Antibacterial Use of Halogenated Salicylanilides
20170258816 · 2017-09-14
Inventors
- Emilie Flora Aurore Delavenne (Le Mans, FR)
- Daniel Jean Jacques SIMON (Søborg, DK)
- Morten Otto Alexander Sommer (Virum, DK)
- Rasmus Vendler TOFT-KEHLER (Kobenhavn Ø, DK)
Cpc classification
A61K47/14
HUMAN NECESSITIES
A61K9/06
HUMAN NECESSITIES
A61K47/10
HUMAN NECESSITIES
A61K9/0014
HUMAN NECESSITIES
A61K47/26
HUMAN NECESSITIES
International classification
Abstract
The invention relates to a halogenated salicylanilide selected from closantel, rafoxanide, oxyclozanide and niclosamide and derivatives thereof including salts, hydrates, esters and the like for use in the topical treatment or prevention of infections caused by Gram-positive bacteria such as Staphylococcus, in particular Staphylococcus aureus, and Streptococcus, in particular Streptococcus pyogenes. Gram positive bacteria treated with the halogenated salicylanilides exhibit a very low frequency of appearance of resistant mutants compared to commonly used topical antibiotics.
Claims
1-35. (canceled)
36. A method of treating a disease or infection caused by Gram positive bacteria in a subject, the method comprising topically administering to the subject an effective amount of a halogenated salicylanilide selected from niclosamide or a salt or hydrate thereof; wherein the Gram positive bacteria is not a propionibacteria.
37. The method according to claim 36, wherein the Gram-positive bacteria develops spontaneous mutations which confer resistance to the halogenated salicylanilide at a frequency of less than 10.sup.−6 at the MIC of the halogenated salicylanilide to the Gram positive bacteria.
38. The method according to claim 37, wherein the Gram-positive bacteria develops spontaneous mutations which confer resistance to the halogenated salicylanilide at a frequency of less than 4×10.sup.−9 at the MIC of the halogenated salicylanilide to the Gram positive bacteria.
39. The method according to claim 36, wherein the Gram-positive bacteria wherein the Gram positive bacteria is selected from Staphylococcus spp or Streptococcus spp.
40. The method according to claim 36, wherein the infection or disease is selected from the group consisting of impetigo, infected eczema, rosacea, bacterial conjunctivitis, atopic dermatitis and related infections, sycosis barbae, superficial folliculitis, paronychia erythrasma, secondary infected dermatoses, carbuncles, furonculosis, ecthyma, cellulitis, erysipelas, necrotising fasciitis, secondary skin infections of wounds, dermatitis, scabies and diabetic ulcers.
41. The method according to claim 36, wherein the infection or disease is selected from the group consisting of impetigo, infected eczema, rosacea, bacterial conjunctivitis and atopic dermatitis.
42. The method according to claim 36, wherein the topical administration is for 2 weeks or less.
43. The method according to claim 36, wherein the halogenated salicylanilide is comprised in a formulation the components of which are selected such that the formulation provides a local pH of less than 6 at the site of infection.
44. The method according to claim 36, wherein the subject is a human.
45. A method of treating or preventing a disease or infection caused by Gram positive bacteria in a subject, the method comprising topically administering to the subject an effective amount of a halogenated salicylanilide selected from the group consisting of closantel, rafoxanide, oxyclozanide and niclosamide or a salt or hydrate or ester thereof; wherein the Gram-positive bacteria is resistant to a drug selected from fusidic acid, mupirocin and retapamulin.
46. The method according to claim 45, wherein the Gram positive bacteria is not a propionibacteria.
47. The method according to claim 45, wherein the Gram-positive bacteria develops spontaneous mutations which confer resistance to the halogenated salicylanilide at a frequency of less than 10.sup.−6 at the MIC of the halogenated salicylanilide to the Gram positive bacteria.
48. The method according to claim 45, wherein the Gram-positive bacteria develops spontaneous mutations which confer resistance to the halogenated salicylanilide at a frequency of less than 10.sup.−9 at the MIC of the halogenated salicylanilide to the Gram positive bacteria.
49. The method according to claim 45, wherein the Gram-positive bacteria wherein the Gram positive bacteria is selected from Staphylococcus spp or Streptococcus spp.
50. The method according to claim 45, wherein the infection or disease is selected from the group consisting of impetigo, infected eczema, rosacea, bacterial conjunctivitis, atopic dermatitis and related infections, sycosis barbae, superficial folliculitis, paronychia erythrasma, secondary infected dermatoses, carbuncles, furonculosis, ecthyma, cellulitis, erysipelas, necrotising fasciitis, secondary skin infections of wounds, dermatitis, scabies and diabetic ulcers.
51. The method according to claim 45, wherein the infection or disease is selected from the group consisting of impetigo, infected eczema, rosacea, bacterial conjunctivitis and atopic dermatitis.
52. The method according to claim 45, wherein the topical administration is for 2 weeks or less.
53. The method according to claim 45, wherein the halogenated salicylanilide is comprised in a formulation the components of which are selected such that it provides a local pH of less than 6 at the site of infection.
54. The method according to claim 45, wherein the salicylanilide is niclosamide or a salt or hydrate thereof.
55. (canceled)
56. A method of treating or preventing or eliminating bacterial colonization by Gram positive bacteria in a subject, the method comprising topically administering to the subject an effective amount a halogenated salicylanilide selected from the group consisting of closantel, rafoxanide, oxyclozanide and niclosamide, or a salt or hydrate or ester thereof.
57. The method of claim 56 wherein the halogenated salicylanilide is topically administered to the subject prior to performing a surgical procedure on the subject.
58. The method of claim 56, wherein the halogenated salicylanilide is administered to the nose of the subject, either intranasally or to the external skin of the nose.
59. The method of claim 56, wherein the halogenated salicylanilide is topically administered to the nose of the subject prior to performing a surgical procedure on the nose or face of the subject.
60. The method of claim 36, wherein the subject is an animal.
61. The method of claim 45, wherein the subject is a human.
62. The method of claim 45, wherein the subject is an animal.
63. The method of claim 56, wherein the subject is a human.
64. A topical pharmaceutical formulation comprising a halogenated salicylanilide selected from the group consisting of closantel, rafoxanide, oxyclozanide and niclosamide and derivatives thereof including salts, hydrates and esters, wherein the components of the formulation are selected such that it provides a local pH of less than 6 at when topically applied to a site of infection in a subject.
Description
BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES
[0106]
[0107]
[0108]
[0109]
[0110]
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0111] In the context of the present application and invention, the following definitions apply:
[0112] The term “Staphylococcus aureus” or “S. aureus” as used herein, without further description, relates to any strain of the gram-positive bacteria classified as Staphylococcus aureus, and which have been associated with a number of infections, including pneumonia, osteomyelitis, arthritis, endocarditis, sepsis and toxic shock syndrome, as well as cause less severe infections of the skin and soft tissues.
