) or M . fortuitum infections, swimming bacteria that is able to resist a given concentration of the pool (or fish tank ) granuloma, lymphadenitis and Buruli antibiotic . For example the rate of resistance development ulcer ( Bairnsdale ulcer, Searles ' ulcer, Kakerifu ulcer or may by 10 - if on average 1 cell in 109 cells is able to survive Toro ulcer ) ) ; as well as infected eczema, burns, abrasions a concentration of antibiotic corresponding to 1xMIC . and skin wounds . Particular fungal conditions that may be 101341. In microbiology , colony - forming unit (CFU ) is a treated by topical pharmaceutical compositions of the pres rough estimate of the number of viable bacteria or fungal ent invention also include the skin - and membrane -related cells in a sample . Viable is defined as the ability to multiply conditions disclosed hereinbefore, as well as: candidiasis ; via binary fission under the controlled conditions . sporotrichosis ; ringworm ( e . g . tinea pedis , tinea cruris , tinea [0135 ] Also disclosed are the following numbered clauses capitis , tinea unguium or tinea corporis ) ; tinea versicolor; [0136 ] 1. Halogenated salicylanilides selected from the and infections with Trichophyton , Microsporum , Epidermo group consisting of closantel, rafoxanide , oxyclozanide phyton or Pityrosporum ovale fungi . and niclosamide and derivatives thereof including salts , [0126 ] In some embodiments of the invention the topical hydrates and esters for use in topical prevention or pharmaceutical compositions of the present invention is not treatment of an infection or disease caused by Gram used to treat a fungal infection , for example the composition positive bacteria , characterized by a rate of developing is not used to treat candidiasis ; sporotrichosis ; ringworm spontaneous mutation frequency less than 10 - . ( e . g . tinea pedis , tinea cruris , tinea capitis , tinea unguium or [0137 ] 2 . The halogenated salicylanilides according to tinea corporis ) ; tinea versicolor ; and infections with clause 1 , characterized by a rate of developing spon Trichophyton , Microsporum , Epidermophyton or Pityrospo taneous mutation frequency less than 10 - 7 or 10 -8 . rum ovale fungi. , [0138 ] 3. The halogenated salicylanilides according to [0127 ] The application regimen will depend on a number clause 2 , characterized by a rate of developing spon of factors that may readily be determined , such as the taneous mutation frequency less than 4x10 - 9 . severity of the condition and its responsiveness to initial [0139 ] 4 . The halogenated salicylanilides according to treatment, but will normally involve one or more applica any of clauses 1 - 3 , selected from the group consisting tions per day on an ongoing basis . The effective dosage of of closantel, rafoxanide, oxyclozanide and niclosamide the pharmaceutical composition of the present invention and salts thereof for use in topical prevention or treat varies from the formulation , administration pathway , age , ment of an infection or disease caused by Gram weight and gender of animal or human with a disease caused positive bacteria , characterized by a rate of developing by Staphylococcus aureus , severity of a disease , diet, admin spontaneous mutation frequency less than 10 - 9 or less istration frequency and pathway, excretion and sensitivity . than 10 - 7 or 10 - or less than 4x10 - 9 . [ 0128 ] Generally , the amount of the halogenated salicy [0140 ] 5 . The halogenated salicylanilides according to lanilide or a derivative thereof to be administered topically any of clauses 1- 4 , selected from the group consisting is in the range of 0 ,01 - 10000 mg/ cm², preferably between of closantel , rafoxanide , oxyclozanide and niclosamide 0 , 1 - 1000 mg/ cm ' and even more preferably between 1 - 100 for use in topical prevention or treatment of an infec mg/ cm² using a pharmaceutical formulation containing tion or disease caused by Gram -positive bacteria , char US 2017 /0326160 A1 Nov . 16 , 2017

acterized by a rate of developing spontaneous mutation treatment of an infection caused by bacteria , wherein frequency less than 10 - or less than 10 - 7 or 10 - 8 or less the medicament is niclosamide and hydrates of salts than 4x10 - 9 . thereof or hydrates of such salts and characterized by a [0141 ] 6 . The halogenated salicylanilides according to rate ofdeveloping spontaneous mutation frequency less any of clauses 1- 5 , wherein the halogenated salicy than 10 - 8 or less than 4x10 - 9 . lanilides is niclosamide and derivatives thereof includ [0148 ] 13 . A pharmaceutical composition comprising ing salts , hydrates and esters for use in topical preven niclosamide and derivatives thereof including salts , tion or treatment of an infection or disease caused by hydrates , esters and hydrates of such salts as the active Gram - positive bacteria , characterized by a rate of ingredient wherein the pharmaceutical composition developing spontaneous mutation frequency less than reduces or eliminates resistance development by the 10 - or less than 10 - 7 or 10 - 8 or less than 4x10 - 9 . bacteria , Staphylococcus aureus or Streptococcus pyo [0142 ] 7. The halogenated salicylanilides according to genes against the used niclosamide by a rate of devel any of clauses 1 - 6 , wherein the halogenated salicy oping spontaneous miss than 10 - 8 or less than 4x10 - 9 . lanilides is niclosamide and hydrates of salts thereof or [0149 ] Following examples are intended to illustrate , but hydrates of such salts for use in topical prevention or not to limit , the invention in any manner, shape , or form , treatment of an infection or disease caused by Gram either explicitly or implicitly . positive bacteria , characterized by a rate of developing spontaneous mutation frequency less than 10 - or less than 4x10 - 9 . EXAMPLES [0143 ] 8 . The halogenated salicylanilides according to any of clauses 1 - 7 , wherein the infection or disease is Example 1 selected from the group consisting of impetigo , bacte [0150 ] Experimental tests were conducted to determine rial conjunctivitis , atopic dermatitis and related infec the antibacterial activity and the mutation rate conferring tions, nasal eradication , sycosis barbae , superficial fol resistance for halogenated salicylanilides and reference liculitis , paronychia erythrasma , secondary infected compounds . dermatoses , carbuncles , furonculosis , ecthyma, celluli tis , erysipelas, necrotising fasciitis , secondary skin [0151 ] Microorganisms infections of wounds , dermatitis , scabies and diabetic [0152 ] Chosen for its relevance regarding bacterial skin ulcers . infections , the methicillin - resistant S. aureus (MRSA ) 01 [0144 ] 9 . The halogenated salicylanilides according to strain was used as the primary test microorganism . This clauses 8 , wherein the infection or disease is selected strain is a community -acquired MRSA clinical isolate of from the group consisting of impetigo , bacterial con USA 300 type , from a skin abscess. junctivitis , atopic dermatitis and infections associated [ 0153 ] Twenty -one other MRSA and methicillin - sensitive with different skin conditions. S . aureus strains , and 4 Streptococcus pyogenes strains , were 10145 ] 10 . The halogenated salicylanilides according to also included in the study ( Table 1 ) . These covered fusidic any of clauses 1 - 9, wherein the bacteria is Staphylo acid - and mupirocin - resistant strains, these two types of coccus aureus or Streptococcus pyogenes. resistance being of clinical relevance . [0146 ] 11 . A method for manufacturing a medicament 101541 Strains were conserved in Luria Bertani (LB ) Broth for use in topical prevention or treatment of an infec ( S . aureus) or Brain Heart Infusion ( BHI) ( S . pyogenes ) tion caused by bacteria , wherein the medicament is supplemented with glycerol 15 % ( v / v ) at - 80° C . , and niclosamide and derivatives thereof including salts , reactivated by isolation on LB (S . aureus ) or BHI ( S . hydrates and esters and characterized by a rate of pyogenes ) agar plates. Strains were cultivated in Mueller developing spontaneous mutation frequency less than Hinton (MH ) Broth — cation adjusted ( S . aureus ) supple 10 - 6 or less than 10 - 7 or 10 - 8 or less than 4x10 - 9 . mented with lysed horse blood 2 .5 % ( v /v ) ( S . pyogenes) . All [0147 ] 12 . Themethod for manufacturing a medicament strains were cultivated at 37° C . , aerobically for S . aureus according to clause 11 for use in topical prevention or strains . TABLE 1 Strains list and information Mupirocin and fusidic acid USA MLSTSCC spa Species Strains Resistance gene type mec type Origin S . aureus Newman MRSA 01 USA 300 ST8 - IV t008 SSI MRSA 02 ST30 - IV t019 SSI MRSA 03 * ND USA 400 ST1 - IV t127 SSI MRSA 04 ST772 - V t657 SSI MRSA 05 ST130 - XI t843 SSI MRSA 06 SSI CC97 - 5C2 ( V ) SSI MRSA 07 ST398 KU MRSA 08 1 NDND USA 300 STS KU MRSA 09 USA600 ST45 KU MRSA 10 ST22 - IV KU MRSA 11 ST36 - II KU US 2017 /0326160 A1 Nov . 16 , 2017

