WO2020089467A1

WO2020089467A1 - Dosage regimen

Info

Publication number: WO2020089467A1
Application number: PCT/EP2019/079996
Authority: WO
Inventors: Morten Otto Alexander SOMMER; Rasmus Vendler TOFT-KEHLER
Original assignee: Union Therapeutics AS; Ceva Sante Animale SA
Current assignee: Union Therapeutics AS; Ceva Sante Animale SA
Priority date: 2018-11-02
Filing date: 2019-11-01
Publication date: 2020-05-07
Anticipated expiration: 2021-05-02

Abstract

A dosage regimen for the topical prevention or treatment of a skin infection or an inflammatory skin condition in a non-human subject using a halogenated salicylanilide. The topical dosage regimen provides high concentrations of the halogenated salicylanilide in skin tissue which are retained for a prolonged period without the need for further topical dosing of the halogenated salicylanilide.

Description

DOSAGE REGIMEN

[0001] This invention relates to a dosage regimen for the topical prevention or treatment of a skin infection or inflammatory skin condition in a non-human subject using a halogenated salicylanilide. Also disclosed are topical compositions suitable for use in the dosage regimen.

BACKGROUND

[0002] Pyoderma is a bacterial infection of the skin. In dogs, in the vast majority of cases, the causative organism is Staphylococcus pseudintermedius, a normal resident of the skin in healthy dogs, though other staphylococcal species (e.g. S. aureus, S. schleiferi) may also be involved.

[0003] Most cases of chronic or recurrent canine pyoderma are associated with underlying causes, such as allergic dermatitis, parasitic skin infestations,

endocrinopathies, follicular dysplasia disorders, or keratinization disorders. In one prospective study of 30 cases of canine recurrent pyoderma, atopic dermatitis was found to be the underlying cause in 60% of cases, food allergy, flea allergy and hypothyroidism in 7% of cases respectively, and hyperestrogenism, demodicosis and zinc responsive dermatosis each accounted for 4% of cases. Only two dogs were an underlying cause not identified.

[0004] Atopic dogs are prone to recurrent skin infections due to increased adherence of staphylococcal bacteria to atopic canine skin cells, alterations in normal skin barrier function, and altered skin immune system function.

[0005] As such, even following complete resolution, pyoderma has a tendency to recur if the underlying disease is not properly addressed. A number of different clinical manifestations of canine pyoderma are recognized based on lesion type and distribution. Classification by lesion depth is considered useful, because choice of therapy may vary according to the cutaneous tissue layers affected.

[0006] Treatment of canine pyoderma has been traditionally based on systemic antibacterial administration for 3-4 weeks, with topical antimicrobial therapy suggested as an adjunctive treatment. Treatment guidelines recommend amoxicillin-clavulanic acid, cephalexin or clindamycin as first-line empirical agents for systemic antibiotic therapy. [0007] Antibiotic resistant strains of Staphylococcus pseudintermedius associated with canine pyoderma are increasing and as a result it is becoming more challenging to treat pyoderma.

[0008] The topical treatment of skin infections such as canine pyoderma often requires regular and prolonged topical application of the treatment to be effective and to eliminate the bacteria causing the infection. Similarly, the topical treatment of inflammatory skin conditions such as atopic dermatitis may require prolonged and frequent topical administration of a drug to provide a therapeutic effect on the dermatitis.

[0009] Prolonged topical treatment periods for skin conditions are inconvenient to apply to animals and may lead to poor compliance with the treatment regime and a sub-optimal therapeutic effect on the condition being treated. In the case of skin infections poor compliance with the dosage regimen may result is failure to eradicate the infection and/or increase the risk of bacterial resistance emerging.

[0010] There is therefore a need for topical treatments for skin conditions such as bacterial skin infections or inflammatory skin conditions, which are effective and

convenient for users to administer to animals, thereby maximising compliance with the dosage regimen and the efficacy of the treatment.

[0011] Halogenated salicylanilides are a series of compounds including niclosamide, closantel, rafoxanide and oxyclozanide.

[0012] Niclosamide is approved for use as an anthelmintic drug for human and veterinary medicine. Niclosamide is a known taenicide effective against several parasitic tapeworms of livestock and pets (e.g. Taenia spp., Moniezia spp.) and also against rumen flukes (Paramphistomum spp.) and blood flukes ( Schistosoma spp.). Niclosamide has also been shown to prevent the penetration of Schistosoma mansoni through the human skin. As well as used as an anticancer drug, pesticide and as an anti-trypanosoma drug. Niclosamide has also been shown to inhibit viral replication in human cells. (Ofori-Adjei et al; The International Journal of Risk & Safety in Medicine. 2008;20:1 13-22; and Pearson et al; Annals of Internal Medicine. 1985;102(4):550-1 ).

[0013] Oxyclozanide (CAS no. 2277-92-1 ) is used for the oral treatment and control of fascioliasis in cattle, sheep and goats (European Medicines Agency outcome or referral procedure report EMA 586006/2017, dated 28 September 2017).

[0014] GB 2,456,376 and WO 2008/155535 describes the use of halogenated

salicylanilides for the treatment of acne caused by propionibacteria. [0015] WO 2016/038035 discloses the use of halogenated salicylanilides for the topical treatment of diseases or infections caused by Gram-positive bacteria.

[0016] Wu et al. (“Antihelminthic niclosamide modulates dendritic cells activation and function”, Cellular Immunology, 288(1-2): 15-23 (2014)) discloses that niclosamide has an inhibitory action on lipopolysaccharide (LPS)-induced dendritic cell maturation and cytokine costimulatory molecule and MHC molecule expression in-vitro. It was also found that niclosamide-treated dendritic cells inhibited antigen specific T cell responses. The reference postulates that niclosamide may be useful for the treatment of chronic inflammatory disorders or dendritic cell mediated autoimmune disease, however, no clinical data is provided and the conclusions of the paper indicate that further studies are required to better understand the molecular mechanisms associated with the compound.

[0017] WO2019/053180, published after the priority date of this patent application, discloses a topical composition comprising oxyclozanide or niclosamide and dimethyl sulfoxide. The compositions are stated to be useful for the topical treatment of pyoderma or dermatitis in non-human mammals.

BRIEF SUMMARY OF THE DISCLOSURE

[0018] The inventors have found that halogenated salicylanilides, for example

niclosamide and oxyclozanide, are readily absorbed by skin tissues when applied topically and that high concentrations of the halogenated salicylanilide are retained in the skin for a prolonged period after the initial topical application. Thus, topical application of a halogenated salicylanilide offers the possibility of a very short (for example 1 or 2 days) initial topical treatment period which provides a long-lasting therapeutic effect on skin conditions. The short initial topical treatment period provides a high concentration of the halogenated salicylanilide in the skin acts as a depot of drug and provides therapeutically effective concentrations of the drug in the skin tissues without the need for further topical application of the halogenated salicylanilide. For some skin conditions (e.g. a skin infection) the duration of the therapeutic effect following initial topical dosing may be sufficiently long that further topical application of halogenated salicylanilide is not required to treat or eradicate the skin condition. Alternatively, for more chronic skin conditions further topical application of halogenated salicylanilide may not be required for many days after completion of the initial topical treatment period. The short initial topical treatment period followed by a prolonged period without the need for additional topical application will be very convenient for users to administrate to non-human subjects and is expected to result in good compliance with the dosage regimen. [0019] In accordance with the present invention there is provided a halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof, for use in the topical treatment or prevention of a skin condition in a non-human subject; wherein the skin condition is selected from (i) a skin condition or skin infection caused by or associated with Gram-positive bacteria; and (ii) an inflammatory skin condition;

wherein the halogenated salicylanilide is topically administered to the subject for an initial treatment period of 5 days or less followed by a treatment-free period of at least 2 days (preferably at least 3 days) during which no further halogenated salicylanilide is topically administered to the subject.

[0020] Also provided is a method of treating a skin condition in a non-human subject, wherein the skin condition is selected from (i) a skin condition or skin infection caused by or associated with Gram-positive bacteria; and (ii) an inflammatory skin condition, the method comprising topically administering to the subject a therapeutically effective amount of a halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof for an initial treatment period of 5 days or less followed by a treatment-free period of at least 2 days (preferably at least 3 days) during which no further halogenated salicylanilide is topically administered to the subject.

[0021] It is intended that the method of treatment is applicable to all the dosage regimens described in this application. Accordingly, any reference to a halogenated salicylanilide for use in a dosage regimen is also applicable to the equivalent method of treatment.

[0022] Also provided is the use of a halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof in the manufacture of a medicament for the topical treatment or prevention of a skin condition in a non-human subject; wherein the skin condition is selected from (i) a skin condition or skin infection caused by or associated with Gram-positive bacteria; and (ii) an inflammatory skin condition;

[0023] It is intended that the use of the halogenated salicylanilide is applicable to all the dosage regimens described in this application. Accordingly, any reference to a

halogenated salicylanilide for use in a dosage regimen is also applicable to the equivalent use of the halogenated salicylanilide for the manufacture of a medicament for use in that dosage regimen.

[0024] In some embodiments the initial treatment period is less than 5 days, thereby providing a very short initial treatment period, followed by a prolonged“drug free” or“drug holiday” period during which no further topical halogenated salicylanilide is topically applied to the subject. For example, the initial treatment period may be 4 days or less, 3 days or less, 2 days or less, or 1 day. The duration of the initial treatment period will depend upon the condition being treated and the concentration of halogenated

salicylanilide required in the skin to provide a therapeutic effect. For certain skin conditions an initial topical treatment period of 1 day or 2 days may be sufficient to provide a concentration of the halogenated salicylanilide in skin tissue that is in excess of the minimum therapeutically effective concentration required to treat the skin condition and to maintain a therapeutic concentration of the halogenated salicylanilide in the skin tissue for a prolonged period without the need for further topical application of the halogenated salicylanilide.

[0025] During the initial treatment period the halogenated salicylanilide is topically applied to provide a therapeutically effective dose of the halogenated salicylanilide in the skin tissue. For example, the halogenated salicylanilide is topically applied 1 , 2, 3 or 4 times per day during the initial treatment period. In some embodiments the halogenated salicylanilide is topically applied once or twice per day during the initial treatment period. In some embodiments the initial treatment period is 1 day and the halogenated salicylanilide is topically applied only once during the initial treatment period. Accordingly, in

embodiments, the halogenated salicylanilide is topically applied once or twice per day for one or two days followed by a treatment free period during which no further halogenated salicylanilide is topically applied to the subject. In certain embodiments the halogenated salicylanilide (e.g. oxyclozanide) is topically applied once followed by a treatment free period during which no further halogenated salicylanilide is topically applied to the subject i.e. the initial treatment consists of a single topical application of the halogenated salicylanilide (e.g. oxyclozanide) followed by a treatment free period (e.g. 1 week or more) during which no further halogenated salicylanilide is topically applied to the subject.