[0113] The term “methicillin-resistant Staphylococcus aureus” or “MRSA” as used herein includes strains of Staphylococcus aureus that are resistant to methicillin and can also broadly relate to Gram-positive bacteria strains (e.g. beta-lactamase-producing bacteria) that are resistant to antibiotics falling within the general classification of penicillins. Methicillin is the common name for (2S,5R,6R)-6-[(2,6-dimethoxybenzoyl)amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, which is a narrow spectrum beta-lactam antibiotic that has been used to treat infections caused by susceptible Gram-positive bacteria (e.g. including Staphylococcus aureus).
[0114] The term “derivative” refer to, but shall not be limited to metabolites, pro-drugs (converted into active drugs), esters, hydrates and/or a pharmaceutically acceptable salt and/or hydrates of such salts of the halogenated salicylanilides of the present invention. Also within the term “derivatives” are crystalline forms of the compounds and co-crystals formed between the halogenated salicylanilide and a suitable co-former(s). A person skilled in the art is well aware of various chemical methods and techniques to render a chemical substance to generate a derivate, which still comprises the chemical basis, such as addition, deletion or substitution of a group or functional group and thus it would be easy to generate a similar compound as niclosamide which has the same effect as the original.
[0115] The term “pharmaceutically acceptable salt” refers to salts (e.g. ethanolamine or piperazine salts) that retain the biological effectiveness and properties of the compounds described herein and, which are not biologically or otherwise undesirable. Pharmaceutically acceptable salts are well known to skilled persons in the art. Accordingly, it may be that a reference to a salt of a halogenated salicylanilide herein may refer to a pharmaceutically acceptable salt of the halogenated salicylanilide.
[0116] The term “solvate” is used herein to refer to a complex of solute, such as a compound or salt of the compound, and a solvent. If the solvent is water, the solvate may be termed a hydrate, for example a monohydrate, dihydrate, trihydrate etc., depending on the number of water molecules present per molecule of substrate.
[0117] The term ‘treatment’ herein indicates (i) the prevention of the disease caused by bacteria, such as Staphylococcus aureus and/or Streptococcus pyogenes; (ii) the suppression of the disease caused by bacteria, such as Staphylococcus aureus and/or Streptococcus pyogenes; and (iii) the relief of the disease caused by bacteria, such as Staphylococcus aureus and/or Streptococcus pyogenes; v) the eradication of a non-symptomatic colonization by Staphylococcus aureus from an area on the body, (v) the eradication of Gram-positive bacteria such as Staphylococcus aureus and/or Streptococcus pyogenes symptomatic infection, (vi) the eradication of Gram-positive bacteria such as Staphylococcus aureus and/or Streptococcus pyogenes; from an area of the body affected by another disease that could enable establishment of an infection more readily, than in a non-disease affected area—e.g. an area of the skin affected by eczema or atopic dermatitis, (vii) the suppression of the disease caused by Gram-positive bacteria such as Staphylococcus aureus and/or Streptococcus pyogenes; from an area of the body affected by another non-infectious disease that enables establishment of an infection more readily, than in a non-disease affected area—e.g. an area of the skin affected by eczema or atopic dermatitis.
[0118] Thus, in the context of the present invention, treatment of a condition encompasses both therapeutic and prophylactic treatment, of either an infectious or a non-infectious condition, in a mammal such as a human or animal, but in particular a human. It may involve complete or partial eradication of the condition, removal or amelioration of associated symptoms, arresting subsequent development of the condition, and/or prevention of, or reduction of risk of, subsequent occurrence of the condition. The bacterial strain may be characterized by a rate of developing spontaneous mutation frequency less than 10.sup.−6, such as less than 10.sup.−7 or 10.sup.−8, such as less than 4×10.sup.−9. The treatment will typically involve the use of the halogenated salicylanilides closantel, rafoxanide, oxyclozanide or niclosamide or derivatives thereof against Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pyogenes. One example which is of special interest is niclosamide.
[0119] It may be that the infection or disease which is to be treated is in an animal, e.g. a mammal. In particular, the halogenated salicylanilide can be used to treat commercial animals such as livestock (e.g. cows, sheep, chickens, pigs, geese, ducks, goats, etc.). Alternatively, the compounds of the present invention can be used to treat companion animals such as cats, dogs, horses, etc.
[0120] A “topical medication” is a medication that is applied to body surfaces such as the skin or mucous membranes to treat ailments via a large range of classes including but not limited to creams, foams, gels, droplets, lotions, and ointments. Topical medications differ from many other types of drugs because mishandling them can lead to certain complications in a patient or administrator of the drug. Many topical medications are epicutaneous, meaning that they are applied directly to the skin. Topical medications may also be inhalational, such as asthma medications, or applied to the surface of tissues other than the skin, such as eye drops applied to the conjunctiva, or ear drops placed in the ear, or medications applied to the surface of a tooth.
[0121] In topical application, a suitable pharmaceutical composition, for example a cream, lotion, gel, ointment, paste, drops or the like, may be spread on the affected surface and gently rubbed in. A solution may be applied in the same way, but more typically will be applied with a dropper, swab, or the like, and carefully applied to the affected areas.
[0122] Topical application of the halogenated salicylanilide according to the present invention enables the halogenated salicylanilide to be delivered selectively to a specific site, and avoids inter- and intra-patient variations which may be associated with alternative routes of drug administration.
[0123] Topical pharmaceutical compositions according to the present invention may be used to treat a variety of skin or membrane disorders, such as infections of the skin or membranes (e.g. infections of nasal membranes, axilla, groin, perineum, rectum, dermatitic skin, skin ulcers, and sites of insertion of medical equipment such as i.v. needles, catheters and tracheostomy or feeding tubes) with any of the bacteria described above, (e.g. any of the Staphylococci, Streptococci such as S. aureus (e.g. methicillin resistant S. aureus (MRSA)). Particular bacterial conditions that may be treated by topical pharmaceutical compositions of the present invention also include the skin- and membrane-related conditions disclosed hereinbefore, as well as: rosacea (including erythematotelangiectatic rosacea, papulopustular rosacea, phymatous rosacea and ocular rosacea); erysipelas; erythrasma; ecthyma; ecthyma gangrenosum; impetigo; paronychia; cellulitis; folliculitis (including hot tub folliculitis); furunculosis; carbunculosis; staphylococcal scalded skin syndrome; surgical scarlet fever; streptococcal perianal disease; streptococcal toxic shock syndrome; pitted keratolysis; trichomycosis axillaris; pyoderma; external canal ear infections; green nail syndrome; spirochetes; necrotizing fasciitis; Mycobacterial skin infections (such as lupus vulgaris, scrofuloderma, warty tuberculosis, tuberculides, erythema nodosum, erythema induratum, cutaneous manifestations of tuberculoid leprosy or lepromatous leprosy, erythema nodosum leprosum, cutaneous M. kansasii, M. malmoense, M. szulgai, M. simiae, M. gordonae, M. haemophilum, M. avium, M. intracellular, M. chelonae (including M. abscessus) or M. fortuitum infections, swimming pool (or fish tank) granuloma, lymphadenitis and Buruli ulcer (Bairnsdale ulcer, Searles' ulcer, Kakerifu ulcer or Toro ulcer)); as well as infected eczema, burns, abrasions and skin wounds. Particular fungal conditions that may be treated by topical pharmaceutical compositions of the present invention also include the skin- and membrane-related conditions disclosed hereinbefore, as well as: candidiasis; sporotrichosis; ringworm (e.g. tinea pedis, tinea cruris, tinea capitis, tinea unguium or tinea corporis); tinea versicolor; and infections with Trichophyton, Microsporum, Epidermophyton or Pityrosporum ovale fungi.