TABLE 1 -continued Strains list and information Mupirocin and fusidic acid USA MLSTSCC spa Species Strains Resistance gene type mec type Origin EEFIC 01 * CC123 t171 SSI EEFIC 02 CC123 t171 SSI MRSA 12 fusB CC80 t044 SSI MRSA 13 fusB CC80 t044 SSI MSSA 01 fusc CC1 t127 SSI MSSA 02 fusc CC1 t127 SSI MRSA 14 fusA CC22 t2006 SSI MRSA 15 fusA CC30 t166 SSI MRSA 161 mupA CC30 t019 SSI MRSA 17 mupB CC509 t375 SSI S . pyogenes 01 SSI CCUG 25571 ATCC 19615 ATCC 12385

All Staphylococcus aureus strains but one MRSA 07 ) are human clinical isolates ; MRSA 07 is a Livestock -associated MRSA ; MRSA 02 and MRS 04 are Community -associated MRSA ; * strains resistant to fusidic acid ; + strains resistant to mupirocin ; ND : Not determined ; EEFIC : Epidemic European Fusidic acid - resistant Impetigo Clone ; MLST: Multilocus Sequence Typing ; SSCmec: staphylococcal cassette chromosome mec ; spa: S . aureus protein A ; KU : Copenhagen University ; SSI: National Reference Laboratory for Staphylococci, Statens Serum Instityt, Copenhagen , Denmark . [0155 ] Antibacterial Activity experiment. This experiment was performed with the pri [ 0156 ] The following tests were performed to assess the mary test microorganism indicated above . antibacterial activity in vitro ( FIG . 1 ) : [0162 ] The overnight culture was stopped and OD600 was [0157 ] 1 . Minimum Inhibitory Concentration (MIC ) measured . Culture was then diluted in indicated medium to Assay obtain an OD600 of 0 .25 in order to have about 5x108 cfu /ml . [0158 ] TheMIC was determined using 96 - well plates, and Two hundred ul of this diluted culture were then added in all serial two -fold dilutions of niclosamide (from Sigma ) ( from conditions except in the negative control. Initial bacterial 51 . 2 to 0 .025 ug /ml ) in above indicated medium , with 150 concentration was about 5x100 cfu /ml . Tubes were incu ul per well . Bacterial cultures were stopped in their expo bated aerobically at 37° C . for 24 hours . nential growth phase and plates were inoculated with the 0163 ] Bacteria were enumerated before the incubation , approximate concentration of 10 cells per well. Plates were after 30 minutes , 1 , 2 , 4 , 8 and 24 hours of incubation by incubated at 37° C . for 18 hours ( S . aureus ) or 24 hours ( S . serial dilutions in NaCl 0 . 9 % and plating on LB agar, with pyogenes ) . Optical density at a wavelength of 600 nm was 2 plates per dilution . Plates were incubated at 37° C . and measured at the end of the incubation time. Inhibition was colonies enumerated after 24 hours . calculated as ( Inhibition = 1 - OD ODo no treatmetreatment ) and MIC [0164 The compound was considered bactericidal if the values were determined as the minimum concentration giv reduction of the bacterial inoculum was superior or equal to ing 100 % inhibition . Experiments were performed at least as 3 logio cfu /ml , bacteriostatic if reduction was inferior to 3 triplicate biological replicates with all strains . log10 cfu /ml . [ 0159 ] The inhibition could be either due to a bactericidal [ 0165 ] 3 . Mutational Frequency Evaluation or a bacteriostatic activity , which is not known from this [0166 ] The frequency of spontaneous single -step muta experiment. The following assay was thus carried out in tions was determined on 3 different strains (MRSA , fusidic order to determine if niclosamide kills or inhibits growth of acid - resistant and mupirocin -resistant ) as described by S . aureus. Drago et al. (2005 ) (Drago , L . , De Vecchi, E . , Nicola , L . , [0160 ] 2 . Time- Kill Assay Tocalli , L . , & Gismondo , M . R . ( 2005 ) . In vitro selection of [0161 ] Assay was performed in 20 ml of medium . It resistance in Pseudomonas aeruginosa and Acinetobacter included a negative control (medium without bacteria ) , a spp . by levofloxacin and ciprofloxacin alone and in combi positive control (bacteria grown without niclosamide ) and nation with beta -lactams and amikacin . The Journal of the assay (bacteria grown with niclosamide ) . Niclosamide Antimicrobial Chemotherapy, 56 ( 2 ) , 353 - 359 ) . One hundred was tested at 10 fold its MIC , determined in the previous ul of an initial inoculum of 10 % cfu /ml from an overnight US 2017 /0326160 A1 Nov . 16 , 2017