[0026] The halogenated salicylanilide is suitably applied to the same area of skin during the initial treatment period thereby maximising the concentration of the salicylanilide in the skin tissue at that location. Generally, the halogenated salicylanilide will be applied directly to the skin affected by the skin condition, for example the halogenated salicylanilide may be applied directly to an inflamed area of skin affected by an inflammatory skin condition (e.g. a skin lesion associated with dermatitis) or directly to the site of a skin infection.

However, it is also contemplated that the halogenated salicylanilide is topically applied to skin that is not directly affected by the skin condition. This may be useful to, for example, decolonise skin of bacteria to prevent or minimise the risk of infection of skin affected by a skin condition and/or to minimise the risk of developing an infection associated with surgical procedures (e.g. surgical incisions or the insertion of medical devices such as intravenous lines or cannulas). Topical application of the halogenated salicylanilide to a local area of the body of an animal may provide therapeutic concentrations of the halogenated salicylanilide in areas of the skin remote from the point of application of the halogenated salicylanilide as a result of diffusion of the halogenated salicylanilide into dermal tissues. For example, local topical application to a particular location (spot on treatment) or as a line or stripe (line-on treatment) on the animal may provide a therapeutic concentration the halogenated salicylanilide in all, or substantially all of the skin of the animal, thereby providing treatment of the whole animal.

[0027] After the initial treatment period there is a treatment-free period during which no further topical halogenated salicylanilide is applied to the subject. The treatment-free period is at least 2 days. Preferably the treatment-free period is at least 3 days. In some embodiments the treatment-free period is at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least 28 days, at least 2 months, at least 3 months, at least 4 months, at least 6 months, at least 9 months or at least 1 year. In embodiments the treatment-free period is selected from 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 21 days, 28 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 9 months and 1 year. For example, the treatment-free period may be from 2 days to 1 month, 3 days to 3 months, 4 days to 1 month, 5 days to 1 month, 2 days to 21 days, 2 days to 14 days, 2 days to 10 days, 2 days to 7 days, 3 days to 21 days, 3 days to 14 days, 3 days to 10 days, 3 days to 7 days, 4 days to 21 days, 4 days to 14 days, 4 days to 10 days, 4 days to 7 days, 5 days to 21 days, 5 days to 14 days or 5 days to 10 days.

[0028] In some embodiments further halogenated salicylanilide is topically applied to the subject at the end of the treatment-free period. This may be required for the treatment of certain skin conditions wherein the condition has not completely resolved and/or wherein it is desirable to provide additional halogenated salicylanilide to maintain the symptoms of the condition at a desirable level (i.e. to provide a maintenance therapy) and/or to prevent recurrence and/or a flare-up of the skin condition. When additional halogenated salicylanilide is topically applied at the end of the treatment free period, the additional halogenated salicylanilide may be topically applied using any dosage regimen. For example, the additional halogenated salicylanilide may be topically applied to the subject regularly (e.g. once or twice per day). However, conveniently the additional halogenated salicylanilide is topically administered to the subject for a second initial treatment period followed by a second treatment-free period during which no further halogenated salicylanilide is topically administered to the subject. The second initial treatment period and the second treatment-free periods may be any of the initial treatment periods and treatment-free periods described herein. The treatment cycle of an initial period followed by a treatment-free period may be repeated numerous times as required to treat or manage the skin condition over a prolonged period of time. For example, the dosage regimen may be repeated 2, 3, 4, 5, 6 or more times.

[0029] In certain embodiments the halogenated salicylanilide (e.g. oxyclozanide) is topically applied once followed by a treatment-free period of at least 7 days. In certain embodiments the halogenated salicylanilide (e.g. oxyclozanide) is topically applied once followed by a treatment-free period of 1 or 2 weeks. It may be that the halogenated salicylanilide (e.g. oxyclozanide) is topically applied once every 7 days (i.e. one time per week). It may be that the halogenated salicylanilide (e.g. oxyclozanide) is topically applied once every 7 days (i.e. one time per week) for 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks.

[0030] Certain skin conditions treated in accordance with the dosage regimen of the invention will resolve during the treatment-free period. Accordingly, it is also contemplated that no further topical halogenated salicylanilide is applied after the initial treatment period. For example, when the skin condition is a skin infection (e.g. canine pyoderma), the initial treatment period provides a concentration of the halogenated salicylanilide in the skin at the site of infection which exceeds the minimum inhibitory concentration (MIC) of the bacteria responsible for the infection for a sufficient period of time that the bacteria are eradicated or reduced to a level such that the infection resolves during the treatment-free period. Accordingly, in these embodiments the halogenated salicylanilide is topically applied to the subject only during the initial treatment period, thereby providing a very short topical treatment dosage regimen of, for example 1 or 2 days, preferably a single topical application, without the need for additional topical application of the halogenated salicylanilide. [0031] The skin condition may be one that affects skin tissue at different depths. For example, the skin condition may affect the dermis and/or the epidermis. In embodiments the skin condition affects the epidermis, for example one or more of the stratum basale, stratum spinosum, stratum granulosum, stratum corneum and/or stratum lucidum. In embodiments the skin condition (e.g. skin infection) affects the stratum corneum. In some embodiments the skin condition (e.g. skin infection) affects the dermis, for example one or more of the papillary layer and the reticular layer of the epidermis. In some embodiments the skin condition affect the dermis and the epidermis. In some embodiments of the invention the skin condition (e.g. skin infection) may affect deeper tissues such as the hypodermis (subcutaneous tissue) and/or underlying muscle tissue. In some

embodiments the skin condition (e.g. skin infection) affects tissue from the outer surface of the skin to a depth of about 20 mm, for example from about 1 mm to about 10 mm.

[0032] The topical application of the halogenated salicylanilide suitably provides a therapeutically effective concentration of the halogenated salicylanilide in the skin tissue (e.g. the dermis and/or the epidermis) at the site of administration following completion of the initial treatment period. The halogenated salicylanilide is retained in the skin at the site of topical application for a prolonged period of time and can therefore maintain therapeutic concentrations of the halogenated salicylanilide for a significant time without the need for further topical administration of halogenated salicylanilide. For example the concentration of halogenated salicylanilide in the skin at the site of topical application is maintained at or in excess of a therapeutically effective concentration for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 1 1 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days or at least 28 days following completion of the initial treatment period (i.e. during the treatment-free period when no additional halogenated salicylanilide is administered to the subject).

[0033] The concentration of the halogenated salicylanilide in the skin at the completion of the initial treatment period may be determined using routine analytical methods. For example, the concentration in one or more layers of the skin can be determined from a skin biopsy taken from the site of topical administration. Suitably the concentration of halogenated salicylanilide is determined shortly after the last topical administration of the halogenated salicylanilide in the initial treatment period. For example, a skin biopsy should be taken (or other concentration analysis performed) within 12 hours, 8 hours, 6 hours, 4 hours, 2 hours or 1 hour or 30 minutes after the last topical application of the halogenated salicylanilide of the initial treatment period.

[0034] In some embodiments the skin condition is a skin infection caused by Gram- positive bacteria. In some embodiments the skin condition is a skin infection caused by or associated with Gram-positive bacteria. In some embodiments the skin condition is an inflammatory skin condition. In some embodiments the skin condition is an inflammatory skin condition that is caused by or associated with the presence of Gram-positive bacteria. In some embodiments the skin condition is an inflammatory skin condition that is colonised by Gram-positive bacteria. In some embodiments the skin condition is an inflammatory skin condition that is infected by Gram-positive bacteria (e.g. infected dermatitis, infected diabetic ulcers or an infected burn or wound). In some embodiments the skin condition is inflammation caused by a Gram-positive skin infection.

[0035] In embodiments where the skin condition is a skin infection, the concentration of the halogenated salicylanilide in the skin at the site of the skin infection upon completion of the initial treatment period is suitably in excess of the MIC of the bacteria responsible for or associated with the skin infection. In some embodiments the concentration of the halogenated salicylanilide in the skin at the site of infection upon completion of the initial treatment period is at least 3 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 15 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times the MIC of the bacteria responsible for or associated with the skin infection. In some embodiments the

concentration of the of the halogenated salicylanilide in the skin at the site of the skin infection upon completion of the initial treatment period is from 3 to at least 10 times, from 5 times to at least 20 times of from at least 5 times to at least 10 times the MIC of the bacteria responsible for or associated with the skin infection.

[0036] In embodiments where the skin condition is an inflammatory skin condition, the concentration of the halogenated salicylanilide in the skin at the site affected by the skin condition upon completion of the initial treatment period suitably exceeds the minimum concentration necessary to treat the skin condition (e.g. to provide an anti-inflammatory effect on the condition and/or to treat one or more of the symptoms associated with the inflammatory skin condition such as pruritus (itching), erythema and/or induration).

[0037] Suitably in any of the embodiments described herein, the concentration of halogenated salicylanilide in the skin at the location where the halogenated salicylanilide is applied, or in the skin at the site affected by the skin condition, at the end of the initial treatment period is at least 0.01 pg/g, for example at least 0.02 pg/g, 0.05 pg/g, at least 0.1 pg/g, at least 1.0 pg/g, at least 10 pg/g, at least 15 pg/g, at least 20 pg/g, at least 25 pg/g, at least 30 pg/g, at least 35 pg/g, at least 40 pg/g, at least 45 pg/g, at least 50 pg/g, at least 55 pg/g, at least 60 pg/g, at least 65 pg/g, at least 70 pg/g, at least 75 pg/g, at least 80 pg/g, at least 85 pg/g, at least 90 pg/g, at least 95 pg/g, at least 100 pg/g, at least 1 10 pg/g, at least 120 pg/g, at least 130 pg/g, at least 140 pg/g, at least 150 pg/g, at least 200 pg/g, at least 250 pg/g, at least 300 pg/g, at least 350 pg/g, at least 400 pg/g at least 450 pg/g at least 500 pg/g, at least 1.0 mg/g, at least 100 mg/g or at least 500 mg/g is present in the skin tissue at the completion of the initial treatment period. In some embodiments the concentration of halogenated salicylanilide in the skin at the completion of the initial treatment period is from about 0.01 pg/g to about 500mg/g, for example from about 0.1 pg/g to about 100 mg/g, about 0.1 pg/g to about 1 mg/g, about 0.1 pg/g to about 500 pg/g, from about 0.1 pg/g to about 100 pg/g, about 0.1 pg/g to about 50 pg/g, from about 0.1 pg/g to about 10 pg/g, about 1 pg/g to about 200 pg/g, about 1 pg/g to about 150 pg/g, about 1 pg/g to about 100 pg/g, about 2 pg/g to about 200 pg/g, about 2 pg/g to about 150 pg/g, or about 2 pg/g to about 100 pg/g. In certain embodiments reference to

concentration of the halogenated salicylanilide in the skin refers to the mean concentration in a skin sample (e.g. a skin biopsy). In certain embodiments reference to concentration of the halogenated salicylanilide in the skin refers to the mean concentration in the dermis, epidermis and stratum corneum. In certain embodiments reference to concentration of the halogenated salicylanilide in the skin refers to the mean concentration in the dermis and epidermis. In certain embodiments reference to concentration of the halogenated salicylanilide in the skin refers to the mean concentration in the stratum corneum.