[0124] In some embodiments of the invention the topical pharmaceutical compositions of the present invention is not used to treat a fungal infection, for example the composition is not used to treat candidiasis; sporotrichosis; ringworm (e.g. tinea pedis, tinea cruris, tinea capitis, tinea unguium or tinea corporis); tinea versicolor; and infections with Trichophyton, Microsporunm, Epidermophyton or Pityrosporum ovale fungi.
[0125] The application regimen will depend on a number of factors that may readily be determined, such as the severity of the condition and its responsiveness to initial treatment, but will normally involve one or more applications per day on an ongoing basis. The effective dosage of the pharmaceutical composition of the present invention varies from the formulation, administration pathway, age, weight and gender of animal or human with a disease caused by Staphylococcus aureus, severity of a disease, diet, administration frequency and pathway, excretion and sensitivity.
[0126] Generally, the amount of the halogenated salicylanilide or a derivative thereof to be administered topically is in the range of 0.01-10000 mg/cm.sup.2, preferably between 0.1-1000 mg/cm.sup.2 and even more preferably between 1-100 mg/cm.sup.2 using a pharmaceutical formulation containing between 1-20%, preferably 2-10%, more preferably 3-8% and even more preferably 4-6% of active ingredient (all numbers given by weight).
[0127] In microbiology, minimum inhibitory concentration (MIC) is the lowest concentration of an antibacterial that will inhibit the visible growth of a microorganism after overnight incubation. Minimum inhibitory concentrations are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents. A MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism.
[0128] In toxicology, the median lethal dose, LD.sub.50 (abbreviation for “lethal dose, 50%”) of a toxin, radiation, or pathogen is the dose required to kill half the members of a tested population after a specified test duration. LD.sub.50 figures are frequently used as a general indicator of a substance's acute toxicity.
[0129] Therapeutic index (therapeutic ratio) is defined as the amount of a therapeutic agent causing the therapeutic effect measured as MIC to the amount that causes death in animal studies measured as LD.sub.50. Some antibiotics require monitoring to balance efficacy with minimizing adverse effects, including: gentamicin, vancomycin, amphotericin B (nicknamed ‘ampho-terrible’ for this very reason), and polymyxin B. Other MIC values could be used as well.
[0130] The rate of resistance development is quantified as the frequency of spontaneous mutants in a population of bacteria that is able to resist a given concentration of the antibiotic. For example the rate of resistance development may by 10.sup.−9 if on average 1 cell in 10.sup.9 cells is able to survive a concentration of antibiotic corresponding to 1×MIC.
[0131] In microbiology, colony-forming unit (CFU) is a rough estimate of the number of viable bacteria or fungal cells in a sample. Viable is defined as the ability to multiply via binary fission under the controlled conditions.
[0132] Also disclosed are the following numbered clauses [0133] 1. Halogenated salicylanilides selected from the group consisting of closantel, rafoxanide, oxyclozanide and niclosamide and derivatives thereof including salts, hydrates and esters for use in topical prevention or treatment of an infection or disease caused by Gram-positive bacteria, characterized by a rate of developing spontaneous mutation frequency less than 10.sup.−6. [0134] 2. The halogenated salicylanilides according to clause 1, characterized by a rate of developing spontaneous mutation frequency less than 10.sup.−7 or 10.sup.−8. [0135] 3. The halogenated salicylanilides according to clause 2, characterized by a rate of developing spontaneous mutation frequency less than 4×10.sup.−9. [0136] 4. The halogenated salicylanilides according to any of clauses 1-3, selected from the group consisting of closantel, rafoxanide, oxyclozanide and niclosamide and salts thereof for use in topical prevention or treatment of an infection or disease caused by Gram-positive bacteria, characterized by a rate of developing spontaneous mutation frequency less than 10.sup.−6 or less than 10.sup.−7 or 10.sup.−8 or less than 4×10.sup.−9. [0137] 5. The halogenated salicylanilides according to any of clauses 1-4, selected from the group consisting of closantel, rafoxanide, oxyclozanide and niclosamide for use in topical prevention or treatment of an infection or disease caused by Gram-positive bacteria, characterized by a rate of developing spontaneous mutation frequency less than 10.sup.−6 or less than 10.sup.−7 or 10.sup.−8 or less than 4×10.sup.−9. [0138] 6. The halogenated salicylanilides according to any of clauses 1-5, wherein the halogenated salicylanilides is niclosamide and derivatives thereof including salts, hydrates and esters for use in topical prevention or treatment of an infection or disease caused by Gram-positive bacteria, characterized by a rate of developing spontaneous mutation frequency less than 10.sup.−6 or less than 10.sup.−7 or 10.sup.−8 or less than 4×10.sup.−9. [0139] 7. The halogenated salicylanilides according to any of clauses 1-6, wherein the halogenated salicylanilides is niclosamide and hydrates of salts thereof or hydrates of such salts for use in topical prevention or treatment of an infection or disease caused by Gram-positive bacteria, characterized by a rate of developing spontaneous mutation frequency less than 10.sup.−8 or less than 4×10.sup.−9. [0140] 8. The halogenated salicylanilides according to any of clauses 1-7, wherein the infection or disease is selected from the group consisting of impetigo, bacterial conjunctivitis, atopic dermatitis and related infections, nasal eradication, sycosis barbae, superficial folliculitis, paronychia erythrasma, secondary infected dermatoses, carbuncles, furonculosis, ecthyma, cellulitis, erysipelas, necrotising fasciitis, secondary skin infections of wounds, dermatitis, scabies and diabetic ulcers. [0141] 9. The halogenated salicylanilides according to clauses 8, wherein the infection or disease is selected from the group consisting of impetigo, bacterial conjunctivitis, atopic dermatitis and infections associated with different skin conditions. [0142] 10. The halogenated salicylanilides according to any of clauses 1-9, wherein the bacteria is Staphylococcus aureus or Streptococcus pyogenes. [0143] 11. A method for manufacturing a medicament for use in topical prevention or treatment of an infection caused by bacteria, wherein the medicament is niclosamide and derivatives thereof including salts, hydrates and esters and characterized by a rate of developing spontaneous mutation frequency less than 10.sup.−6 or less than 10.sup.−7 or 10.sup.−8 or less than 4×10.sup.−9. [0144] 12. The method for manufacturing a medicament according to clause 11 for use in topical prevention or treatment of an infection caused by bacteria, wherein the medicament is niclosamide and hydrates of salts thereof or hydrates of such salts and characterized by a rate of developing spontaneous mutation frequency less than 10.sup.−8 or less than 4×10.sup.−9. [0145] 13. A pharmaceutical composition comprising niclosamide and derivatives thereof including salts, hydrates, esters and hydrates of such salts as the active ingredient wherein the pharmaceutical composition reduces or eliminates resistance development by the bacteria, Staphylococcus aureus or Streptococcus pyogenes against the used niclosamide by a rate of developing spontaneous mutation frequency less than 10.sup.−8 or less than 4×10.sup.−9.