culture were plated on LB agar plates supplemented with the [ 0183 ] Results and Conclusions test compound (Ox , 1x , 2x , 4x and 10x MIC ) . Adequate [0184 ] Microbiology: MIC & Kill Curves — FIGS. 1 & 2 dilutions were plated on plates without the compound . and Tables 2 and 3 [0167 ] Viable cell growth was enumerated after 48 hours of incubation at 37° C . TABLE 2 [0168 ] Ten replicates were carried out for each strain and fusidic acid , mupirocin and retapamulin were used as con in - vitro susceptibility of S. aureus clinical isolates trols for the MRSA 01 strain . and S . aureus ATCC 29213 reference strain . [0169 ] Colony- Forming Unit (CFU ) in Skin Lesions MIC (ug / ml ) 10170 ] Three animal studies were performed with niclos amide and fusidic acid as comparison with different formu S . aureus strains Newman lations and doses . MRSA 01 0 . 4 [0171 ] In each experiment , female Balb / c mice received a MRSA 02 0 . 2 skin lesion approximately 1 cm² and were challenged with MRSA 03 * 0 . 4 1 .5x108 cfu MRSA 01 topically . Twenty - four hours after the MRSA 04 0 . 4 wound formation and the contamination , mice were treated MRSA 05 0 . 2 topically with 0 .05 ml twice daily for three days . Mice were MRSA 06 0 . 4 sacrificed on day 4 , skin lesions were excised and cfu MRSA 07 0 . 2 quantitated . Fusidin ointment ( 2 % from Leo Pharma ) was MRSA 08* 0 . 4 included as control. MRSA 09 0 .2 [0172 ] Experiment 1 MRSA 10 0 . 4 [0173 ] Sample 5 . — Niclosamide (N ) 5 % modified basis MRSA 11 0 . 1 creme with higher lipid content - Lipocreme _ according to EEFIC 01 * 0 . 4 the description in Danske Laegemiddelsstandarder (DLS ) . EEFIC 02 * 0 . 2 [0174 ] 2 .25 g niclosamide was mixed with 47 .75 g crème MRSA 12 * 0 .4 prepared according to Danske Laegemiddels - standarder MRSA 13 * 0 . 4 (DLS ) ( see FIG . 3 ) . MSSA 01 * 0 . 4 MSSA 02 * 0 . 4 [0175 ] Oil phase : Polysorbate 80 10 g MRSA 14 * 0 . 4 [0176 ] Cetostearyl alcohol 100 g MRSA 15 * 0 . 2 [0177 ] Paraffin oil 100 g MRSA 16 * 0 . 2 MRSA 17 * [0178 ] Glycerol monostearate 40 - 50 120 g S . pyogenes strains 01 [0179 ] Water phase : Methyl parabenzoate 1g CCUG 25571 [0180 ] Glycerol 85 % 40 g ATCC 19615 3 . 2 [0181 ] Sorbitol 70 g ATCC 12385 1 . 6 [0182 ] Water Milli - Q 724 g TABLE 3 Therapeutic indexes of halogenated salicylanilides, calculated from their MICs against S. aureus and S. pyogenes strains and their lethal Dose 50 (LD50 ) in rats and mice . Mic 100 LD50 LD50 Ther. Index S . aureus S. pyogenes rats p . mice p . L Dso, rais! Compound ug/ ml UM ug/ ml UM (mg / kg ) (mg / kg ) MIC100 , S. aureus ) Niclosamide 5050 . 4 <$ 1 . 25 25 <53 3 s353 5000 > 1500 12 ,500 ,000 ClosantelClosantel s $ 1. 7 $ 2 .5 12 .87 198 300 331 176, 000 Oxyclozanide 1 . 6 * 4 * 6 .4 15 .97 980 -3500 300 612, 000 -2187 ,000 Rafoxanide UX * 1. 25 * 6. 47 10 + 1500 270 1, 875 ,000 * Tested against one strain : MRSA 01 * Tested against one strain : S . pyogenes 01 US 2017 /0326160 A1 Nov . 16 , 2017 13

[0185 ] The data shows that halogenated salicylanilides [0194 ] In conclusion the in vivo data shows that niclos such as closantel, oxyclozanide, rafoxanide and particularly a mide is well suited as a topical antibiotic for treatment of niclosamide are strongly potent against Gram -positive infections caused by Gram positive infections. strains such as S . aureus and S . pyogenes. Notably the effect is independent of the resistance profile of the isolates Example 2 towards other currently used antibiotics for topical treatment of these microorganisms, including fucidic acid and mupi [0195 ] Additional More Extensive Screen of Clinical Iso rocin . Accordingly , the halogenated salicylanilides in gen lates Performed with Niclosamide . eral and niclosamide in particular are well suited as a [0196 ] Methods possible treatment for both susceptible and resistant Gram [0197 ] Microorganisms positive strains . [0198 ] Chosen for its relevance regarding bacterial skin [0186 ] Microbiology Resistance Development — Table 4 infections , the methicillin -resistant S . aureus (MRSA ) 01 [0187 ] Spontaneous mutations conferring resistance to strain was used as the primary test microorganism . This halogenated salicylanilides occurred at a very low frequency strain is a community -acquired MRSA clinical isolate of (mutational frequency = 5x10 - 9 , 2x10 - 8 and 1x10 - 7 at MIC USA 300 type , from a skin abscess . x1 for rafoxanide, closantel, and oxyclozanide respectively ) [0199 ] Two - hundred - four other MRSA and methicillin and for niclosamide at an extremely low frequency (Os sensitive S . aureus strains , and 4 Streptococcus pyogenes mutational frequency < 4x10 - 1° at MIC X 1 ) compared to strains , were also included in the study . These covered currently used antibiotics such as fusidic acid , retapamulin fusidic acid - and mupirocin - resistant strains, these two types and mupirocin (mutational frequency: 24x10 -5 at MIC 1) of resistance being of clinical relevance . ( see Table 4 ) . [ 0200 ] Strains were conserved in Luria Bertani (LB ) Broth ( S . aureus ) or Brain Heart Infusion (BHI ) ( S . pyogenes ) TABLE 4 supplemented with glycerol 15 % (v / v ) at - 80° C ., and Mutation rates conferring resistance to halogenated salicylanilides . reactivated by isolation on LB ( S . aureus ) or BHI ( S . pyogenes ) agar plates. Strains were cultivated in Mueller Compound Resistance mutation rate at MIC 1 Hinton (MH ) Broth — cation adjusted ( S . aureus ) or BHI ( S . Niclosamide < 4 x 10 -91 pyogenes) . All strains were cultivated aerobically (mi Closantel = 2 x 10 -8 * croaerobically for S . pyogenes strains) at 37° C . Oxyclozanide = 1 x 10 - 7 * Rafoxanide = 5 x 10 -9 * [ 0201 ] Antibacterial activity Fusidic acid 24 x 10 - 5 * Mupirocin 24 x 10 - 5 * [0202 ] 1 . Minimum Inhibitory Concentration (MIC ) Retapamulin 24 x 10 - 5 * Assay Mutation rate conferring resistance against MRSA 01, MRSA 15 ( fusidic acid - resistant ) [ 0203 ] Minimal inhibitory concentrations (MICs ) of and MRSA 16 (mupirocin -resistant ) niclosamide , fusidic acid , and mupirocin were determined * Tested against one strain : MRSA 01 according to CLSI criteria with a doubling dilution concen tration range ( 16 to 0 .008 ug /ml ) in Mueller Hinton Broth [ 0188 ] A . The mutational frequency data gives the fre cation - adjusted (Fluka Analytical 90922 ), using 96 -well quency of a resistant mutant within a given population . plates , for 205 different S . aureus strains . S . aureus ATCC When the mutational frequency is bellow 10 - 9 means that 29213 was included as a control reference strain and clin there is less than one resistant mutant in a population of 109 damycin and vancomycin were included as control antibi cells . otics. [ 0189 ] Unexpectedly , the resistance development towards [0204 ] Bacterial cultures were stopped in their exponential halogenated salicylanilides in general and niclosamide in growth phase and plates were inoculated with the approxi particular is much slower than resistance development mate concentration of 5x10 cells per well . Plates were towards drugs like fusidic acid , mupirocin and retapamulin incubated at 37° C . for 18 hours (S . aureus ) or 24 hours ( S. being on the market. pyogenes) . Optical density at a wavelength of 600 nm was [0190 ] In combination , the high potency and the imple measured at the end of the incubation time. Inhibition was mentation of the low rate of resistance development makes calculated as ( Inhibition = 1 - OD O D no treatmenttreatment) and MIC niclosamide particularly useful for treatment, especially values were determined as the minimum concentration giv topical treatment of infections caused by Gram positive ing 100 % inhibition . organisms. [0205 ] Due to interference with blood (MIC increased by [0191 ] In view of the unexpected microbiological findings 16 with 5 % lysed horse blood ), the MIC determination that niclosamide has very unique properties that make it an against S . pyogenes strains was performed in BHI. ideal candidate as a topical anti - infective animal experi [0206 ] Results and Discussion ments were performed to test the effect of niclosamide in [ 0207 ] In Vitro MIC Determination and Breadth of Effect vivo . [ 0208 ] TheMIC ofniclosamide was s0. 5 ug/ ml against all 10192 ] The data described herein shows that niclosamide targeted S . aureus and S . pyogenes strains, including the reduces colonization by S . aureus in animal model used for strains resistant to fusidic acid , mupirocin , clindamycin and the tests. retapamulin ( Table 5 , Table 6 , Table 7 and FIG . 4 ). Dose [0193 ] In Experiment 1 (FIG . 3 ) niclosamide in the tested response curves of niclosamide, fusidic acid and mupirocin ointment and cream formulations led to significant bacterial against S . aureus with different resistance profiles are rep reductions compared to the control vehicles. resented in Figure X . US 2017 /0326160 A1 Nov . 16 , 2017 14