[0038] The concentration of the halogenated salicylanilide in the skin tissue will decline during the treatment-free period due to, for example, absorption, metabolism and or/degradation of the halogenated salicylanilide. Further topical application of the halogenated salicylanilide may therefore be required at the end of the treatment-free period to maintain a therapeutically effective concentration of the halogenated

salicylanilide in the skin tissue. In those embodiments where the skin condition is a skin infection further halogenated salicylanilide may be topically administered when the concentration of the halogenated salicylanilide in the skin tissue (e.g. the dermis and/or epidermis) has declined to about the MIC of the bacteria responsible for or associated with the skin infection. Preferably, however, further halogenated salicylanilide is topically administered before the concentration in the skin tissue (e.g. the dermis and/or epidermis) has declined to the MIC to ensure that the bacteria at the site of infection are continuously exposed to concentrations of the halogenated salicylanilide that exceed the MIC of the bacteria. For example, further halogenated salicylanilide is topically administered when the concentration of the halogenated salicylanilide in the skin tissue (e.g. the dermis and/or epidermis) at the site of the skin infection has declined to 2 times, 3 times, 4 times or 5 times the MIC of the bacteria responsible for or associated with the skin infection. In some embodiments the concentration of the halogenated salicylanilide in the skin tissue during the treatment-free period may be sufficient to eradicate or reduce the bacteria responsible for the skin infection such that the skin infection resolves during the treatment-free period. In those embodiments no further topical halogenated salicylanilide would be required at the end of the treatment-free period. In some embodiments a single dose of the halogenated salicylanilide is topically administered to the subject to treat the skin condition (e.g. a skin infection such as pyoderma). In this embodiment a single topical dose of the halogenated salicylanilide is sufficient to treat the skin condition and not further topical administration is required. For example, a single topical administration may be sufficient to eradicate or control a skin infection (e.g. pyoderma).

[0039] The inventors have found that high concentrations of the halogenated

salicylanilide are retained in the skin tissue following completion of the initial dosing period and that high concentrations are retained in the skin for a prolonged period. This provides the possibility of retaining therapeutically effective concentrations of the halogenated salicylanilide in the skin tissues without the need for further topical administration of the halogenated salicylanilide. In some embodiments the concentration of the halogenated salicylanilide in skin tissue at the site of topical administration is greater than about 300 pg/g, (e.g. greater than about 500 pg/g, greater than about 1000 pg/g, greater than 1500 pg/g or greater than 2000 pg/g) for a period of at least 5 days (e.g. at least 7 days, at least 10 days, at least 14 days, at least 21 days or at least 28 days) after completion of the initial dosing period.

[0040] In an embodiment there is provided a halogenated salicylanilide, or a

pharmaceutically acceptable salt or hydrate thereof, for use in the topical treatment of a skin infection caused by or associated with Gram-positive bacteria in a non-human subject, wherein the halogenated salicylanilide is topically administered to the subject at the site of infection for an initial treatment period of 5 days or less (e.g. a single topical treatment) to provide a concentration of the halogenated salicylanilide in the epidermis (e.g. the stratum corneum) and/or the dermis at the site of infection upon completion of the first treatment period of at least 5 times the minimum inhibitory concentration of the Gram-positive bacteria responsible for or associated with the skin infection;

and wherein

A: the bacteria responsible for the infection are eradicated from the site of infection and/or the infection resolves without further topical administration of the halogenated salicylanilide to the subject after the initial treatment period; or

B: no further halogenated salicylanilide is topically administered after the initial treatment period until the concentration of the halogenated salicylanilide in the epidermis (e.g. the stratum corneum) and/or the dermis at the site of administration has reduced to about the minimum inhibitory concentration (MIC) of the Gram-positive bacteria responsible for or associated with the skin infection.

[0041] In this embodiment, where further topical halogenated salicylanilide is topically applied (i.e. B above), further topical halogenated salicylanilide may be applied when the concentration of the halogenated salicylanilide in the epidermis (e.g. the stratum corneum) and/or the dermis at the site of administration has reduced to about 2 times, about 3 times or about 4 times, preferably about 3 times the MIC of the Gram-positive bacteria responsible for or associated with the skin infection.

[0042] The concentration of the halogenated salicylanilide in the skin of the subject may be determined using known methods, for example by tape stripping methods wherein tape is used to strip layers of skin from the site of application, or preferably from a skin biopsy, which are then analysed to assess the concentration of the compound in a particular skin layer. Suitable tape stripping methods are described in the Examples herein, for example an analogous method to that described in Videmont at al.(“Characterization of the canine skin barrier restoration following acute disruption by tape stripping,” Veterinary

Dermatology, vol. 23, pp. 103-123, 2011 ). Alternatively, the concentration of halogenated salicylanilide is determined by analysing a skin biopsy taken from the site of administration of the halogenated salicylanilide. Biopsies can be obtained using well-known methods, for example a punch biopsy. One or more biopsies may be taken. Suitably punch biopsies of about 4mm diameter are taken from the treatment area under local anaesthesia. The concentration of the halogenated salicylanilide in the skin sample may be assessed using well-known analytical methods, for example HPLC or UPLC/MS methods as illustrated in the Examples herein. Suitably a skin biopsy is taken from the subject and this is cut into small pieces or is homogenised. The sample is then placed in a suitable solvent to extract the halogenated salicylanilide present in the sample. Suitably the solvent used for extraction is DMSO, acetonitrile or a mixture thereof, preferably a 1 :1 (v/v) mixture of DMSO and acetonitrile, although other solvents are also suitable. The extraction is conveniently performed at ambient temperature. The extraction is suitably performed for a period of 3 to 24 hours, for example 15 to 24 hours. Following extraction, the

concentration of the halogenated salicylanilide may be determined by analysing the concentration of the halogenated salicylanilide present in the supernatant liquid using a suitable analytical method such as HPLC/MS or UPLC/MS. The concentration in the skin sample may be determined by correlating the concentration measured in the supernatant liquid to the initial volume of skin in the skin biopsy.

[0043] The inventors have found that topical application of the halogenated salicylanilide provides high concentrations of the compound in skin tissue and only a low systemic exposure to the drug. The dosage regimen of the invention is therefore expected to provide an effective local treatment for the skin condition with no or only minimal side effects associated with systemic exposure to the halogenated salicylanilide.

[0044] Suitably the concentration of the halogenated salicylanilide in the skin tissue at the site of topical application of the halogenated salicylanilide upon completion of the initial treatment period is greater than the mean plasma C_max during or after the initial treatment period. For example, in some embodiments the concentration of the halogenated salicylanilide in the skin tissue at the site of topical application of the halogenated salicylanilide upon completion of the initial treatment period is at least 5 times, at least 10 times, at least 20 times, at least 50 times or at least 100 times the mean plasma C_max of the halogenated salicylanilide during or after the initial treatment period.

[0045] In some embodiments the mean plasma C_max of the halogenated salicylanilide during or after completion of the initial dosage period is less than about 2000 pg/l, 1500 pg/l, for example less than 1000 pg/l, less than 500 pg/l, or less than 200 pg/l. The plasma C_{max W}ill vary depending on the dose of the halogenated salicylanilide topically

administered, the area of the skin to which the compound is topically applied and possibly the frequency of dosing during the initial treatment period. In some embodiments the mean plasma C_max of the halogenated salicylanilide during or after the initial treatment period is less than about 2000 pg/l, 1500 pg/l, for example less than 1000 pg/l, less than 500 pg/l, or less than 200 pg/l, wherein the halogenated salicylanilide is topically administered at a dose of 20 mg/kg administered once per day during the initial treatment period, or a mean C_maxdirectly proportional thereto for a topically applied dose other than 20 mg/kg. In some embodiments the mean plasma C_max of the halogenated salicylanilide (e.g. oxyclozanide), is less than about 1500 pg/l, for example less than 1000 pg/l, less than 500 pg/l, or less than 200 pg/l, when topically administered as a single dose of 20 mg/kg applied as a line along the spine of an animal such as a dog.

[0046] In some embodiments the mean plasma concentration of the halogenated salicylanilide (e.g. oxyclozanide) measured over a period of 24 to 96 hours after topically administering a single dose of 20 mg/kg of the halogenated salicylanilide applied as a line along the spine of an animal such as a dog is less than about 1300 pg/l, for example less than about 800 pg/l, less than 700 pg/l, less than 500 pg/l, less than 200 pg/l, or less than 150 pg/l, or a concentration directly proportional thereto for a single dose other than 20 mg/kg. For example, the mean plasma concentration of the halogenated salicylanilide (e.g. oxyclozanide) in the period of 24 to 96 hours after topical administration of the halogenated salicylanilide (e.g. oxyclozanide) may be from about 20 to about 200 pg/l or about 50 pg/l to about 150 pg/l.

[0047] The plasma C_max of the halogenated salicylanilide may be determined using routine methods, for example by HPLC analysis of a blood samples collected at multiple time points during and after the initial topical treatment period. For example, blood samples may be taken at about 1 , 2, 4, 6 and 8 hours following the administration of the last dose of the halogenated salicylanilide in the initial treatment period.

[0048] The plasma C_max in the embodiments described herein is suitably determined in the period after the last topical administration of the halogenated salicylanilide of the initial treatment period. The C_max may be determined by taking regular blood samples after the last dose of the halogenated salicylanilide so as to determine the maximum plasma concentration. Generally, samples taken once per hour or once every 2 hours for 12 hours after the last topical administration of the halogenated salicylanilide will be sufficient to determine the C_max value. The plasma concentration of the halogenated salicylanilide (e.g. oxyclozanide), for example to determine the mean plasma concentration in the period of 24 to 96 hours after topical administration of the halogenated salicylanilide, may be measured using analogous methods to those described above for C_max determination.

[0049] In some embodiments the mean Cmax of the halogenated salicylanilide (e.g. oxyclozanide) in the stratum corneum measured over a period of 1 day to 28 days after topically administering a single dose of 20 mg/kg of the halogenated salicylanilide (e.g. oxyclozanide) applied as a line along the spine of an animal such as a dog is greater than 50 pg/g, or a concentration directly proportional thereto for a single dose other than 20 mg/kg. For example, the mean Cmax in the stratum corneum after topically administering a single dose of 20 mg/kg of the halogenated salicylanilide (e.g. oxyclozanide) is greater than about 60 mg/g, about 70 mg/g, about 80 mg/g, about 90 mg/g, about 100 mg/g, about 1 10 mg/g, about 120 mg/g, about 150 mg/g, about 175 mg/g or about 200 mg/g, or a concentration directly proportional thereto for a single dose other than 20 mg/kg. It may be that the mean Cmax in the stratum corneum after topically administering a single dose of 20 mg/kg of the halogenated salicylanilide (e.g. oxyclozanide) is from about 30 to about 700 pg/g, from about 50 to about 600 pg/g, from about 60 to about 550 pg/g, or from about 60 to about 500 pg/g, or a concentration directly proportional thereto for a single dose other than 20 mg/kg.