[0146] Following examples are intended to illustrate, but not to limit, the invention in any manner, shape, or form, either explicitly or implicitly.
EXAMPLES
Example 1
[0147] Experimental tests were conducted to determine the antibacterial activity and the mutation rate conferring resistance for halogenated salicylanilides and reference compounds.
Microorganisms
[0148] Chosen for its relevance regarding bacterial skin infections, the methicillin-resistant S. aureus (MRSA) 01 strain was used as the primary test microorganism. This strain is a community-acquired MRSA clinical isolate of USA 300 type, from a skin abscess.
[0149] Twenty-one other MRSA and methicillin-sensitive S. aureus strains, and 4 Streptococcus pyogenes strains, were also included in the study (Table 1). These covered fusidic acid- and mupirocin-resistant strains, these two types of resistance being of clinical relevance.
[0150] Strains were conserved in Luria Bertani (LB) Broth (S. aureus) or Brain Heart Infusion (BHI) (S. pyogenes) supplemented with glycerol 15% (v/v) at −80° C., and reactivated by isolation on LB (S. aureus) or BHI (S. pyogenes) agar plates. Strains were cultivated in Mueller Hinton (MH) Broth-cation adjusted (S. aureus) supplemented with lysed horse blood 2.5% (v/v) (S. pyogenes). All strains were cultivated at 37° C., aerobically for S. aureus strains.
TABLE-US-00001 TABLE 1 Strains list and information Mupirocin and fusidic acid Resistance Species Strains gene USA type MLSTSCC mec spa 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 .sup.‡ ND USA 300 ST8 KU MRSA 09 USA600 ST45 KU MRSA 10 ST22-IV KU MRSA 11 ST36-II KU 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 16 .sup.‡ mupA CC30 t019 SSI MRSA 17 .sup.‡ 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; .sup.‡ 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.
Antibacterial Activity
[0151] The following tests were performed to assess the antibacterial activity in vitro (
[0152] 1. Minimum Inhibitory Concentration (MIC) Assay
[0153] The MIC was determined using 96-well plates, and serial two-fold dilutions of niclosamide (from Sigma) (from 51.2 to 0.025 μg/ml) in above indicated medium, with 150 μl per well.
[0154] Bacterial cultures were stopped in their exponential growth phase and plates were inoculated with the approximate concentration of 10.sup.3 cells per well. Plates were incubated at 37° C. for 18 hours (S. aureus) or 24 hours (S. pyogenes). Optical density at a wavelength of 600 nm was measured at the end of the incubation time. Inhibition was calculated as (Inhibition=1−OD.sub.test/OD.sub.no treatment) and MIC values were determined as the minimum concentration giving 100% inhibition. Experiments were performed at least as triplicate biological replicates with all strains.
[0155] The inhibition could be either due to a bactericidal or a bacteriostatic activity, which is not known from this experiment. The following assay was thus carried out in order to determine if niclosamide kills or inhibits growth of S. aureus.
[0156] 2. Time-Kill Assay
[0157] Assay was performed in 20 ml of medium. It included a negative control (medium without bacteria), a positive control (bacteria grown without niclosamide) and the assay (bacteria grown with niclosamide). Niclosamide was tested at 10 fold its MIC, determined in the previous experiment. This experiment was performed with the primary test microorganism indicated above.
[0158] The overnight culture was stopped and OD.sub.600 was measured. Culture was then diluted in indicated medium to obtain an OD.sub.600 of 0.25 in order to have about 5×10.sup.8 cfu/ml. Two hundred μ1 of this diluted culture were then added in all conditions except in the negative control. Initial bacterial concentration was about 5×10.sup.6 cfu/ml. Tubes were incubated aerobically at 37° C. for 24 hours.
[0159] Bacteria were enumerated before the incubation, after 30 minutes, 1, 2, 4, 8 and 24 hours of incubation by serial dilutions in NaCl 0.9% and plating on LB agar, with 2 plates per dilution. Plates were incubated at 37° C. and colonies enumerated after 24 hours.
[0160] The compound was considered bactericidal if the reduction of the bacterial inoculum was superior or equal to 3 log.sub.10 cfu/ml, bacteriostatic if reduction was inferior to 3 log.sub.10 cfu/ml.
[0161] 3. Mutational Frequency Evaluation
[0162] The frequency of spontaneous single-step mutations was determined on 3 different strains (MRSA, fusidic acid-resistant and mupirocin-resistant) as described by Drago et al. (2005) (Drago, L., De Vecchi, E., Nicola, L., Tocalli, L., & Gismondo, M. R. (2005). In vitro selection of resistance in Pseudomonas aeruginosa and Acinetobacter spp. by levofloxacin and ciprofloxacin alone and in combination with beta-lactams and amikacin. The Journal of Antimicrobial Chemotherapy, 56(2), 353-359). One hundred μ1 of an initial inoculum of 10.sup.9 cfu/ml from an overnight culture were plated on LB agar plates supplemented with the test compound (0×, 1×, 2×, 4× and 10×MIC). Adequate dilutions were plated on plates without the compound.
[0163] Viable cell growth was enumerated after 48 hours of incubation at 37° C.
[0164] Ten replicates were carried out for each strain and fusidic acid, mupirocin and retapamulin were used as controls for the MRSA 01 strain.
Colony-Forming Unit (CFU) in Skin Lesions
[0165] Three animal studies were performed with niclosamide and fusidic acid as comparison with different formulations and doses.
[0166] In each experiment, female Balb/c mice received a skin lesion approximately 1 cm.sup.2 and were challenged with 1.5×10.sup.8 cfu MRSA 01 topically. Twenty-four hours after the wound formation and the contamination, mice were treated topically with 0.05 ml twice daily for three days. Mice were sacrificed on day 4, skin lesions were excised and cfu quantitated. Fusidin ointment (2% from Leo Pharma) was included as control.
Experiment 1
[0167] Sample 5.—Niclosamide (N) 5% modified basis creme with higher lipid content—Lipocreme—according to the description in Danske Laegemiddelsstandarder (DLS).