TABLE 5 in -vitro susceptibility of S. aureus clinical isolates and S. aureus ATCC 29213 reference strain . Resistances are indicated in Bold . ND : not determined . MIC (ug /ml ) niclosamide Fusidic acid Mupirocin Retapamulin Clindamycin Vancomycin ATCC 29213 0 . 5 0 .06 0 . 125 0 .03 0 . 125 Newman 0 . 25 0 . 25 0 . 25 0 .03 0 . 125 MRSA 01 0 . 25 0 . 125 0 .25 0 .06 0 . 125 MRSA 02 0 . 25 0 . 5 0 .25 0 . 06 0 . 25 MRSA 03 0 . 25 16 0 .25 0 .03 0 . 125 MRSA 04 0 . 25 0 . 25 0 . 25 0 .06 0 . 125 MRSA 05 0 .25 0 . 125 0 . 125 0 .03 0 . 125 MRSA 06 0 . 25 0 .125 0 . 25 0 . 03 0 . 125 MRSA 07 0 . 125 0 .25 0 .25 0 . 06 0 . 125 MRSA 08 0 . 25 0 . 125 > 16 0 .03 > 16 MRSA 09 0 . 125 0 .25 0 .25 0 . 06 0 . 25 MRSA 10 0 .25 0 . 25 0 .06 0 . 25 MRSA 11 0 . 25 0 . 5 0 .06 > 16 EEFIC 01 0 . 25 0 . 125 0 . 03 0 .125 EEFIC 02 0 .25 0 . 125 0 .03 0 . 125 MRSA 12 0 . 5 0 . 125 0 .03 ND MRSA 13 0 . 25 0 . 25 0 . 06 0 . 125 MSSA 01 0 . 5 0 . 125 0 .03 0 . 125 MSSA 02 0 . 25 0 . 125 0 . 03 0 . 125 ZAPANANANANANA MRSA 14 0 . 25 > 16TTTTTT 0 . 125 0 . 03 > 16 MRSA 15 0 .25 > 16 0 . 25 0 . 06 0 . 125 MRSA 16 0 . 25 0 .25 > 16 0 . 06 0 .25 MRSA 17 0 . 25 > 16 0 . 03 > 16 MRSA 18 0 . 25 0 .25 0 . 125 0 . 06 0 . 125 MRSA 19 0 . 25 0 . 125 0 . 25 0 .03 0 . 125 MRSA 20 0 . 25 0 . 125 0 . 25 0 .06 0 . 06 MRSA 21 0 . 25 0 . 125 0 .125 0 . 125 0 . 03 MRSA 22 0 . 25 > 16 0 . 25 0 . 06 0 . 06 0 . 5 MRSA 23 0 . 25 0 .25 0 .25 0 . 06 0 . 125 PAPANANARA MRSA 24 0 . 25 0 . 25 0 .25 0 . 06 0 . 125 MRSA 25 0 . 25 16 0 .25 0 . 06 0 . 125 MRSA 26 0 . 5 0 . 5 0 . 5 0 . 06 > 16 MRSA 27 0 . 5 0 . 25 0 . 25 0 . 06 0 . 125 MRSA 28 0 . 25 0 . 06 0 . 5 0 . 03 0 . 06 MRSA 29 0 . 5 0 . 25 0 . 06 0 . 125 MRSA 30 0 . 25 0 . 125 0 . 125 0 .03 0 .06 MRSA 31 0 . 25 0 . 25 0 . 25 16 > 16 MRSA 32 0 . 25 0 . 5 0 .25 0 . 06 0 . 125 MRSA 33 0 . 25 0 . 5 0 . 25 0 . 06 0 . 125 MRSA 34 0 . 25 0 . 25 0 . 25 0 .06 0 . 125 MRSA 35 0 . 25 16 0 . 5 0 . 03 > 16 MRSA 36 0 . 25 0 . 25 0 .06 0 . 06 MRSA 37 0 . 25 0 . 5 0 . 25 0 .06 0 . 125 MRSA 38 0 . 25 0 .125 0 . 06 0 . 125 MRSA 39 0 . 25 0 . 25 0 . 06 0 . 06 MRSA 40 0 . 25 0 .125 0 .25 0 . 03 0 .06 MRSA 41 0 . 25 > 16 0 . 125 0 .02 0 .06 MRSA 42 0 . 25 0 .25 0 . 25 0 . 06 > 16 MRSA 43 0 . 25 0 . 125 0 . 25 0 .06 0 .06 MRSA 44 0 . 25 0 . 25 0 . 02 0 .03 MRSA 45 0 . 25 0 . 06 0 . 13 0 .02 0 .06 MRSA 46 0 .25 0 . 06 0 . 13 0 .02 0 .03 MRSA 47 0 . 25 0 .06 0 . 25 0 .02 > 16 0 . 5 MRSA 48 0 . 25 0 .13 0 . 02 0 .03 0 . 5 MRSA 49 0 . 25 0 .25 0 .25 0 .02 0 .03 MRSA 50 0 . 25 0 .03 0 . 25 0 .02 0 .02 MRSA 51 0 . 125 0 . 13 0 .25 0 .03 0 .06 MRSA 52 0 . 25 0 .06 0 . 25 0 . 02 0 .03 MRSA 53 0 . 25 0 . 25 0 . 25 0 . 03 0 .06 MRSA 54 0 . 25 0 . 125 0 . 25 0 . 03 0 . 06 MRSA 55 0 . 25 0 .25 0 . 02 0 . 03 ORHANOHRAHONNAO MRSA 56 0 . 25 0 .06 0 .25 < 0 .01 0 . 03 MRSA 57 0 . 25 0 . 125 0 . 125 0 . 02 0 .03 MRSA 58 0 . 25 0 . 125 0 .02 0 .06 MRSA 59 0 . 25 0 . 06 0 . 125 0 . 02 0 . 03 MRSA 60 0 . 25 0 . 25 0 .02 0 .06 MRSA 61 0 . 25 0 . 06 0 .25 0 .02 0 . 03 MRSA 62 0 . 25 0 .06 0 . 25 0 .02 0 .06 MRSA 63 0 . 25 0 . 125 0 . 125 0 .02 0 .06 MRSA 64 0 .25 0 . 25 0 .25 0 .02 0 .06 US 2017 /0326160 A1 Nov . 16 , 2017 15