[0050] In some embodiments the mean Cmax of the halogenated salicylanilide (e.g. oxyclozanide) in the dermis and epidermis measured over a period of 1 day to 28 days after topically administering a single dose of 20 mg/kg of the halogenated salicylanilide (e.g. oxyclozanide) applied as a line along the spine of an animal such as a dog is greater than 1 pg/g, or a concentration directly proportional thereto for a single dose other than 20 mg/kg. For example, the Cmax in the dermis and epidermis after topically administering a single dose of 20 mg/kg of the halogenated salicylanilide (e.g. oxyclozanide) is greater than about 1.5 pg/g, about 2 pg/g, about 2.5 pg/g, about 3 pg/g, about 3.5 pg/g, about 4 pg/g, about 4.5 pg/g or about 5 pg/g, or a concentration directly proportional thereto for a single dose other than 20 mg/kg. It may be that the Cmax in the dermis and epidermis after topically administering a single dose of 20 mg/kg of the halogenated salicylanilide (e.g. oxyclozanide) is from about 1 to about 13 pg/g, from about 1.5 to about 10 pg/g or from about 2 to about 8 pg/g, or a concentration directly proportional thereto for a single dose other than 20 mg/kg.

[0051] In some embodiments the area under the curve of the halogenated salicylanilide (e.g. oxyclozanide) in the stratum corneum (AUC28) 28 days after topically administering a single dose of 20 mg/kg of the halogenated salicylanilide (e.g. oxyclozanide) applied as a line along the spine of an animal such as a dog is greater than 500 day^*pg/g, or a value directly proportional thereto for a single dose other than 20 mg/kg. For example the AUC28 is greater than 600 day^*pg/g, greater than 700 day^*pg/g, greater than 800 day^*pg/g, greater than 900 day^*pg/g, greater than 1000 day^*pg/g, greater than 1200 day^*pg/g or greater than 1500 day^*pg/g. It may be that the AUC28 is from 800 day^*pg/g to 7000 day^*pg/g, from 850 day^*pg/g to 6000 day^*pg/g, or from 850 day^*pg/g to 5500 day^*pg/g.

[0052] In some embodiments, the time to reach maximum concentration of the halogenated salicylanilide (e.g. oxyclozanide) in the skin, T_max, (e.g. the stratum corneum, or the dermis and epidermis) at sites remote from the point of application after topically administering a single dose of, for example, 20 mg/kg of the halogenated salicylanilide (e.g. oxyclozanide) applied as a line along the spine of an animal such as a dog, is from about 1 to 10 days, for example from 2 to 10 days, from 3 to 8 days or from 4 to 8 days. The site remote from the point of topical application of the halogenated salicylanilide may be, for example the belly, chest, ear, fore leg, hind leg or shoulder of the animal (e.g. dog).

[0053] The concentration of the halogenated salicylanilide in the skin (or individual skin layers) may be assessed by measuring the concentration in a skin biopsy taken from the subject following topical administration of the halogenated salicylanilide, for example using the methods described herein and in the Examples (e.g. a tape stripping method). The Cmax, AUC and T_max values may be calculated using well known methods based on the measured concentrations of the halogenated salicylanilide in the skin sample, such methods are illustrated in the Examples herein.

[0054] In embodiments where the skin condition is a skin infection, the skin infection is caused by or associated with Gram-positive bacteria. In some embodiments the Gram- positive bacteria are selected from Staphylococcus spp., Streptococcus spp.,

Propionibacterium spp. or Corynebacterium spp. In some embodiments the Gram- positive bacteria are selected from Staphylococcus spp.. In some embodiments the Gram- positive bacteria are selected from selected from Staphylococcus aureus, Streptococcus pyogenes and Propionibacterium acnes. In some embodiments the Gram-positive bacteria are selected from Streptococcus uberis, Staphylococcus aureus, Staphylococcus pseudin termedius, Staphylococcus intermedius, Staphylococcus schleiferi, and coagulase- positive staphylococci. In some embodiments the Gram-positive bacteria are selected from Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus intermedius, Staphylococcus schleiferi and Staphylococcus hyicus. In a preferred embodiment the bacteria are Staphylococcus pseudintermedius.

[0055] In some embodiments, the Gram-positive bacteria are from the Streptococcus genus. It may be that the Gram-positive bacteria are Streptococcus selected from

Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus suis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis or Streptococcus viridans. In some embodiments, the Gram-positive bacteria are Streptococcus pyogenes.

[0056] In some embodiments, the Gram-positive bacteria are from the Staphylococcus genus. It may be that the Gram-positive bacteria are Staphylococcus selected from

Staphylococcus epidermidis, Staphylococcus pseudintermedius, Staphylococcus aureus, Staphylococcus saprophyticus and Staphylococcus lugdunensis. In some embodiments, the coccus Gram- positive bacteria are Staphylococcus aureus and/or Staphylococcus pseudintermedius.

[0057] In some embodiments the Gram-positive bacteria are selected from

Corynebacterium spp. for example, Corynebacterium bovis.

[0058] In some embodiments the Gram-positive bacteria are not a Propionibacterium spp. For example, the Gram-positive bacteria are not Propionibacterium acnes.

[0059] In some embodiments the Gram-positive bacteria is not an antibiotic resistant strain.

[0060] In some embodiments the Gram-positive bacteria is an antibiotic resistant strain.

It may be that the Gram-positive bacteria are resistant to an antibiotic other that the halogenated salicylanilide. In some embodiments the Gram-positive bacteria are resistant to a drug selected from fusidic acid, mupirocin, retapamulin, erythromycin, clindamycin, lincomycin, a tetracycline, a b-lactam (e.g. methicillin, amoxicillin, oxacillin, dicloxacillin, flucloxacillin or a cephem (e.g. cefazolin, cefalexin or cefixime)), a cephalosprin (e.g. cephalexin or cefovecin), a fluoroquinolone (e.g. enrofloxacin, orbifloxacin, marbofloxacin or ciprofloxacin), trimethoprim, sulfamethoxazole and sulfadiazine or a combination of two or more thereof.

[0061] It may be that the Gram-positive bacteria are resistant to a topical antibiotic other than the halogenated salicylanilide. In some embodiments the Gram-positive bacteria are resistant to a drug selected from fusidic acid, mupirocin and retapamulin. In a particular embodiment the Gram-positive bacteria are methicillin resistant Staphylococcus aureus (MRSA), methicillin resistant Staphylococcus, pseudintermedius (MRSP) or methicillin resistant Staphylococcus intermedius (MRSI).

[0062] In some embodiments where the skin condition is a skin infection caused by or associated with Gram-positive bacteria susceptibility testing of the bacteria to the halogenated salicylanilide may be carried out. Susceptibility testing may be performed prior to initiation of the topical treatment to ensure that the bacteria will respond to the halogenated salicylanilide. If the bacteria are susceptible to the halogenated salicylanilide the topical application is initiated.

[0063] Gram-positive bacteria have been shown to exhibit a very low frequency of spontaneous mutations that confer resistance to halogenated salicylanilides such as niclosamide, rafoxanide, closantel, and oxyclozanide (see WO 2016/038035). Accordingly, the emergence of resistance to the halogenated salicylanilide is not expected. Nevertheless, susceptibility testing may be performed during the treatment regimen to ensure that the bacteria continue to respond to the halogenated salicylanilide and may be useful to detect the development of resistance to the halogenated salicylanilide during the treatment regimen. In the event that resistance emerges during the treatment regimen topical treatment with the halogenated salicylanilide may be discontinued.

[0064] Susceptibility testing may be carried out using known methods, for example using standard disk diffusion or broth microdilution tests performed on bacteria collected from the site of infection by e.g. swabbing or from a tissue sample taken from the site of infection. Broth dilution is useful for the determination of the MIC of the halogenated salicylanilide against a particular strain of Gram-positive bacteria and may influence the dose of the halogenated salicylanilide that is topically applied during the initial treatment period. An increase in the MIC observed during the dosage regimen (e.g. at the end of the treatment- free period) may also signal the potential emergence of resistance and can be used by a physician to decide whether or not to continue topical treatment with the halogenated salicylanilide. Examples of susceptibility tests that may be used include analogous methods to those described in Clinical and Laboratory Standards Institute (CLSI),

Performance Standards for Antimicrobial Susceptibility Testing; 25^th Informational

Supplement; CLSI document M100-S25, 2015; CLSI Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard 10^th edition. CLSI document M07-A10, 2015; and CLSI, Performance Standards for Antimicrobial Disk Diffusion Susceptibility Tests; Approved Standard 12^th edition, 2015.

[0065] In some embodiments the skin condition is a superficial skin infection caused by or associated with Gram-positive bacteria.

[0066] In some embodiments the skin condition is a skin condition caused by or associated with Gram-positive bacteria, for example wherein the skin condition is selected from pyoderma (including surface, superficial and deep pyoderma e.g. canine pyoderma or feline pyoderma), otitis, bacterial conjunctivitis, dermatitis, folliculitis, superficial folliculitis, erythrasma, erythroderma, dermatoses, carbuncles, abscesses, cysts, furunculosis, ecthyma, cellulitis, erysipelas, necrotising fasciitis, secondary skin infections, scabies (including sarcoptic mange), chronic ulcers, vesicular eruptions, bullous eruptions, mastitis, chronic rhinosinusitis, traumatic skin lesions and Staphylococcal scalded skin syndrome (SSSS or Ritter’s disease). [0067] In some embodiments the skin condition is superficial or deep pyoderma. In some embodiments the skin condition is superficial pyoderma. In some embodiments the skin condition is deep pyoderma. It may be that the pyoderma is caused by, or associated with, Gram-positive bacteria. It may be that the pyoderma is caused by, or associated with, Staphylococcus spp. bacteria. It may be that the pyoderma is caused by, or associated with, bacteria selected from Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus intermedius, Staphylococcus vitulinus, Staphylococcus albus,

Staphylococcus chromogenes and Staphylococcus hominis spp. It may be that the pyoderma is caused by, or associated with, bacteria selected from Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus intermedius, Staphylococcus schleiferi and Staphylococcus hyicus. In another embodiment the pyoderma is caused by, or associated with, Staphylococcus intermedius or Staphylococcus pseudintermedius.

Preferably the pyoderma is caused by, or associated with Staphylococcus

pseudin termed i us.