[0168] 2.25 g niclosamide was mixed with 47.75 g créme prepared according to Danske Laegemiddels-standarder (DLS) (see
TABLE-US-00002 Oil phase: Polysorbate 80 10 g Cetostearyl alcohol 100 g Paraffin oil 100 g Glycerol monostearate 40-50 120 g Water phase: Methyl parabenzoate 1 g Glycerol 85% 40 g Sorbitol 70 g Water Milli-Q 724 g
Results and Conclusions
Microbiology: MIC & Kill Curves—FIGS. 1 & 2 and Tables 2 and 3
[0169]
TABLE-US-00003 TABLE 2 in-vitro susceptibility of S. aureus clinical isolates and S. aureus ATCC 29213 reference strain. MIC (μg/ml) S. aureus strains Newman 0.2 MRSA 01 0.4 MRSA 02 0.2 MRSA 03* 0.4 MRSA 04 0.4 MRSA 05 0.2 MRSA 06 0.4 MRSA 07 0.2 MRSA 08* 0.4 MRSA 09 0.2 MRSA 10 0.4 MRSA 11 0.1 EEFIC 01* 0.4 EEFIC 02* 0.2 MRSA 12* 0.4 MRSA 13* 0.4 MSSA 01* 0.4 MSSA 02* 0.4 MRSA 14* 0.4 MRSA 15* 0.2 MRSA 16* 0.2 MRSA 17* 0.2 S. pyogenes strains 01 3.2 CCUG 25571 3.2 ATCC 19615 3.2 ATCC 12385 1.6
TABLE-US-00004 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.sub.100 LD.sub.50 LD.sub.50 Ther. Index S. aureus S. pyogenes rats p.o mice p.o LD.sub.50, rats/ Compound μg/ml μM μg/ml μM (mg/kg) (mg/kg) MIC.sub.100, S. aureus) Niclosamide ≦0.4 ≦1.25 ≦3 ≦3 5000 >1500 12,500,000 Closantel ≦1.7 ≦2.5 12.8.sup.‡ 19.sup.‡ 300 331 176,000 Oxyclozanide 1.6* 4* 6.4.sup.‡ 15.9.sup.‡ 980-3500 300 612,000-2187,000 Rafoxanide 0.8* 1.25* 6.4.sup.‡ 10.sup.‡ 1500 270 1,875,000 *Tested against one strain: MRSA 01 .sup.‡Tested against one strain: S. pyogenes 01
[0170] The data shows that halogenated salicylanilides such as closantel, oxyclozanide, rafoxanide and particularly niclosamide are strongly potent against Gram-positive strains such as S. aureus and S. pyogenes. Notably the effect is independent of the resistance profile of the isolates towards other currently used antibiotics for topical treatment of these microorganisms, including fucidic acid and mupirocin. Accordingly, the halogenated salicylanilides in general and niclosamide in particular are well suited as a possible treatment for both susceptible and resistant Gram-positive strains.
Microbiology Resistance Development—Table 4
[0171] Spontaneous mutations conferring resistance to halogenated salicylanilides occurred at a very low frequency (mutational frequency=5×10.sup.−9, 2×10.sup.−8 and 1×10.sup.−7 at MIC×1 for rafoxanide, closantel, and oxyclozanide respectively) and for niclosamide at an extremely low frequency (0≦mutational frequency<4×10.sup.−1° at MIC×1) compared to currently used antibiotics such as fusidic acid, retapamulin and mupirocin (mutational frequency: ≧4×10.sup.−5 at MIC×1) (see Table 4).
TABLE-US-00005 TABLE 4 Mutation rates conferring resistance to halogenated salicylanilides. Resistance mutation rate Compound at MIC × 1 Niclosamide <4 × 10.sup.−9 1 Closantel =2 × 10.sup.−8* Oxyclozanide =1 × 10.sup.−7* Rafoxanide =5 × 10.sup.−9* Fusidic acid ≧4 × 10.sup.−5* Mupirocin ≧4 × 10.sup.−5* Retapamulin ≧4 × 10.sup.−5* .sup.1 Mutation rate conferring resistance against MRSA 01, MRSA 15 (fusidic acid-resistant) and MRSA 16 (mupirocin-resistant) *Tested against one strain: MRSA 01
[0172] A. The mutational frequency data gives the frequency of a resistant mutant within a given population. When the mutational frequency is bellow 10.sup.−9 means that there is less than one resistant mutant in a population of 10.sup.9 cells.
[0173] Unexpectedly, the resistance development towards halogenated salicylanilides in general and niclosamide in particular is much slower than resistance development towards drugs like fusidic acid, mupirocin and retapamulin being on the market.
[0174] In combination, the high potency and the implementation of the low rate of resistance development makes niclosamide particularly useful for treatment, especially topical treatment of infections caused by Gram positive organisms.
[0175] In view of the unexpected microbiological findings that niclosamide has very unique properties that make it an ideal candidate as a topical anti-infective animal experiments were performed to test the effect of niclosamide in vivo.
[0176] The data described herein shows that niclosamide reduces colonization by S. aureus in animal model used for the tests.
[0177] In Experiment 1 (
[0178] In conclusion the in vivo data shows that niclosamide is well suited as a topical antibiotic for treatment of infections caused by Gram positive infections.
Example 2
[0179] Additional More Extensive Screen of Clinical Isolates Performed with Niclosamide.
Methods
Microorganisms
[0180] Chosen for its relevance regarding bacterial skin infections, the methicillin-resistant S. aureus (MRSA) 01 strain was used as the primary test microorganism. This strain is a community-acquired MRSA clinical isolate of USA 300 type, from a skin abscess.
[0181] Two-hundred-four other MRSA and methicillin-sensitive S. aureus strains, and 4 Streptococcus pyogenes strains, were also included in the study. These covered fusidic acid- and mupirocin-resistant strains, these two types of resistance being of clinical relevance.
[0182] Strains were conserved in Luria Bertani (LB) Broth (S. aureus) or Brain Heart Infusion (BHI) (S. pyogenes) supplemented with glycerol 15% (v/v) at −80° C., and reactivated by isolation on LB (S. aureus) or BHI (S. pyogenes) agar plates. Strains were cultivated in Mueller Hinton (MH) Broth—cation adjusted (S. aureus) or BHI (S. pyogenes). All strains were cultivated aerobically (microaerobically for S. pyogenes strains) at 37° C.
Antibacterial Activity
[0183] 1. Minimum Inhibitory Concentration (MIC) Assay
[0184] Minimal inhibitory concentrations (MICs) of niclosamide, fusidic acid, and mupirocin were determined according to CLSI criteria with a doubling dilution concentration range (16 to 0.008 μg/ml) in Mueller Hinton Broth cation-adjusted (Fluka Analytical 90922), using 96-well plates, for 205 different S. aureus strains. S. aureus ATCC 29213 was included as a control reference strain and clindamycin and vancomycin were included as control antibiotics.
[0185] Bacterial cultures were stopped in their exponential growth phase and plates were inoculated with the approximate concentration of 5×10.sup.5 cells per well. Plates were incubated at 37° C. for 18 hours (S. aureus) or 24 hours (S. pyogenes). Optical density at a wavelength of 600 nm was measured at the end of the incubation time Inhibition was calculated as (Inhibition=1−OD.sub.test/OD.sub.no treatment) and MIC values were determined as the minimum concentration giving 100% inhibition.
[0186] Due to interference with blood (MIC increased by 16 with 5% lysed horse blood), the MIC determination against S. pyogenes strains was performed in BHI.