TABLE 5 -continued in -vitro susceptibility of S. aureus clinical isolates and S. aureus ATCC 29213 reference strain . Resistances are indicated in Bold . ND : not determined . MIC (ug /ml ) niclosamide Fusidic acid Mupirocin Retapamulin Clindamycin Vancomycin MRSA 65 0 . 25 0 . 06 0 . 25 0 .02 0 .03 MRSA 66 0 . 25 0 . 06 0 . 25 0 .02 0 . 03 1 MRSA 67 0 . 25 0 . 25 0 . 125 0 . 02 0 .03 - MRSA 68 0 .25 0 . 125 0 . 25 0 .02 0 .03 - MRSA 69 0 .25 0 . 125 < 0 . 01 0 .03 - MRSA 70 0 . 125 0 . 25 0 . 02 0 .06 - MRSA 71 0 .25 0 . 06 0 . 125 < 0 .01 0 .02 - MRSA 72 0 . 25 16 0 . 25 0 .03 0 .06 - MRSA 73 0 . 5 0 . 25 0 . 25 0 . 03 0 . 125 - MRSA 74 0 .25 0 . 25 0 . 25 0 .03 0 . 125 - MRSA 75 0 . 5 0 . 5 0 . 5 0 .03 0 . 125 - MRSA 76 0 . 25 0 . 25 0 .25 0 .03 0 . 125 - MRSA 77 0 . 25 16 0 . 25 0 .03 0 . 125 - MRSA 78 0 . 5 0 . 125 0 . 25 0 . 03 0 .06 - MRSA 79 0 . 25 0 . 5 0 . 25 < 0 .01 0 . 03 - MRSA 80 0 . 5 0 . 125 0 . 25 0 . 02 0 . 06 - MRSA 81 0 .5 0 . 25 0. 25 0 .03 0 . 125 - MRSA 82 0 .25 0 . 25 0 . 03 ?????0 . 125 - MRSA 83 0 . 5 0 .06 0 . 25 0 .03 0 .06 - 0 .02 - MRSA 27b 0 .25 Oooo 0 . 25 0 .06 MRSA 84 0 . 25 0 . 25 0 .03 0 . 125 - MRSA 85 0 . 5 0 . 25 0 .03 0 . 125 - MRSA 86 0 .25 > 16 0 . 25 0 .03 0 .06 - MRSA 87 0 .25 0 . 125 0 . 25 0 .02 0 . 06 - MRSA 88 0 . 25 0 .06 0 . 25 0 .03 0 .06 - MRSA 89 0 .5 0 .06 0 . 25 0 .02 > 16 - MRSA 90 0 . 5 16 0 . 25 0 . 06 0 . 125 - MRSA 91 0 . 25 0 .25 0 . 25 0 .06 0 . 125 - MRSA 92 0 . 25 0 .25 0 .06 0 .06 - MRSA 93 0 . 25 0 .02 0 . 25 0 .02 0 .03 - MRSA 94 0 . 25 0 . 125 0 . 25 0 .03 0 . 125 - MRSA 95 0 . 25 8 0 . 25 0 .03 0 . 125 -

MRSA 96 0 . 25 0 . 125 0 . 125 0 .03 > 16 - MRSA 97 0 . 25 0 . 25 0 .03 ?????????0 . 06 - MRSA 98 0 . 5 0 .06 0 . 5 0 .03 > 16 - MRSA 99 0 . 25 33,289090.323982*2531329331135-4333299910 . 125 0 . 5 0 .03 0 . 125 - MRSA 100 0 .25 0 . 125 0 . 5 0 . 25 - MRSA 101 0 . 5 0 . 25 0 .03 0 . 06 - MRSA 102 0 . 25 0 . 06 0 . 25 0 .03 0 . 06 - MRSA 103 0 . 25 0 . 25 0 .03 0 . 125 - MRSA 104 0 . 5 0 . 25 0 . 25 0 .03 0 . 125 - MRSA 105 0 .25 0 . 125 0 . 25 0 .03 0 . 125 - MRSA 106 0 . 25 0 .125 0 . 25 0 .06 0 . 125 - MRSA 107 0 . 25 0 . 25 0 . 25 0 .06 0 . 125 - MRSA 108 0 . 25 0 . 25 0 .03 0 . 125 - MRSA 109 0 . 25 0 .25 0 . 25 0 .03 0 . 125 - MRSA 110 0 . 25 0 .06 0 . 125 0 .03 0 . 125 -

MRSA 111 0 . 5 0 . 25 0 .03 0 . 125 - MRSA 112 0 . 25 0 .06 0 . 25 0 . 03 0 .06 - MRSA 113 0 . 25 0 . 125 0 . 25 0 .03 0 . 06 - K000796 0 . 25 0 . 5 0 .03 0 . 125 - K115688 0 .25 0 . 125 0 .25 0 .03 0 . 125 -