[0068] In some embodiments the skin condition is pyoderma, preferably canine pyoderma. Reference to pyoderma herein includes for example, pyotraumatic dermatitis, impetigo (superficial pustular dermatitis), superficial bacterial folliculitis, chin pyoderma (canine acne), skin fold dermatitis (intertrigo, skin fold pyoderma), mucocutaneous pyoderma, nasal pyoderma, bacterial pododermatitis, canine pedal furunculosis

(interdigital bullae, interdigital pyogranuloma). In a preferred embodiment the pyoderma is surface or superficial pyoderma.

[0069] In some embodiments the skin condition is a Gram-positive bacterial infection of skin tissue that has been compromised by an underlying condition. For example, the barrier function of the skin may be compromised in conditions such as dermatitis (e.g. atopic dermatitis) or chronic ulcers, thereby enabling bacterial to penetrate and colonise skin tissues resulting in an infection. Similarly, when the skin is damaged as a result of skin lesion (e.g. a traumatic skin lesions, such as, burns, cuts, abrasions, animal bites or insect bites or surgical wound/incisions) the site of the skin lesion can become infected with bacteria. In some embodiments the skin condition is a skin infection caused by or associated with Gram-positive bacteria and is selected from the group consisting of infected dermatitis (including infected atopic dermatitis, infected canine atopic dermatitis, infected flea allergy dermatitis, infected malassezia dermatitis, infected intertrigo (“skin fold dermatitis”), infected pododermatitis, infected demodicosis, infected food allergic dermatitis, and infected contact dermatitis), infected ulcers, infected scabies (including infected sarcoptic mange), otitis and infected traumatic skin lesions (including infected surgical wounds, burns, cuts, abrasions, animal bites or insect bites).

[0070] In some embodiments the skin condition is a skin condition caused by or associated with Gram-positive bacteria and is selected from the group consisting of pyoderma (e.g. canine pyoderma or feline pyoderma), otitis, bacterial conjunctivitis, folliculitis, superficial folliculitis, erythrasma, dermatoses, carbuncles, abscesses, cysts, furunculosis, ecthyma, cellulitis, paronychia, erysipelas, necrotising fasciitis, secondary skin infections, vesicular eruptions, bullous eruptions, mastitis and Staphylococcal scalded skin syndrome (SSSS or Ritter’s disease) and erysipelas.

[0071] Pyoderma, for example canine pyoderma, is often associated with underlying conditions which make the subject more prone to infection and/or recurrence of the infection. Accordingly, in some embodiments the dosage regimen of the invention is for use in the treatment of pyoderma (e.g. superficial pyoderma or deep pyoderma) in a non- human subject, wherein the non-human subject (e.g. a dog) has one or more conditions in addition to pyoderma selected from: dermatitis, (e.g. atopic dermatitis or hypersensitivity dermatitis), a parasitic skin infestation, an endocrinopathy, a follicular dysplasia disorder, and a keratinization disorder, a food allergy, flea allergy, hypothyroidism,

hyperestrogenism and demodicosis, scabies and zinc-responsive dermatosis. In particular embodiments in some embodiments the dosage regimen of the invention is for use in the treatment of pyoderma (e.g. superficial pyoderma or deep pyoderma) in a non-human subject, wherein the non-human subject (e.g. a dog) has dermatitis, especially particularly atopic dermatitis.

[0072] It may be that in any of the embodiments described herein the pyoderma is recurrent pyoderma. That is pyoderma that has recurred following an earlier treatment, for example treatment with an antibiotic agent other than the halogenated salicylanilide (e.g. oxyclozanide). Recurrent pyoderma is common in subjects that have an underlying condition that make the subject more prone to bacterial infection, for example one or more of the underlying conditions described in the paragraph above. In other embodiments the pyoderma is treatment naive prior to topical treatment with the halogenated salicylanilide. For example, wherein the pyoderma has not been previously treated with an antibiotic agent prior to the topical treatment the halogenated salicylanilide. In some embodiments the skin condition is mastitis, preferably mastitis in cattle. [0073] The halogenated salicylanilide provides an antibiotic effect on the gram-positive bacteria colonising the skin tissue at the site of infection and may produce a bacteriostatic effect or a bactericidal effect on the gram-positive infection thereby reducing or eliminating the bacteria and enabling the infection to resolve. The dosage regimen of the invention may reduce, eliminate or prevent one or more of the symptoms or effects associated with a skin infection, for example one or more of blistering, exudate/pus, crusting, itching, pain, erythema, or inflammation associated with the skin infection (for example pyoderma).

[0074] In certain embodiments when the skin condition is pyoderma it may be that the dosage regimen of the invention treats or prevents one or more of papules, pustules, epidermal collarettes, scales, crusts and pruritus associated with the pyoderma. In a particular embodiment the dosage regimen of the invention is for use in the treatment or prevention of pruritus associated with the pyoderma (e.g. canine pyoderma).

[0075] In some embodiments the subject is not a mouse with an infected superficial skin wound. For example, the subject is not a mouse with an infected superficial skin wound, wherein the infected wound is topically treated with niclosamide for three days (for example topically treated twice per day for three days with a composition comprising niclosamide). In a particular embodiment the subject is not a mouse with a superficial skin wound infected by MRSA wherein the wound is topically treated twice per day for three days with a composition comprising niclosamide (for example a composition comprising 2% or 4% by weight of niclosamide).

[0076] It may be that the dosage regimen of the invention is used to decolonize Gram- positive bacteria from skin tissue. Decolonization may be beneficial in preventing the risk of a skin infection developing and/or to reduce or prevent symptoms of the skin condition that may be associated with, or exacerbated by, the presence of the bacteria. For example, the subject may have an underlying skin condition in which the barrier function of the skin is compromised or where the skin is damaged (e.g. in a dermatitis lesion or a traumatic skin injury). Decolonisation of the skin may be beneficial in preventing an infection from developing in the damaged skin. For skin conditions such as atopic dermatitis the presence of bacteria may contribute to or cause one or more of the symptoms of the skin condition. Bacterial decolonisation may therefore be beneficial in preventing, reducing or eliminating one or more of the symptoms of the skin condition (e.g. pruritus (itching), erythema, induration, excoriation, lichenification, scaling, oozing, crusting or xerosis associated with the skin condition, such as atopic dermatitis). Decolonisation is suitably performed by topically applying the halogenated salicylanilide to the skin affected by the skin condition in accordance with the dosage regimen described herein. Also contemplated is the decolonisation of skin on parts of the body that are not affected by the skin condition to prevent or reduce the risk of bacterial colonisation of the skin affected by the skin condition. For example, it is known that a common site for bacterial colonisation such as MRSA is the nose. Accordingly, the halogenated salicylanilide may be applied topically to the nose of an animal, for example a horse. Particularly the halogenated salicylanilide may be applied to the anterior nares (the inner surface of the nostrils) in accordance with the dosage regimen according to the invention.

[0077] Bacterial decolonisation using the dosage regimen of the invention may also be beneficial in preventing or reducing the risk of surgical site infections resulting from surgical or medical procedures carried out on the subject or at the site of medical devices such as catheters or IV lines or cannula. Accordingly, the dosage regimen of the invention may provide a prophylactic effect to prevent an infection of a non-human subject caused by or associated with Gram-positive bacteria. It may be that the halogenated salicylanilide is for use in the decolonisation of a non-human subject prior to or after carrying out a surgical procedure on the subject, wherein the halogenated salicylanilide is applied topically to the subject in accordance with the dosage regimen of the invention.

[0078] In some embodiments the skin condition is an inflammatory skin condition. It may be that the skin condition is an inflammatory skin condition selected from psoriasis, dermatitis, scleroderma, disorders of hair follicles and sebaceous glands, rhinophyma, cutaneous lupus, inflammatory reactions (for example drug eruptions, erythema multiforme, erythema nodosum, and granuloma annulare) and inflammation associated with fungal or yeast infections (e.g. dermatophytosis).

[0079] In a particular embodiment the skin condition is a dermatitis, for example atopic dermatitis.

[0080] It is known that the presence of bacteria in skin affected by an inflammatory skin condition can cause and/or exacerbate an inflammatory response in some skin conditions. For example, in dermatitis, particularly atopic dermatitis, colonisation of skin lesions with bacteria (e.g. S. aureus) is associated with inflammation and the pathogenesis of the disease. Reducing or eradicating bacteria from skin lesions in inflammatory skin conditions may therefore provide an anti-inflammatory effect on the skin condition, for example atopic dermatitis. The invention contemplates topical administration of the halogenated salicylanilide according to the dosage regimen of the invention to provide an anti-bacterial effect and/or an anti-inflammatory effect on the skin condition that is being treated. Many skin infections result in inflammation associated with the infection. The dosage regimen of the invention may provide an anti-inflammatory effect on the infected tissues as well as reducing or eliminating the Gram-positive bacteria present in the skin infection.

[0081] Accordingly the dosage regimen of the invention may be for use in the treatment or prevention of a bacterial infection in skin affected with an inflammatory skin condition (e.g. any one of the inflammatory skin conditions mentioned herein). In particular, pyoderma, especially recurrent pyoderma, is common in animals that have underlying inflammatory skin conditions. Accordingly, the dosage regimen of the invention may be for use in the topical treatment of pyoderma in a non-human subject (e.g. a dog or cat), wherein the subject has an inflammatory skin condition. It may be that the inflammatory skin condition is any one of the inflammatory skin conditions described herein. For example it may be that the inflammatory skin condition is dermatitis. The dermatitis may be any dermatitis described herein, particularly atopic dermatitis. It may be that the pyoderma is present in areas of the skin of the subject affected by the inflammatory skin condition (e.g. dermatitis, especially atopic dermatitis).

[0082] In some embodiments topical administration of the halogenated salicylanilide according to the dosage regimen of the invention provides a direct anti-inflammatory effect on the skin condition that is independent to the anti-bacterial effects of the compound. For example, the halogenated salicylanilide may provide a direct anti-inflammatory effect on an inflammatory skin condition selected from a reduction or elimination of one or more of pruritus, erythema, or induration associated with the inflammatory skin condition.

[0083] In some embodiments the skin condition is selected from pyoderma (including both deep and superficial pyoderma) and a dermatitis (e.g. atopic dermatitis, contact dermatitis, allergic contact dermatitis, irritant contact dermatitis, seborrhoeic dermatitis, lichen simplex chronicus (neurodermatitis), exfoliative dermatitis, carcinomatous dermatitis, perioral dermatitis, dermatomycosis, flea allergy dermatitis, malassezia dermatitis, intertrigo, pododermatitis, demodicosis, and food allergic dermatitis).

[0084] The halogenated salicylanilide may be any halogenated salicylanilide or pharmaceutically acceptable salt or hydrate or ester thereof which provides an

antibacterial and/or anti-inflammatory effect on the skin condition when topically administered to a non-human subject. Representative examples of halogenated salicylanilides include but are not limited to those described herein (e.g. niclosamide and/or oxyclozanide). [0085] In some embodiments the halogenated salicylanilide is selected from rafoxanide, oxyclozanide, closantel and niclosamide or a pharmaceutically acceptable salt, solvate or ester thereof.