Results and Discussion
In Vitro MIC Determination and Breadth of Effect
[0187] The MIC of niclosamide was ≦0.5 μg/ml against all targeted S. aureus and S. pyogenes strains, including the strains resistant to fusidic acid, mupirocin, clindamycin and retapamulin (Table 5, Table 6, Table 7 and
TABLE-US-00006 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 (μg/ml) niclosamide Fusidic acid Mupirocin Retapamulin Clindamycin Vancomycin ATCC 29213 0.5 0.06 0.125 0.03 0.125 1 Newman 0.25 0.25 0.25 0.03 0.125 2 MRSA 01 0.25 0.125 0.25 0.06 0.125 1 MRSA 02 0.25 0.5 0.25 0.06 0.25 2 MRSA 03 0.25 16 0.25 0.03 0.125 1 MRSA 04 0.25 0.25 0.25 0.06 0.125 2 MRSA 05 0.25 0.125 0.125 0.03 0.125 1 MRSA 06 0.25 0.125 0.25 0.03 0.125 2 MRSA 07 0.125 0.25 0.25 0.06 0.125 1 MRSA 08 0.25 0.125 >16 0.03 >16 1 MRSA 09 0.125 0.25 0.25 0.06 0.25 2 MRSA 10 0.25 1 0.25 0.06 0.25 1 MRSA 11 0.25 1 0.5 0.06 >16 1 EEFIC 01 0.25 4 0.125 0.03 0.125 1 EEFIC 02 0.25 4 0.125 0.03 0.125 1 MRSA 12 0.5 4 0.125 0.03 ND ND MRSA 13 0.25 4 0.25 0.06 0.125 1 MSSA 01 0.5 4 0.125 0.03 0.125 1 MSSA 02 0.25 4 0.125 0.03 0.125 1 MRSA 14 0.25 >16 0.125 0.03 >16 1 MRSA 15 0.25 >16 0.25 0.06 0.125 2 MRSA 16 0.25 0.25 >16 0.06 0.25 1 MRSA 17 0.25 4 >16 0.03 >16 2 MRSA 18 0.25 0.25 0.125 0.06 0.125 1 MRSA 19 0.25 0.125 0.25 0.03 0.125 1 MRSA 20 0.25 0.125 0.25 0.06 0.06 1 MRSA 21 0.25 0.125 0.125 0.125 0.03 1 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 1 MRSA 24 0.25 0.25 0.25 0.06 0.125 2 MRSA 25 0.25 16 0.25 0.06 0.125 1 MRSA 26 0.5 0.5 0.5 0.06 >16 1 MRSA 27 0.5 0.25 0.25 0.06 0.125 1 MRSA 28 0.25 0.06 0.5 0.03 0.06 1 MRSA 29 0.5 4 0.25 0.06 0.125 1 MRSA 30 0.25 0.125 0.125 0.03 0.06 1 MRSA 31 0.25 0.25 0.25 16 >16 1 MRSA 32 0.25 0.5 0.25 0.06 0.125 1 MRSA 33 0.25 0.5 0.25 0.06 0.125 1 MRSA 34 0.25 0.25 0.25 0.06 0.125 1 MRSA 35 0.25 16 0.5 0.03 >16 1 MRSA 36 0.25 8 0.25 0.06 0.06 1 MRSA 37 0.25 0.5 0.25 0.06 0.125 1 MRSA 38 0.25 8 0.125 0.06 0.125 1 MRSA 39 0.25 8 0.25 0.06 0.06 1 MRSA 40 0.25 0.125 0.25 0.03 0.06 1 MRSA 41 0.25 >16 0.125 0.02 0.06 1 MRSA 42 0.25 0.25 0.25 0.06 >16 1 MRSA 43 0.25 0.125 0.25 0.06 0.06 1 MRSA 44 0.25 4 0.25 0.02 0.03 1 MRSA 45 0.25 0.06 0.13 0.02 0.06 1 MRSA 46 0.25 0.06 0.13 0.02 0.03 1 MRSA 47 0.25 0.06 0.25 0.02 >16 0.5 MRSA 48 0.25 2 0.13 0.02 0.03 0.5 MRSA 49 0.25 0.25 0.25 0.02 0.03 2 MRSA 50 0.25 0.03 0.25 0.02 0.02 1 MRSA 51 0.125 0.13 0.25 0.03 0.06 1 MRSA 52 0.25 0.06 0.25 0.02 0.03 1 MRSA 53 0.25 0.25 0.25 0.03 0.06 1 MRSA 54 0.25 0.125 0.25 0.03 0.06 2 MRSA 55 0.25 4 0.25 0.02 0.03 0.5 MRSA 56 0.25 0.06 0.25 <0.01 0.03 1 MRSA 57 0.25 0.125 0.125 0.02 0.03 1 MRSA 58 0.25 4 0.125 0.02 0.06 1 MRSA 59 0.25 0.06 0.125 0.02 0.03 1 MRSA 60 0.25 8 0.25 0.02 0.06 0.5 MRSA 61 0.25 0.06 0.25 0.02 0.03 2 MRSA 62 0.25 0.06 0.25 0.02 0.06 2 MRSA 63 0.25 0.125 0.125 0.02 0.06 1 MRSA 64 0.25 0.25 0.25 0.02 0.06 1 MRSA 65 0.25 0.06 0.25 0.02 0.03 1 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 1 MRSA 68 0.25 0.125 0.25 0.02 0.03 1 MRSA 69 0.25 4 0.125 <0.01 0.03 1 MRSA 70 0.125 8 0.25 0.02 0.06 1 MRSA 71 0.25 0.06 0.125 <0.01 0.02 1 MRSA 72 0.25 16 0.25 0.03 0.06 1 MRSA 73 0.5 0.25 0.25 0.03 0.125 1 MRSA 74 0.25 0.25 0.25 0.03 0.125 2 MRSA 75 0.5 0.5 0.5 0.03 0.125 1 MRSA 76 0.25 0.25 0.25 0.03 0.125 1 MRSA 77 0.25 16 0.25 0.03 0.125 2 MRSA 78 0.5 0.125 0.25 0.03 0.06 1 MRSA 79 0.25 0.5 0.25 <0.01 0.03 1 MRSA 80 0.5 0.125 0.25 0.02 0.06 1 MRSA 81 0.5 0.25 0.25 0.03 0.125 1 MRSA 82 0.25 8 0.25 0.03 0.125 2 MRSA 83 0.5 0.06 0.25 0.03 0.06 1 MRSA 27b 0.25 8 0.25 0.02 0.06 1 MRSA 84 0.25 8 0.25 0.03 0.125 2 MRSA 85 0.5 4 0.25 0.03 0.125 1 MRSA 86 0.25 >16 0.25 0.03 0.06 2 MRSA 87 0.25 0.125 0.25 0.02 0.06 1 MRSA 88 0.25 0.06 0.25 0.03 0.06 1 MRSA 89 0.5 0.06 0.25 0.02 >16 1 MRSA 90 0.5 16 0.25 0.06 0.125 1 MRSA 91 0.25 0.25 0.25 0.06 0.125 1 MRSA 92 0.25 8 0.25 0.06 0.06 1 MRSA 93 0.25 0.02 0.25 0.02 0.03 2 MRSA 94 0.25 0.125 0.25 0.03 0.125 2 MRSA 95 0.25 8 0.25 0.03 0.125 2 MRSA 96 0.25 0.125 0.125 0.03 >16 1 MRSA 97 0.25 8 0.25 0.03 0.06 1 MRSA 98 0.5 0.06 0.5 0.03 >16 1 MRSA 99 0.25 0.125 0.5 0.03 0.125 1 MRSA 100 0.25 0.125 0.5 1 0.25 1 MRSA 101 0.5 8 0.25 0.03 0.06 1 MRSA 102 0.25 0.06 0.25 0.03 0.06 2 