K000866 0 . 25 8 0 . 5 0 .03 0 . 125 - K000864 0 . 25 0 . 25 0 . 25 0 .03 0 . 125 - K000863 0 .25 0 . 5 0 .03 0 . 125 - K115689 0 . 25 0 . 125 0 . 25 0 .03 0 . 125 - K000772 0 . 25 > 16 0 . 125 0 .06 0 . 125 - K115498 0 . 25 0 .125 0 . 5 0 .03 0 . 125 - R000024 0 . 25 16 0 . 5 0 .06 0 . 125 - R000020 0 . 5 0 . 125 0 . 5 0 .03 0 . 125 - R000019 0 . 5 0 . 125 0 .5 0 . 06 0 . 125 - U115579 0 . 25 0 . 25 0 . 5 0 .06 0 . 125 - 1153700 0 . 25 0 . 5 0 . 125 0 . 06 0 . 125 - 114660U 0 . 25 0 .25 0 . 25 0 .06 0 . 125 - 115584D 0 . 25 0 . 25 0 . 5 0 .06 0 . 125 - 115740E 0 . 5 0 . 25 CCCCCCCCCCCCCCECCEZEGGEREGGEGCCLEEGSEGGGGGGGGGGGG0 . 25 0 . 06 0 . 125 - 115810E 0 .25 0 .25 0 . 25 0 .06 0 . 125 - 115628T 0 .25 0 .25 0 .03 0 .06 - 0002741 0 .25 0 . 5 0 . 5 0 . 06 0 . 125 - 115691T 0 . 5 0 . 25 0 . 25 0 .03 0 . 125 - US 2017 / 0326160 A1 Nov . 16 , 2017 16

TABLE 5 -continued in -vitro susceptibility of S. aureus clinical isolates and S. aureus ATCC 29213 reference strain . Resistances are indicated in Bold . ND : not determined . MIC ( ug/ ml ) niclosamide Fusidic acid Mupirocin Retapamulin Clindamycin Vancomycin

115903T 0 . 5 0 . 5 0 . 03 (0 . 125 — 116122T 0 . 25 0 .125 0 . 5 0 . 03 (0 . 125 — 115015T 0 .5 (0 .25 (0 . 5 () .06 (0 . 125 115273C 0 . 5 (0 .25 0 .25 0 .03 . 125 — (000040C (0 .5 0 .25 0 . 25 0 . 03 ( . 125 — 115690C 0 .25 0 . 125 0 .2 (0 . 5 115561C 0 .25 (0 .125 0 . 5 0 .03 ( . 125 — 115445C 0 . 5 (0 .25 (0 .25 0 . 03 ( 1 , 125 — 115263C 0 .25 (0 . 125 (0 .25 0 . 03 (1 . 6 115303C (0 . 5 (0 .25 (0 . 25 ( . 03 > 16 — 115268C ( . 5 (0 . 25 0 . 5 ( . 03 () . 125 — 115295C 0 .25 (0 .125 0 .25 0 . 03 ( . 125 — 115242C 0 . 5 8 . (0 .25 0 . 03 (1 . 06 — 115427C (0 . 25 () . 125 0 .25 ( . 03 (1 . 6 — (000041C 0 . 25 (0 . 25 (0 .25 0 . 03 0 . 06 — E5 -148654 0 .25 (0 . 5 0 . 25 () .06 (0 . 125 — 9 - 29 - 245 (0 .25 (0 . 25 0 . 25 0 .06 (0 . 125 — E - 1046019 (0 . 25 0 .25 (0 . 25 0 . 03 (7 . 125 — E5 - 1046020 ( . . 0 .25 0 . 25 ( . 06 . 125 — E5 - 1047585 (0 .25 0 .25 (0 .25 0 . 03 (0 . 125 E5 - 1038294 (0 . 5 (0 .25 (0 . 2 0 . 03 (0 . 125 E5 - 103579 0 . 5 0 . 125 0 . 5 0 . 03 (0 . 125 — 9 - 1 862936 (0 .5 () . 125 (0 . 25 0 .03 ( .12 — E5 - 1033091 0 . 5 0 .03 0 . 25 ( . 02 ( 1 , 06 — 9 -26422166 ( . 5 0 .25 0 . 03 (0 .125 — 9 - 2642158 (0 . 25 ( . ? (0 . 25 (0 . 06 0 .125 — E5 - 103575 0 . 5 > 16 (0 .25 0 . 06 0 . 125 — E - 1029558 0 .25 16 (0 . 5 0 . 03 (0 . 125 — E5 - 1038279 (0 . 5 0 .25 () . 03 . 125 — E - 139697 (0 . 25 0 . 5 (0 .25 () .06 ( . 12 — E5 - 1041979 (0 . 5 (0 . 25 (0 . 5 0 . 03 ( . 125 — E5 - 103284 0 . 25 (0 .25 0 . 25 0 .03 . 125 — E5 -130469 0. 25 . 125 (0 . 25 0 .03 (0 . 125 E5 - 1030472 ( (0 . 25 0 . 5 0 . 06 (0 .125 — E5 -14197 } 0 .5 16 . S 0 .03 () . 12 ? E5 - 1041987 . 16 0 .25 0 . 03 0 . 125 — E - 139684 (0 .5 16 (0 . 25 0 . 06 (0 . 125 — E5 - 1041980 (0 .25 0 . 25 (0 . 25 () . 3 . 125 —

— E -133088 0 .25 (0 .25 |0 . 25 0 . 03 () . 12 ? E5 - 10352 } } (0 . 5 16 (0 . 5 0 .03 (0 . 125 — E5 -146096 0 . 5 0 . 5 0 . 5 0 . 06 () .12 — E - 1046085 0. 5 8 . 0. 5 0 . 06 (0 . 125 9 - 2625962 0 . 5 (0 .25 (0 . 5 0 . 03 ( . 125 — E5 - 1043668 0 . 25 (0 . 2 ( . 06 (0 .25 — E5 - 1048428 (0 .25 () . 5 0 . 25 () .06 0 .25 — E - 1 (047924 0 .5 (0 . 25 (0 . 5 0 . 03 . 125 — E5 - 1047606 0 .5 0 . 5 0 . 03 () . 125 — E5 - 1046070 0 .25 (0 . 25 (0 . 5 0 . 03 () . 12 ? — E -146298 0 . 25 . 125 (0 . 25 ( . 03 (1 , 125 — E5 -146296 (0 . 5 0 .125 0 . 03 (1 .15 — E5 - 1046297 (0 . 5 . 125 (0 .25 0 .06 . 125 — ES - 1043184 . 5 16 0 . 5 0 . 03 (0 . 125 — E5 - 1038286 (0 . 25 (0 .25 () .06 (0 . 125 — E5 - 1037958 (0 . 5 16 0 .06 . 125 — E5 - 1037971 (0 .25 (0 .25 (0 . 25 0 .03 ( . 125 — E5 - 1033076 (0 .5 (0 .25 (0 . 25 0 . 03 . 125 — E5 - 1033076 (0 . 5 0 . 25 (0 .25 0 . 03 (0 . 125 — E5 - 1029252 ??(0 . 25 0 .25 (0 . 5 0 . 03 (1 .15 — E5 - 1030440 0 . 25 (0 . 25 0 .25 0 . 06 (1 . 06 E - 130482 0 . 125 16 (0 .25 0 .02 (1 . 06 E - 1 (046014 (0 . 25 (0 . 125 (0 . 25 0 . 06 ( 0 . 125 E -14824 0 . 25 (0 .25 0 .25 0 . 03 ( . 06 E - 1 (048670 ( . 125 0 . 5 0 . 5 0 . 06 (0 .125 E5 - 1046039 (0 . 25 (0 .25 (0 . 25 0 .06 (0 . 125 E5 - 1045179 (0 .25 (0 . 25 0 .25 ( . 06 (0 . 125 E5 -146723 0 .25 ( 0 . 5 (0 .25 0 . 06 (7 . 12 US 2017 /0326160 A1 Nov . 16 , 2017 17