[0086] In some embodiments the halogenated salicylanilide is selected from niclosamide or a pharmaceutically acceptable salt or hydrate thereof, optionally niclosamide or a pharmaceutically acceptable salt thereof, e.g. wherein the halogenated salicylanilide is niclosamide.

[0087] In some embodiments the halogenated salicylanilide is selected from

oxyclozanide, or a pharmaceutically acceptable salt or hydrate thereof, optionally oxyclozanide or a pharmaceutically acceptable salt thereof, e.g. wherein the halogenated salicylanilide is oxyclozanide.

[0088] The halogenated salicylanilide is suitably topically administered in the form of a pharmaceutical composition suitable for topical administration, for example as a cream, ointment, gel, foam, or aqueous, non-aqueous or oily solution or suspension. In some embodiments the halogenated salicylanilide is formulated as a non-aqueous

pharmaceutical composition suitable for topical administration, for example a non-aqueous cream, ointment, gel, lotion, solution, suspension or foam comprising the halogenated salicylanilide (for example niclosamide or a pharmaceutically acceptable salt or hydrate thereof). In some embodiments the halogenated salicylanilide is formulated as an aqueous pharmaceutical composition suitable for topical administration, for example an aqueous cream, ointment, gel, lotion, or foam comprising the halogenated salicylanilide (for example niclosamide or a pharmaceutically acceptable salt or hydrate thereof or oxyclozanide or a pharmaceutically acceptable salt or hydrate thereof). In some embodiments the halogenated salicylanilide (e.g. niclosamide or a pharmaceutically acceptable salt or hydrate thereof or oxyclozanide or a pharmaceutically acceptable salt or hydrate thereof) is formulated as a spot-on or line-on formulation.

[0089] In certain embodiments the halogenated salicylanilide is formulated as a topical composition comprising the halogenated salicylanilide (for example, selected from niclosamide, rafoxanide, oxyclozanide and closantel, or a pharmaceutically acceptable salt thereof of hydrate thereof); and polyethylene glycol (PEG).

[0090] In certain embodiments the halogenated salicylanilide is formulated as a topical composition comprising the halogenated salicylanilide (for example, selected from niclosamide, rafoxanide, oxyclozanide and closantel, or a pharmaceutically acceptable salt thereof of hydrate thereof) and a non-polymeric glycol (for example an alkylene glycol, e.g. a C alkylene glycol such as propylene glycol).

[0091] In certain embodiments the halogenated salicylanilide is formulated as a topical composition comprising the halogenated salicylanilide (for example, selected from niclosamide, rafoxanide, oxyclozanide and closantel, or a pharmaceutically acceptable salt thereof of hydrate thereof) and a glycol ether, for example, 2-(2-ethoxyethoxy)ethanol (also known as diethylene glycol monoethyl ether, commercially available as Transcutol).

[0092] In certain embodiments the halogenated salicylanilide is formulated as a non- aqueous topical composition comprising:

(i) a halogenated salicylanilide (for example, selected from niclosamide, rafoxanide, oxyclozanide and closantel, or a pharmaceutically acceptable salt thereof of hydrate thereof); and

(ii) polyethylene glycol (PEG).

[0093] In certain embodiments the halogenated salicylanilide is formulated as a non- aqueous topical gel composition comprising a halogenated salicylanilide (for example, selected from niclosamide, rafoxanide, oxyclozanide and closantel, or a pharmaceutically acceptable salt thereof of hydrate thereof) and a gel forming agent. The gel-forming agent may be any of the gel-forming agents disclosed herein. Suitably the topical gel composition further comprises a PEG.

[0094] Suitably the PEG in the composition is selected such that the composition together with any other components of the composition (e.g. in the form of a liquid, semi solid or gel composition) can easily be applied to, spread over and/or rubbed into the skin. It may be that the PEG has a melting point that is less than 35°C. In certain embodiments the PEG is selected such that it is soft or, suitably molten at body temperature. For example, the PEG may have a melting point of 32°C or less, or less than 30°C, or less than 25°C.

It may be that the halogenated salicylanilide is present in an amount of up to 15% by weight of the compositions described herein, for example from 0.05% to 10% by weight of the composition, from 0.05% to 4.5% by weight, 0.01 % to 7.5%, from 1% to 15% by weight, from 1% to 12% by weight, from 1 % to 3% by weight, from 1.5% to 4.5% by weight, from 2% to 15% by weight, from 2% to 12% by weight, from 3% to 12% by weight, from 4% to 12% by weight, from 7% to 12% by weight or from 8 to 1 1 % by weight of the composition It may be that the halogenated salicylanilide (e.g. oxyclozanide or niclosamide) is present in an amount of about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 1 1%, about 12%, about 13%, about 14% or about 15% by weight of the composition. For example, the

halogenated salicylanilide (e.g. oxyclozanide or niclosamide) is present in the composition at about 2% by weight of the composition or at about 4% by weight of the composition. It may be that the halogenated salicylanilide (e.g. oxyclozanide or niclosamide) is present in an amount of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11 %, about 12%, about 13%, about 14% or about 15% by weight/volume of the composition. In a particular embodiment the halogenated salicylanilide in oxyclozanide and is present in the composition in an amount of from 7 to 12% by weight/volume of the composition or about 9% to 11 % by weight/volume of the composition, for example wherein the oxyclozanide is present in an amount of about 10% by weight/volume of the composition.

[0095] In certain embodiments the composition comprising the halogenated salicylanilide does not comprise dimethyl sulfoxide (DMSO).

[0096] It may be that the topical composition comprising the halogenated salicylanilide provides a local pH of greater than 4.5 at the site of application of the composition (for example a skin lesion associated with the skin condition or the site of a skin infection).

[0097] It may be that the non-aqueous composition provides a local pH of less than 6 at the site of application following topical application of the composition. Suitably the non- aqueous composition provides a local pH in the range of from about 4.5 to about 6 at the site of topical application of the composition.

[0098] Also provided is a spot-on or line-on composition comprising the halogenated salicylanilide or a pharmaceutically acceptable salt or solvate thereof. Examples of spot- on or line-on compositions are set out in the detailed description herein.

[0099] Further aspects and features of the invention are set out in the detailed description.

BRIEF DESCRIPTION OF DRAWINGS

[00100] Figure 1 shows the changes in biomarker expression that correlated with TSS/TAA and were found to have significantly changed compared to vehicle and baseline (S100A12, S100A9, PI3, CXCL1 and S100A7) as analysed in skin biopsies taken at Day 22 in the study of Example 5. [00101] Figures 2-5 show the changes in biomarker expression (KRT16, MMP12, IL13, CCL17, CCL22, IL8, LOR, FLG, CD1 1c Dermis, S100A8, S100A12, S100A7, S100A9, IL22, PI3, CXCL1 , IL17A, IL19, CAMP and DEFB4A/DEFB4B) that were found to correlate with TSS and were found to have significantly changed compared to baseline as analysed in skin biopsies taken at Day 22 in the study of Example 5.

[00102] Figure 6 shows the correlation between individual scores (erythema, edema/papulation, oozing/crusting, excoriation, lichenification and dryness) and TSS as found in the study of Example 5.

[00103] Figure 7 shows the changes in expression of biomarkers (IL13, S100A7, S100A8, KRT16, IL22, S100A9, S100A12, CCL17, MMP12, PI3, CCL22, DEFB4A/ DEFB4B, IL19 and LOR) that correlated with edema/papulation and were found to have significantly changed compared to baseline as analysed in skin biopsies taken at Day 22 in the study of Example 5.

[00104] Figure 8 shows the changes in expression of biomarkers (S100A7, S100A9, KRT16, IL13, S100A8, DEFB4A/DEFB4B, PI3, CCL17, S100A12, IL22 and MMP12) that correlated with erythema and were found to have significantly changed compared to baseline as analysed in skin biopsies taken at Day 22 in the study of Example 5.

[00105] Figure 9 shows the changes in expression of biomarkers (IL22, S100A7, S100A8, S100A12, DEFB4A/DEFB4B, S100A9 and LOR) that correlated with lichenification and were found to have significantly changed compared to baseline as analysed by in skin biopsies taken at Day 22 in the study of Example 5.

[00106] Figure 10 shows the changes in expression of biomarkers (I L13) that correlated with dryness and were found to have significantly changed compared to baseline as analysed in skin biopsies taken at Day 22 in the study of Example 5.

[00107] Figure 1 1 shows the changes in expression of biomarkers (IL8) that correlated with excoriation and were found to have significantly changed compared to baseline as analysed in skin biopsies at Day 22 in the study of Example 5.

[00108] Figures 12-15 show the changes in biomarker expression (KRT16, MMP12, IL13, CCL17, CCL22, IL8, LOR, FLG, S100A8, S100A12, S100A7, S100A9, IL22, PI3, DEFB4A/DEFB4B, IL19) that were found to correlate with TAA and were found to have significantly changed compared to baseline as analysed in skin biopsies taken at Day 22 in the study of Example 5. [00109] Figures 16-25 show changes in biomarker expression (IL6, IL8, IL17C, IL1 B, I L15, IL15RA, IL2, CCL5, IFNG, CXCL9, I L12A/I L12p35, CXCL10, IL13, IL10, IL33, TSLP-R, IL31 , IL5, CCL17, CCL18, CCL22, CCL26, IL17A, IL17F, I L23A/I L23p19, CAMP/LL37,

I L19, IL12B/IL23p40, DEFB4A/DEFB4B, CXCL1 , CXCL2, CCL20, PI3, IL22, S100A7, S100A8, S100A9, S100A12, FLG, PPL, LOR, KRT16, MMP12, IL9 and FOXP3) for vehicle (A) and niclosamide (B) compared to baseline as analysed in skin biopsies taken at Day 22 in the study of Example 5. FCH stands for fold change.

[00110] Figures 26-29 show changes in cell markers (CD3, langerin, CD1 1c and FceR1 ) for vehicle (A) and niclosamide (B) compared to baseline as analysed in skin biopsies taken at Day 22 in the study of Example 5.

[00111] Figure 30 illustrates the arithmetic profile of oxyclozanide (pg/g) in stratum corneum of skin flank following the oral or topical administration in dogs in the study of Example 6.

[00112] Figure 31 illustrates the mean plasma concentration-time of oxyclozanide (pg/L) obtained following topical administration in dogs in the study of Example 7.

[00113] Figure 32 illustrates the mean (pg/g) skin biopsies concentration-time of oxyclozanide obtained following topical administration in dogs in the study of Example 7.

[00114] Figure 33 illustrates mean (pg/g) stratum corneum (strips) concentration-time of oxyclozanide obtained following topical administration in dogs on 6 zones in the study of Example 7.

[00115] Figure 34 illustrates the distribution of total skin score in dogs with superficial pyoderma treated with oxyclozanide in the study of Example 8.