MRSA 103 0.25 8 0.25 0.03 0.125 1 MRSA 104 0.5 0.25 0.25 0.03 0.125 1 MRSA 105 0.25 0.125 0.25 0.03 0.125 1 MRSA 106 0.25 0.125 0.25 0.06 0.125 1 MRSA 107 0.25 0.25 0.25 0.06 0.125 2 MRSA 108 0.25 4 0.25 0.03 0.125 1 MRSA 109 0.25 0.25 0.25 0.03 0.125 1 MRSA 110 0.25 0.06 0.125 0.03 0.125 1 MRSA 111 0.5 8 0.25 0.03 0.125 1 MRSA 112 0.25 0.06 0.25 0.03 0.06 1 MRSA 113 0.25 0.125 0.25 0.03 0.06 1 K000796 0.25 8 0.5 0.03 0.125 1 K115688 0.25 0.125 0.25 0.03 0.125 2 K000866 0.25 8 0.5 0.03 0.125 1 K000864 0.25 0.25 0.25 0.03 0.125 1 K000863 0.25 1 0.5 0.03 0.125 1 K115689 0.25 0.125 0.25 0.03 0.125 2 K000772 0.25 >16 0.125 0.06 0.125 1 K115498 0.25 0.125 0.5 0.03 0.125 2 R000024 0.25 16 0.5 0.06 0.125 1 R000020 0.5 0.125 0.5 0.03 0.125 1 R000019 0.5 0.125 0.5 0.06 0.125 2 U115579 0.25 0.25 0.5 0.06 0.125 1 115370U 0.25 0.5 0.125 0.06 0.125 1 114660U 0.25 0.25 0.25 0.06 0.125 1 115584D 0.25 0.25 0.5 0.06 0.125 2 115740E 0.5 0.25 0.25 0.06 0.125 1 115810E 0.25 0.25 0.25 0.06 0.125 1 115628T 0.25 8 0.25 0.03 0.06 2 000274T 0.25 0.5 0.5 0.06 0.125 1 115691T 0.5 0.25 0.25 0.03 0.125 1 115903T 0.5 8 0.5 0.03 0.125 1 116122T 0.25 0.125 0.5 0.03 0.125 1 115015T 0.5 0.25 0.5 0.06 0.125 2 115273C 0.5 0.25 0.25 0.03 0.125 1 000040C 0.5 0.25 0.25 0.03 0.125 1 115690C 0.25 8 0.125 0.25 0.5 2 115561C 0.25 0.125 0.5 0.03 0.125 1 115445C 0.5 0.25 0.25 0.03 0.125 1 115263C 0.25 0.125 0.25 0.03 0.06 2 115303C 0.5 0.25 0.25 0.03 >16 1 115268C 0.5 0.25 0.5 0.03 0.125 1 115295C 0.25 0.125 0.25 0.03 0.125 1 115242C 0.5 8 0.25 0.03 0.06 1 115427C 0.25 0.125 0.25 0.03 0.06 1 000041C 0.25 0.25 0.25 0.03 0.06 1 E5-1048654 0.25 0.5 0.25 0.06 0.125 1 9-2955245 0.25 0.25 0.25 0.06 0.125 1 E5-1046019 0.25 0.25 0.25 0.03 0.125 1 E5-1046020 0.5 0.25 0.25 0.06 0.125 1 E5-1047585 0.25 0.25 0.25 0.03 0.125 1 E5-1038294 0.5 0.25 0.25 0.03 0.125 1 E5-1035779 0.5 0.125 0.5 0.03 0.125 1 9-1862936 0.5 0.125 0.25 0.03 0.125 1 E5-1033091 0.5 0.03 0.25 0.02 0.06 1 9-26422166 0.5 8 0.25 0.03 0.125 1 9-2642158 0.25 0.25 0.25 0.06 0.125 1 E5-1035775 0.5 >16 0.25 0.06 0.125 1 E5-1029558 0.25 16 0.5 0.03 0.125 1 E5-1038279 0.5 4 0.25 0.03 0.125 1 E5-1039697 0.25 0.5 0.25 0.06 0.125 1 E5-1041979 0.5 0.25 0.5 0.03 0.125 1 E5-1035284 0.25 0.25 0.25 0.03 0.125 1 E5-1030469 0.25 0.125 0.25 0.03 0.125 2 E5-1030472 0.5 0.25 0.5 0.06 0.125 1 E5-1041977 0.5 16 0.5 0.03 0.125 2 E5-1041987 0.5 16 0.25 0.03 0.125 1 E5-1039684 0.5 16 0.25 0.06 0.125 1 E5-1041980 0.25 0.25 0.25 0.03 0.125 1 E5-1033088 0.25 0.25 0.25 0.03 0.125 1 E5-1035277 0.5 16 0.5 0.03 0.125 1 E5-1046096 0.5 0.5 0.5 0.06 0.125 1 E5-1046085 0.5 8 0.5 0.06 0.125 2 9-2625962 0.5 0.25 0.5 0.03 0.125 1 E5-1043668 0.25 1 0.25 0.06 0.25 1 E5-1048428 0.25 0.5 0.25 0.06 0.25 1 E5-1047924 0.5 0.25 0.5 0.03 0.125 1 E5-1047606 0.5 8 0.5 0.03 0.125 1 E5-1046070 0.25 0.25 0.5 0.03 0.125 1 E5-1046298 0.25 0.125 0.25 0.03 0.125 1 E5-1046296 0.5 0.125 1 0.03 0.125 1 E5-1046297 0.5 0.125 0.25 0.06 0.125 1 E5-1043184 0.5 16 0.5 0.03 0.125 1 E5-1038286 0.25 0.25 0.5 0.06 0.125 1 E5-1037958 0.5 16 0.5 0.06 0.125 1 E5-1037971 0.25 0.25 0.25 0.03 0.125 1 E5-1033076 0.5 0.25 0.25 0.03 0.125 1 E5-1033076 0.5 0.25 0.25 0.03 0.125 1 E5-1029252 0.25 0.25 0.5 0.03 0.125 1 E5-1030440 0.25 0.25 0.25 0.06 0.06 1 E5-1030482 0.125 16 0.25 0.02 0.06 1 E5-1046074 0.25 0.125 0.25 0.06 0.125 1 E5-1048204 0.25 0.25 0.25 0.03 0.06 2 E5-1048670 0.125 0.5 0.5 0.06 0.125 2 E5-1046039 0.25 0.25 0.25 0.06 0.125 1 E5-1045179 0.25 0.25 0.25 0.06 0.125 1 E5-1046723 0.25 0.5 0.25 0.06 0.125 2
TABLE-US-00007 TABLE 6 MIC distribution of niclosamide against Staphylococcus aureus and Streptococcus pyogenes strains (percentage and ratio) MIC (μg/ml) 0.0625 0.125 0.25 0.5 Staphylococcus 3% (6/205) 70% (144/205) 27% (55/205) aureus strains Streptococcus 25% 25% (1/4) 25% (1/4) 25% (1/4) pyogenes (1/4) strains
TABLE-US-00008 TABLE 7 MIC90, MIC50 and MIC ranges of niclosamide for Staphylococcus aureus strains. MIC90 MIC50 Range values 0.5 μg/ml 0.25 μg/ml 0.125-0.5 μg/ml
[0188] Niclosamide was inhibitory at a concentration equal or below 0.5 μg/ml. for all targeted S. aureus and S. pyogenes strains, including fusidic acid- and mupirocin-resistant strains.