TABLE 6 [0215 ] Mutational Frequency Evaluation [0216 ] The frequency of spontaneous single -step muta MIC distribution of niclosamide against Staphylococcus aureus tions was determined on the 3 different strains as described and Streptococcus pyogenes strains (percentage and ratio ) by Drago et al. (2005 ) and Pannu et al . ( 2011 ) . One hundred MIC (ug /ml ) 0 . 0625 0 .125 0 . 25 0 . 5 ul of an initial inoculum of about 10º cfu /ml from an overnight culture were plated on LB agar plates supple Staphylococcus 3 % 70 % 27 % mented with the test compound (Ox , 1x , 2x , 4x and 10x aureus strains ( 6 /205 ) ( 144 /205 ) ( 55 / 205 ) Streptococcus 25 % 25 % 25 % 25 % MIC ) . Adequate dilutions were plated on plates without the pyogenes strains ( 1 / 4 ) ( 1 / 4 ) ( 1 / 4 ) ( 1 / 4 ) compound . [0217 ) Viable cell growth was enumerated after 48 hours of incubation at 37° C . [0218 ] The spontaneous resistance frequency for an iso TABLE 7 late - drug combination was calculated from the number of MIC90 , MIC50 and MIC ranges of niclosamide colonies that grew on plates containing drug versus the for Staphylococcus aureus strains . number of colonies that grew on drug - free agar. [02191 . Ten replicates were carried out for each strain and MIC90 MIC50 Range values fusidic acid , mupirocin and retapamulin were used as con 0 .5 ug/ ml 0 . 25 ug/ ml 0. 125 - 0. 5 ug/ ml trols for the MRSA 01 strain . [ 0220 ] Results and Discussion [0221 ] Spontaneous mutations conferring resistance to [0209 ] Niclosamide was inhibitory at a concentration niclosamide occurred at an extremely low frequency (below equal or below 0 . 5 ug/ ml . for all targeted S . aureus and S . the detection limit ) ( 0s mutational frequency < 4 . 10 - 9 at pyogenes strains, including fusidic acid - and mupirocin MIC 1 ) for all tested strains (MRSA 01, MRSA 15 ( fusidic resistant strains . acid - resistant) and MRSA 16 (mupirocin - resistant) ) com pared to fusidic acid ( mutational frequency : 3 . 10 - at MIC Example 3 x 10 and 24 . 10 - 5 at MIC * 1 ) and to mupirocin and [0210 ] A further study was carried out to examine the retapamulin . Results with the strain MRSA 01 are shown in frequency of spontaneous mutation conferring resistance to Table 8 . TABLE 8 Frequencies of spontaneous mutations conferring resistance to niclosamide, fusidic acid , mupirocin and retapamulin with the strain MRSA 01 . Average of 10 replicates. Niclosamide Fusidic acid Mupirocin Retapamulin Concentration MIC X 1 < 4 x 10 -9 * 24 · 10 -5 24 · 10 - 5 24 . 10 -5 MIC X 2 < 4 x 10 - 9 * 2 · 10 - 5 8 · 10 - 8 3 · 10 - 7 MIC X4 14 x 10 - 9 * 1 . 10 -6 1 . 10 - 8 2 . 10 - 8 MIC ~ 10 < 4 x 10 - 9 * 3 10 - 7 54 · 10 - 9 < 4 · 10 - 9 * * Below the detection limit (no colony on plates ) niclosamide in 3 methicillin - resistant Staphylococcus [0222 ] As for MRSA 01 , no colony grew on plates with aureus strains , including a fusidic acid - and a mupirocin niclosamide with the strains MRSA 15 and MRSA 16 . These resistant strains . This frequency was compared with the led to a mutation frequency s3x10 - 8 for MRSA 15 and frequencies of spontaneous mutation conferring resistance < 1x10 - 7 for MRSA 16 at MIC x 1 (0 . 25 ug /ml ) , these to fusidic acid , mupirocin and retapamulin in one MRSA differences in the detection limits being due to differences in strain . the bacterial concentrations of overnight cultures. 10223 ] Conclusions [ 0211 ] Methods [0224 ] Frequencies of spontaneous mutations conferring [ 0212 ] Microorganisms resistance to niclosamide in Staphylococcus aureus were much lower than frequencies of spontaneous mutations [0213 ] Three methicillin - resistant Staphylococcus aureus conferring resistance to fusidic acid ,mupirocin and retapa (MRSA ) clinical isolates , with different resistance profiles mulin in Staphylococcus aureus . This supports the use of (MRSA 01 , MRSA15 [ fusidic acid -resistant strain ] and niclosamide for cutaneous decolonization of S . aureus . MRSA 16 [mupirocin - resistant strain ]) were chosen for their [0225 ] Example 4 relevance regarding bacterial skin infections The MRSA 01 [0226 ] A study was carried out to determine the effect of strain was used as the primary test microorganism . This pH on the antibacterial activity of niclosamide against strain is a community -acquired MRSA clinical isolate of Staphylococcus aureus in order to assess whether niclos USA 300 type , from a skin abscess . amide is still active against S . aureus at pH close to the pH [0214 ] Strains were conserved in Luria Bertani (LB ) Broth of the skin . supplemented with glycerol 15 % ( v / v ) at - 80° C ., and [ 0227 ] Methods reactivated by isolation on LB agar plates . Strains were [ 0228 ] Microorganisms cultivated aerobically in Mueller Hinton (MH ) Broth [0229 ] Chosen for its relevance regarding bacterial skin cation adjusted at 37° C . infections, the methicillin - resistant S . aureus (MRSA ) 01 US 2017 /0326160 A1 Nov . 16 , 2017 strain was used . This strain is a community - acquired MRSA control wells ) . However, the strain was inhibited by the clinical isolate of USA 300 type , from a skin abscess . lowest pH (pH 4 to pH 5 ) (FIG . 5 ) . [0230 ] This strain was conserved in Luria Bertani (LB ) [0239 ] MIC determinations showed that the inhibitory Broth supplemented with glycerol 15 % ( v / v ) at - 80° C . , and activity of niclosamide against MRSA 01 was increased reactivated by isolation on LB agar plates . It was then when pH was decreased , with lower MICs ( Table 9 ) . TABLE 9 pH and related MICs of niclosamide against MRSA 01 with the 3 different replicates . Replicate 1 pH after Replicate 2 Replicate 3 addition of MIC actual MIC actual MIC actual pH niclosamide (ug / ml ) pH (ug / ml ) pH (ug /ml ) Not adjusted 7 . 4 7 . 4 0 . 5 7 .4 0 . 25 7. 4 0 . 5 pH 7 . 0 6 . 921 6 . 938 0 . 25 7 .071 0 . 125 7. 025 0 .125 pH 6 . 5 6 .512 6 . 543 0 . 125 6 .566 0 .06 6 .540 0 .03 pH 6 .0 5 . 893 5 . 954 0 .06 5 . 955 0 .06 5 .980 0 .016 pH 5 . 5 5 . 569 5 .616 50 .03 5 . 547 50 .008 5 .530 50 . 008 pH 5 . 0 5 . 070 5 .095 No growth 5 .068 No growth 5 .008 No growth pH 4 . 5 4 . 569 4 . 589 in positive 4 .568 in positive 4 .550 in positive pH 4 .0 4 . 076 4 . 105 control. 4 .044 control . 4 .023 control . Strain Strain Strain inhibited inhibited inhibited by acidic by acidic by acidic PH PH PH Initial 2 . E + 05 cfu /ml 2 . E + 05 cfu /ml 3 . E + 05 cfu /ml bacterial concentration cultivated aerobically in Mueller Hinton (MH ) Broth — [0240 ] Conclusions cation adjusted at 37° C . [0241 ] The maximal inhibitory effect of niclosamide was [ 0231 ] Assessmentof the Effect of pH on the Antibacterial observed at pH 5 . 5 , which is close to the pH of the skin . Activity of Niclosamide 1 . A method of treating atopic dermatitis and related [ 0232 ] The pH of Mueller -Hinton Broth cation -adjusted infections in a subject, the method comprising topically was adjusted with HC1 2M to 7 , 6 . 5 , 6 , 5 . 5 , 5 , 4 . 5 and 5 . Ten administering to the subject an effective amount of niclos ml of medium for each pH were prepared . pH of non amide or a pharmaceutically acceptable salt or hydrate adjusted MHBII was equal to 7 . 4 . thereof. [ 0233 ] Each pH - adjusted samples were filtered on 0 . 2 um 2 . The method of claim 1 , wherein the treatment prevents filters before being used for the MIC determination assay . or ameliorates symptoms of the atopic dermatitis and related For each pH , minimal inhibitory concentrations (MICs ) of infections caused by Gram -positive bacteria . niclosamide were determined according to CLSI criteria 3 . The method of claim 2 , wherein the Gram - positive with a doubling dilution concentration range ( 16 to 0 . 008 bacteria are from the Staphylococcus genus or the Strepto ug/ ml ) . coccus genus. [ 0234 Bacterial culture was stopped in its exponential 4 . The method of claim 2 , wherein the Gram - positive growth phase and plates were inoculated with the approxi bacteria are selected from Staphylococcus aureus and Strep mate concentration of 5x10 cells per well. Plates were tococcus pyogenes. incubated at 37° C . for 18 hours ( S . aureus ) . Optical density 5 . The method of claim 2 , wherein the Gram - positive at a wavelength of 600 nm was measured at the end of the bacteria are resistant to an antibiotic other than niclosamide . incubation time. Inhibition was calculated as ( Inhibition = 1 6 . The method of claim 2 , wherein the Gram - positive OD OD treatment) and MIC values were determined as bacteria are resistant to an antibiotic selected from fusidic the minimum concentration giving 100 % inhibition . acid , mupirocin and retapamulin . [0235 ] The experiment was performed in triple biological 7. The method of claim 2 , wherein the Gram -positive replicates. bacteria are methicillin -resistant Staphylococcus aureus. [ 0236 ] Results and Discussion 8 . The method of claim 1 , wherein the niclosamide is [0237 ] The pH of different pH - adjusted media were administered topically to the subject in the form of a checked after the addition of niclosamide in order to check pharmaceutical formulation comprising the niclosamide , or that the addition of niclosamide did not have any influence a hydrate thereof. on the pH . Measurements showed that the addition of 9 . The method of claim 1 , wherein the niclosamide is niclosamide ( 16 ug/ ml ) in the pH -adjusted media had no administered topically to the subject in the form of a influence on the pH ( Table 9 ). pharmaceutical formulation comprising from 2 to 10 % by [0238 ] MRSA 01 grew equally well from pH 6 to pH 7 . 4 weight of niclosamide . (OD600 ~ 0 . 2 in average in positive control wells ) and 10 . The method of claim 1 , wherein the subject is a slightly less in pH 5 . 5 (OD600 ~ 0 . 1 in average in positive human . US 2017 /0326160 A1 Nov . 16 , 2017 19