[00116] Figure 35 illustrates the distribution of total skin score in dogs with deep pyoderma treated with oxyclozanide in the study of Example 8.

[00117] Figure 36 shows evolution of mean pruritus score in dogs with superficial pyoderma treated with oxyclozanide in the study of Example 8.

[00118] Figure 37 shows evolution of mean pruritus score in dogs with deep pyoderma treated with oxyclozanide in the study of Example 8.

[00119] Figure 38 shows the % reduction in mean severity score for each lesion following topical treatment with oxyclozanide in the study of Example 8. The figure also shows the % of dogs that showed a 100% reduction in severity score for each lesion.

DETAILED DESCRIPTION Definitions

[00120] Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

[00121] The terms“treating” or“treatment” refers to any indicia of success in the treatment or amelioration of a disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a subject’s physical or mental well-being. Symptoms of inflammatory skin conditions and skin infections are known or may be readily determined by a person of ordinary skill in the art. The term "treating" and conjugations thereof, include prevention of a pathology, condition, or disease (e.g. preventing the development of one or more symptoms of the skin condition).

[00122] By way of example, in the context of inflammatory skin conditions“treatment” may include, relief, eradication or prevention of one or more symptoms of the inflammatory skin condition, for example pruritus, erythema, induration, lichenification, scaling or crusting.

[00123] By way of further example, in the context of skin infections“treatment” may include (i) the prevention of the disease or infection caused by Gram-positive bacteria; (ii) the suppression or relief of one or more of the symptoms of a disease or infection caused by Gram-positive bacteria; iii) the reduction or eradication of a non-symptomatic Gram- positive bacterial colonisation from an area on the body, (iv) the reduction or eradication of Gram-positive bacteria from a symptomatic skin infection, (v) the reduction or eradication of Gram-positive bacteria colonising an area of the body affected by a skin condition other than a skin infection ( e.g. colonisation of an inflammatory skin condition such as an area of skin affected by dermatitis e.g. atopic dermatitis); (vi) the suppression or relief of one or more symptoms of disease caused by Gram-positive bacteria from an area of the body affected by another non-infectious disease (e.g. an inflammatory skin condition such as an atopic dermatitis skin lesion); (vii) prevention of Gram-positive bacterial infection of skin affected by an inflammatory skin condition (e.g. prevention of infection of a dermatitis lesion); and (vii) prevention of Gram-positive bacterial infection of skin damaged by trauma (e.g. wounds, burns, stings or bites), by surgery, by medical devices (e.g. needles, catheters or cannulas etc.) or skin affected by a condition which compromises the barrier function of the skin. In each case the Gram-positive bacteria include but are not limited to any of the Gram-positive bacteria disclosed herein (e.g. Staphylococcus aureus or Streptococcus pyogenes, Staphylococcus pseudintermedius and Staphylococcus intermedius, including antibiotic resistant strains thereof (e.g. MRSA, MRSP, and MRSI).

[00124] In the context of skin infections, particularly pyoderma,“treatment” may include, relief, eradication or prevention of one or more symptoms of the skin infection, for example pruritus, papules, pustules, epidermal collarettes, scaling or crusts associated with the pyoderma.

[00125] Superficial pyoderma refers to a bacterial skin infection restricted to the epidermis that do not penetrate below the basement membrane. Superficial pyodermas are typically exudative and lesions include papules, pustules, epidermal collarettes, scales and crusts. Pruritus is often present.

[00126]“Deep pyoderma” refers to a serious skin bacterial infection below the basement membrane into the dermis and deeper tissues.

[00127] The term“subject” or“patient” used herein refers to a non-human subject unless expressly stated otherwise.

[00128] The term“associated” or“associated with” in the context of a substance or substance activity or function associated with a disease (e.g. a skin infection or an inflammatory skin condition such as atopic dermatitis) means that the disease is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function.

[00129] When a compound or salt described in this specification is administered to treat a disorder, a“therapeutically effective amount” is an amount sufficient to reduce or completely alleviate symptoms or other detrimental effects of the disorder; cure the disorder; reverse, completely stop, or slow the progress of the disorder; or reduce the risk of the disorder getting worse.

[00130] Colony-forming unit (CFU) is an approximate estimate of the number of viable bacterial cells in a sample. Viable is defined as the ability of the cell to multiply via binary fission under the controlled conditions.

[00131] The term“pharmaceutically acceptable salt” refers to salts that retain the biological effectiveness and properties of the compounds described herein and, which are not biologically or otherwise undesirable. Reference to pharmaceutically acceptable salts is intended to encompass all salt forms that are suitable for administration to a non-human subject and as such encompasses veterinarially acceptable salts. Pharmaceutically acceptable salts are well known to skilled persons in the art. Particular salts include ethanolamine or piperazine salts. 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.

[00132] 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. Reference to“a halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof” includes hydrates of the halogenated salicylanilide and hydrates of a salt of the

halogenated salicylanilide.

[00133] The term“halo” or“halogen” refers to one of the halogens, group 17 of the periodic table. In particular the term refers to fluorine, chlorine, bromine and iodine.

Preferably, the term refers to fluorine, chlorine or bromine and particularly fluorine.

[00134] The term C_m-n refers to a group with m to n carbon atoms.

[00135] The term“C-i-₆ alkyl” refers to a linear or branched hydrocarbon chain containing 1 , 2, 3, 4, 5 or 6 carbon atoms, for example methyl, ethyl, n-propyl, iso- propyl, n-butyl, sec- butyl, tert- butyl, n-pentyl and n-hexyl.“C1-4 alkyl” similarly refers to such groups containing up to 4 carbon atoms. The alkyl groups may be unsubstituted or substituted by one or more substituents. Substituents for the alkyl group may be halogen, e.g. fluorine, chlorine, bromine and iodine, OH, Ci-4 alkoxy.

[00136] The term“Ci-₆-haloalkyl” refers to a C1-6 alkyl group that is substituted by at least one halogen atom independently chosen at each occurrence, for example fluorine, chlorine, bromine and iodine. The halogen atom may be present at any position on the hydrocarbon chain. For example, C1-6 haloalkyl may refer to chloromethyl, fluoromethyl, trifluoromethyl, chloroethyl e.g. 1 -chloromethyl and 2-chloroethyl, trichloroethyl e.g. 1 ,2,2- trichloroethyl, 2,2,2-trichloroethyl, fluoroethyl e.g. 1-fluoroethyl and 2-fluoroethyl, trifluoroethyl e.g. 1 ,2,2-trifluoroethyl and 2,2,2-trifluoroethyl, chloropropyl, trichloropropyl, fluoropropyl, trifluoropropyl. A haloalkyl group may be a fluoroalkyl group, i.e. a Ci-₆ alkyl group substituted with at least one fluorine atom, for example Ci-₆ alkyl.

[00137] Reference to an“ester” of the halogenated salicylanilide refers to an ester (RC(O)O-or ROC(O)-) formed with an available hydroxy or carboxy group on the halogenated salicylanilide. For example, an ester formed by the esterification of the 2- hydroxy group of the benzamide in a halogenated salicylanilide. The ester may be cleavable following topical application of the salicylanilide to provide the free hydroxy or carboxy group of the parent molecule thereby providing a prodrug of the halogenated salicylanilide. The ester may be for example a Ci-₆-alkyl ester.

[00138] Reference to an“alkyl monohydroxy alcohol” refers to an alkyl alcohol which has one hydroxyl group, representative examples of alkyl monohydroxy alcohols include short chain alkyl monohydroxy alcohols, particularly Ci-₆-monohydroxy alcohols or Ci_-4- monohydroxy alcohols, for example methanol, ethanol, propanol or isopropanol.

[00139] Reference to an“alkanol amine” refers to an amine N-substituted by one, two or three alkyl alcohol moieties (for example one, two or three Ci_-4-alkyl alcohol moieties). Representative examples of alkanol amine include ethanolamine, diethanolamine, triethanolamine, isopropanolamine and diisopropanolamine.

[00140] Reference to“PEG xOO” herein means a polyethylene glycol with an average molecular weight of xOO. For example, PEG 400 refers to a PEG with an average molecular weight of 400. Unless stated otherwise reference herein to the molecular weight of polymer, such as a PEG is a reference to number average molecular weight (Mn) of the polymer. The number average molecular weight can be measured using well known methods, for example by gel permeation chromatography or 1 H NMR end-group analysis. Such methods include GPC analysis as described in Guadalupe et al (Handbook of Polymer Synthesis, Characterization, and Processing, First Edition, 2013) and end group analysis described in e.g. Page et al Anal. Chem., 1964, 36 (10), pp 1981-1985.

[00141] The halogenated salicylanilide may be administered to the subject in the form of a prodrug of the halogenated salicylanilide. As used herein, the term“prodrug” refers to covalently bonded moiety on the halogenated salicylanilide which modifies the biological and/or physical properties of the compound. The active halogenated salicylanilide is released following administration (for example topical administration) of the prodrug compound. Prodrugs may be formed by, for example, modification of a suitable functional group in the parent compound, for example a carboxylic or hydroxy group may be modified to form an ester which is cleaved following topical application of the prodrug. Various prodrug strategies are known and are described in, for example, the following documents: a) Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985);

b) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985); c) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen d) H. Bundgaard, Chapter 5“Design and Application of Pro-drugs”, p. 1 13-191 (1991 ); and

e) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992).

[00142] Unless stated otherwise, reference herein to a“% by weight of a halogenated salicylanilide or a pharmaceutically acceptable salt thereof” is intended to refer to the amount of the free acid (i.e. non-salt form) of the halogenated salicylanilide. For example, reference to a composition comprising“5% by weight of niclosamide or a pharmaceutically acceptable salt thereof” refers to a composition comprising 5% by weight of the

niclosamide as the free acid. Accordingly, where such a composition comprises a salt of niclosamide, the absolute amount of the niclosamide salt in the composition will be higher than 5% by weight in view of the salt counter ion that will be also be present in the composition. Similarly reference to“% by weight/volume (% w/v) of a halogenated salicylanilide or a pharmaceutically acceptable salt thereof refer to the concentration of the free acid (i.e. non-salt form) of the halogenated salicylanilide per unit volume and is calculated as the %w/v = (weight of the salicylanilide in g/per ml. of the

compositional 00%.