Example 3
[0189] A further study was carried out to examine the frequency of spontaneous mutation conferring resistance to niclosamide in 3 methicillin-resistant Staphylococcus aureus strains, including a fusidic acid- and a mupirocin-resistant strains. This frequency was compared with the frequencies of spontaneous mutation conferring resistance to fusidic acid, mupirocin and retapamulin in one MRSA strain.
Methods
Microorganisms
[0190] Three methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates, with different resistance profiles (MRSA 01, MRSA15 [fusidic acid-resistant strain] and MRSA 16 [mupirocin-resistant strain]) were chosen for their relevance regarding bacterial skin infections The MRSA 01 strain was used as the primary test microorganism. This strain is a community-acquired MRSA clinical isolate of USA 300 type, from a skin abscess.
[0191] Strains were conserved in Luria Bertani (LB) Broth supplemented with glycerol 15% (v/v) at −80° C., and reactivated by isolation on LB agar plates. Strains were cultivated aerobically in Mueller Hinton (MH) Broth—cation adjusted at 37° C.
Mutational Frequency Evaluation
[0192] The frequency of spontaneous single-step mutations was determined on the 3 different strains as described by Drago et al. (2005) and Pannu et al. (2011). One hundred μ1 of an initial inoculum of about 10.sup.9 cfu/ml from an overnight culture were plated on LB agar plates supplemented with the test compound (0×, 1×, 2×, 4× and 10×MIC). Adequate dilutions were plated on plates without the compound.
[0193] Viable cell growth was enumerated after 48 hours of incubation at 37° C.
[0194] The spontaneous resistance frequency for an isolate-drug combination was calculated from the number of colonies that grew on plates containing drug versus the number of colonies that grew on drug-free agar.
[0195] Ten replicates were carried out for each strain and fusidic acid, mupirocin and retapamulin were used as controls for the MRSA 01 strain.
Results and Discussion
[0196] Spontaneous mutations conferring resistance to niclosamide occurred at an extremely low frequency (below the detection limit) (0≦mutational frequency<4.10.sup.−9 at MIC×1) for all tested strains (MRSA 01, MRSA 15 (fusidic acid-resistant) and MRSA 16 (mupirocin-resistant)) compared to fusidic acid (mutational frequency: 3.10.sup.−7 at MIC×10 and ≧4.10.sup.−5 at MIC×1) and to mupirocin and retapamulin. Results with the strain MRSA 01 are shown in Table 8.
TABLE-US-00009 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 Con- MIC × 1 <4 × 10.sup.−9* ≧4 .Math. 10.sup.−5 ≧4 .Math. 10.sup.−5 ≧4 .Math. 10.sup.−5 cen- MIC × 2 <4 × 10.sup.−9* 2 .Math. 10.sup.−5 8 .Math. 10.sup.−8 3 .Math. 10.sup.−7 tra- MIC × 4 <4 × 10.sup.−9* 1 .Math. 10.sup.−6 1 .Math. 10.sup.−8 2 .Math. 10.sup.−8 tion MIC × 10 <4 × 10.sup.−9* 3 .Math. 10.sup.−7 ≦4 .Math. 10.sup.−9 <4 .Math. 10.sup.−9* *Below the detection limit (no colony on plates)
[0197] As for MRSA 01, no colony grew on plates with niclosamide with the strains MRSA 15 and MRSA 16. These led to a mutation frequency<3×10.sup.−8 for MRSA 15 and <1×10.sup.−7 for MRSA 16 at MIC×1 (0.25 μg/ml), these differences in the detection limits being due to differences in the bacterial concentrations of overnight cultures.
Conclusions
[0198] Frequencies of spontaneous mutations conferring resistance to niclosamide in Staphylococcus aureus were much lower than frequencies of spontaneous mutations conferring resistance to fusidic acid, mupirocin and retapamulin in Staphylococcus aureus. This supports the use of niclosamide for cutaneous decolonization of S. aureus.
Example 4
[0199] A study was carried out to determine the effect of pH on the antibacterial activity of niclosamide against Staphylococcus aureus in order to assess whether niclosamide is still active against S. aureus at pH close to the pH of the skin.
Methods
Microorganisms
[0200] Chosen for its relevance regarding bacterial skin infections, the methicillin-resistant S. aureus (MRSA) 01 strain was used. This strain is a community-acquired MRSA clinical isolate of USA 300 type, from a skin abscess.
[0201] This strain was conserved in Luria Bertani (LB) Broth supplemented with glycerol 15% (v/v) at −80° C., and reactivated by isolation on LB agar plates. It was then cultivated aerobically in Mueller Hinton (MH) Broth—cation adjusted at 37° C.
Assessment of the Effect of pH on the Antibacterial Activity of Niclosamide
[0202] The pH of Mueller-Hinton Broth cation-adjusted was adjusted with HCl 2M to 7, 6.5, 6, 5.5, 5, 4.5 and 5. Ten ml of medium for each pH were prepared. pH of non-adjusted MHBII was equal to 7.4.
[0203] Each pH-adjusted samples were filtered on 0.2 μm filters before being used for the MIC determination assay. For each pH, minimal inhibitory concentrations (MICs) of niclosamide were determined according to CLSI criteria with a doubling dilution concentration range (16 to 0.008 μg/ml).
[0204] Bacterial culture was stopped in its exponential growth phase and plates were inoculated with the approximate concentration of 5×10.sup.5 cells per well. Plates were incubated at 37° C. for 18 hours (S. aureus). Optical density at a wavelength of 600 nm was measured at the end of the incubation time Inhibition was calculated as (Inhibition=1−OD.sub.test/OD.sub.no treatment) and MIC values were determined as the minimum concentration giving 100% inhibition.
[0205] The experiment was performed in triple biological replicates.
Results and Discussion
[0206] The pH of different pH-adjusted media were checked after the addition of niclosamide in order to check that the addition of niclosamide did not have any influence on the pH. Measurements showed that the addition of niclosamide (16 μg/ml) in the pH-adjusted media had no influence on the pH (Table 9).
[0207] MRSA 01 grew equally well from pH 6 to pH 7.4 (OD.sub.600≈0.2 in average in positive control wells) and slightly less in pH 5.5 (OD.sub.600≈0.1 in average in positive control wells). However, the strain was inhibited by the lowest pH (pH 4 to pH 5) (
[0208] MIC determinations showed that the inhibitory activity of niclosamide against MRSA 01 was increased when pH was decreased, with lower MICs (Table 9).
TABLE-US-00010 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 (μg/ml) pH (μg/ml) pH (μg/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 ≦0.03 5.547 ≦0.008 5.530 ≦0.008 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
CONCLUSIONS
[0209] The maximal inhibitory effect of niclosamide was observed at pH 5.5, which is close to the pH of the skin.