11 . The method of claim 1 , wherein the subject is not 19 . The method of claim 12 , wherein the niclosamide is being treated for acne vulgaris with niclosamide . administered topically to the subject in the form of a 12 . A method of treating atopic dermatitis and related pharmaceutical formulation comprising from 2 to 10 % by infections colonized by Gram -positive bacteria in a subject, weight of niclosamide . the method comprising topically administering to the subject 20 . The method of claim 12 , wherein the subject is a an effective amount of niclosamide or a pharmaceutically human . acceptable salt or hydrate thereof. 21 . The method of claim 12 , wherein the subject is not 13 . The method of claim 12 , wherein the Gram -positive being treated for acne vulgaris with niclosamide . bacteria are from the Staphylococcus genus or the Strepto 22 . A method of treating , preventing or eliminating bac coccus genus . terial colonization by Gram positive bacteria in a subject 14 . The method of claim 12 , wherein the Gram - positive affected by atopic dermatitis and related infections , the bacteria are selected from Staphylococcus aureus and Strep method comprising topically administering to the subject an tococcus pyogenes . effective amount of niclosamide or a pharmaceutically 15 . The method of claim 12 , wherein the Gram - positive acceptable salt or hydrate thereof . bacteria are resistant to an antibiotic other than niclosamide . 23 . The method of claim 22 , wherein the Gram -positive 16 . The method of claim 12 , wherein the Gram -positive bacteria are from the Staphylococcus genus or the Strepto bacteria are resistant to an antibiotic selected from fusidic coccus genus . acid , mupirocin and retapamulin . 24 . The method of claim 22 , wherein the Gram -positive 17 . The method of claim 12 , wherein the Gram - positive bacteria are resistant to an antibiotic other than niclosamide . bacteria are methicillin - resistant Staphylococcus aureus. 25 . The method of claim 22 , wherein the Gram - positive 18 . The method of claim 12 , wherein the niclosamide is bacteria are methicillin - resistant Staphylococcus aureus. administered is administered topically to the subject in the 26 . The method of claim 22 , wherein the subject is not form of a pharmaceutical formulation comprising the niclos being treated for acne vulgaris with niclosamide . amide in free form , or a hydrate thereof. * * * * *