[00143] The term "gel" is used herein refers to a semi-solid, apparently homogeneous substance that may be elastic and jelly-like (as in gelatin). The gel comprises a three- dimensional polymeric or inorganic matrix within which is dispersed a liquid phase. The matrix of the gel comprises a network of physically or chemical cross-linked polymers or copolymers that swell but do not dissolve in the presence of a solvent (for example the low molecular weight PEG). The cross-linking within the gel matrix may be physical cross linking (for example by hydrogen bonding or ionic cross-linking) or may be covalently cross-linked. In some embodiments the gel composition is a non-aqueous gel

compositions wherein the halogenated salicylanilide is dissolved or dispersed in a suitable non-aqueous medium (e.g. PEG). The non-aqueous medium/halogenated salicylanilide solution or dispersion is then dispersed within the polymeric cross-linked network of the gel. Alternatively, the halogenated salicylanilide may be dissolved or dispersed within the polymeric cross-linked network of the gel. The gels are preferably clear in appearance; however, turbid gels are also contemplated. Generally, the gel-forming agent, for example gel-forming polymer is present in the gel in an amount of from about 0.5-15% by weight, typically 0.5-2% by weight. The U.S.P. defines gels as a semi-solid system consisting of dispersion made up of either small inorganic particles or large organic molecule enclosing and interpenetrated by liquid.

[00144] Reference to a“non-aqueous” composition (e.g. a non-aqueous topical composition), means that the composition is anhydrous and therefore substantially water free. For example, the compositions disclosed herein including the gel, cream and foam compositions contain less than 5%, less than 1% or suitably less than 0.01 %, preferably less than 0.001% by weight water. Preferred non-aqueous compositions are those which are anhydrous and contain no detectable water.

[00145] Protic organic solvents are those that are capable of hydrogen bonding. The most common examples of protic organic solvents include but are not limited to alcohols and carboxylic acids.

[00146] Aprotic organic solvents are those that are not capable of hydrogen bonding. Common aprotic organic solvents include but are not limited to ethers, dimethylformamide (DMF), dimethylsulfoxide (DMSO) and acetonitrile.

[00147] Reference to“about” in the context of a numerical is intended to encompass the value +/- 10%. For example, about 20% includes the range of from 18% to 22%.

[00148] Reference to“the dosage regimen according to the invention” or“dosage regimen of the invention” encompasses the dosage regimens described herein comprising an initial treatment period wherein the halogenated salicylanilide is administered to the subject followed by a treatment-free period during which no further halogenated salicylanilide is administered to the subject.

[00149] Throughout the description and claims of this specification, the words“comprise” and“contain” and variations of them mean“including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[00150] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments.

The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[00151] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

Skin Infections

[00152] The dosage regimen of the invention may be for use in the treatment or prevention of a skin infection caused by or associated with Gram-positive bacteria. WO 2016/038035 discloses that halogenated salicylanilides (e.g. niclosamide, rafoxanide, closantel, and oxyclozanide) are potent antibacterial compounds that are active against a wide range of Gram-positive bacteria including Staphylococcus spp., Streptococcus spp. (e.g. Staphylococcus aureus, Streptococcus pyogenes) as well as antibiotic resistant strains, including MRSA. WO 2016/038035 also discloses that Gram-positive bacteria, especially niclosamide and oxyclozanide, exhibit very low frequencies of spontaneous mutations that confer resistance to the halogenated salicylanilide. Accordingly, halogenated salicylanilides are expected to provide a highly effective topical treatment for skin infections caused by or associated with Gram-positive bacteria.

[00153] The topical dosage regimen of the invention provides high concentrations of the halogenated salicylanilide in skin tissue which are well in excess of the MIC of the Gram- positive bacteria and sustains the high concentration over a prolonged period of time without the need to apply additional topical halogenated salicylanilide after completion of the initial topical treatment period. The very low frequency of spontaneous resistance conferring mutation associated with halogenated salicylanilides suggests that the risk of bacterial resistance to the halogenated salicylanilide emerging during the dosage regimen of the invention is low.

[00154] The skin condition may be any skin condition in a non-human subject, especially a skin infection, that is caused by or associated with Gram-positive bacteria, including but not limited to any of the skin infections disclosed herein. Examples of skin conditions caused by or associated with Gram-positive bacteria that can be treated using the dosage regimen of the invention include pyoderma (e.g. canine or feline pyoderma), otitis, bacterial conjunctivitis, dermatitis (including topic dermatitis, contact dermatitis, allergic contact dermatitis, irritant contact dermatitis, atopic dermatitis, seborrhoeic dermatitis, actinic dermatitis, pododermatitis, lichen simplex chronicus (including Canine acral lick dermatitis and neurodermatitis), digital dermatitis (including bovine digital dermatitis), exfoliative dermatitis (erythroderma), carcinomatous dermatitis, nummular dermatitis, stasis dermatitis, perioral dermatitis, dermatomyositis, eczematous dermatitis, photoallergic dermatitis, phototoxic dermatitis, phytophotodermatitis and radiation-induced dermatitis, flea allergy dermatitis, malassezia dermatitis, intertrigo and food allergic dermatitis), folliculitis, superficial folliculitis, erythrasma, dermatoses, carbuncles, abscesses, cysts (including pilonidal and sebaceous cysts), furunculosis, ecthyma, cellulitis, paronychia, erysipelas, necrotising fasciitis, secondary skin infections, scabies, chronic ulcers

(including varicose ulcers and decubitus ulcers), vesicular eruptions, bullous eruptions, mastitis, chronic rhinosinusitis, traumatic skin lesions (including infected surgical wounds, burns, cuts, abrasions, animal bites or insect bites) and Staphylococcal scalded skin syndrome (SSSS or Ritter’s disease).

[00155] In some embodiments the dosage regimen of the invention is used to reduce or preferably eradicate the Gram-positive bacteria that cause a skin infection in the subject. The effect of the halogenated salicylanilide on the bacteria causing the skin infection may be assessed by taking a sample from a lesion (e.g. a swab or a tissue sample) and culturing the sample to determine the bacteria present (the baseline bacteria). At the completion of the dosage regimen, a further sample is taken from the area of the skin that has been treated and in cultured. The bacteria are considered to have been eradicated if no growth of the baseline bacteria is observed. The post-treatment sample is suitably taken at the end of the treatment-free period, for example 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 or 14 days after the completion of the initial treatment period.

[00156] In some embodiments the dosage regimen of the invention is used to reduce or eliminate one or more of the symptoms of a skin infection in a non-human animal. For example, the dosage regimen may be for use in reducing or eliminating one or more of blistering, exudate/pus, crusting, itching, pain or erythema/inflammation associated with the skin infection.

[00157] In certain embodiments the dosage regimen of the invention is for use in the topical treatment or prevention of one or more of erythema, lichenification/hyperpigmentation, excoriation, alopecia, keratoseborrheic condition, papules, pustules, macules and epidermal collarettes caused by or associated with the skin condition (e.g. a skin infection such as pyoderma).

[00158] In certain embodiments the dosage regimen of the invention is for use in the topical treatment or prevention of one or more of papules and pustules caused by or associated with pyoderma.

[00159] In certain embodiments the dosage regimen of the invention is for use in the topical treatment of pyoderma to reduce or eliminate one or more of erythema, lichenification/hyperpigmentation, excoriation, alopecia, keratoseborrheic condition, papules, pustules, macules and epidermal collarettes.

[00160] In certain embodiments the dosage regimen of the invention is for use in the topical treatment or prevention of one or more of papules and pustules caused by or associated with pyoderma.

[00161] In certain embodiments the dosage regimen of the invention is for use in the topical treatment or prevention of inflammation associated with a skin infection (e.g.

pyoderma). For example, it may be that the dosage regimen of the invention reduces or eliminates one or more of erythema, excoriation, alopecia, and epidermal collarettes associated with or caused by the skin infection (e.g. pyoderma). It may be that the dosage regimen of the invention eliminates erythema associated with or caused by the skin infection (e.g. pyoderma).

[00162] The severity of skin lesions and extent of infection in an animal may be assessed using a suitable scoring system. For example, by scoring the severity of each type of lesion and the parts of the animals body affected to give a total skin score as follows:

• Lesion types (9): erythema, lichenification/hyperpigmentation, excoriation, alopecia, keratoseborrheic condition, papules, pustules, macules and epidermal collarettes.

• Body areas (14): face, ears, neck, axilliae (L +R), thorax, abdomen & groin, lumber & flanks, front leg & paw (L +R), hind leg & paw (L +R), perineum, tail

• Severity score: none (0); mild (1 ); moderate (2); severe (3);

• wherein the total skin score the sum of the severity score from each lesion and each body area.

[00163] The efficacy of treatment may be assessed by comparing the total skin score after treatment (e.g. 1 week, 2 weeks, 3 weeks or 4 weeks) after topically treating the subject with the halogenated salicylanilide with that prior to treatment. It may be that the mean total skin score after topical treatment is reduced by at least 10, 20 , 30 , 40, 50 or 60 points compared to the scope prior to starting treatment (baseline score). It may be that the skin score is reduced by at least 10%, 20%, 30%, 40% , 50%, 60%, 70%, 80%, 90% or 100% relative to the baseline skin score.

[00164] Pruritus (itching) is common is subjects with many skin infections and/or inflammatory skin infections. However, when subjects scratch the lesions this can lead to the spread of the condition to other areas of the body. Accordingly, there is a particular need for a treatment which can reduce or eliminate the pruritus associated with a skin infection caused by Gram-positive bacteria or an inflammatory skin infection in a non- human subject. The severity of pruritus in a subject can be assessed using a suitable scoring system, for example a Visual Analog Scale (VAS) suitable for use in a non-human subject such that described in Hill et al. Development of an owner-assessed scale to measure the severity of pruritus in dogs, Vet Dermatol. 2007 Oct;18(5):301-8. Suitably the dosage regimen of the invention reduces the VAS score by 2 or more points, preferably 4 or more points compared to the score prior to initiation of the topical treatment. A suitable pruritis scoring system is the PVAS scoring system described in the Examples . It may be that the pruritis score after topical treatment with the halogenated salicylanilide (e.g. 1 week, 2 weeks, 3 weeks or 4 weeks after treatment) is reduced by at least 1 , 2, 3, 4, 5, 6 or 7 points compared to the score prior to topical treatment.

[00165] In some embodiments the skin condition, for example skin infection such as pyoderma is eradicated or controlled without the need for further topical administration of the halogenated salicylanilide at the completion of the treatment-free period. In this embodiment the skin condition is treated by topical application of the halogenated salicylanilide in the initial treatment period only. For example, the skin condition is treated by topical application of the halogenated salicylanilide for 1 day, 2 days or 3 days, preferably by topical treatment for 1 day or 2 days, even more preferably by topical treatment for 1 day. The halogenated salicylanilide is suitably administered at a frequency of, for example once or twice per day during the initial treatment period. In a preferred embodiment the initial treatment period consists of a single topically applied dose of the halogenated salicylanilide to the subject.

Inflammatory skin conditions [00166] The topical application of the halogenated salicylanilide may provide an anti- inflammatory effect on the skin condition. For example, by reducing or eliminating one of more inflammatory symptoms of an inflammatory skin condition.

[00167] In some embodiments topical administration of the halogenated salicylanilide in the dosage regimen of the invention for the treatment of an inflammatory skin condition provides an anti-inflammatory effect on the skin condition. For example, the halogenated salicylanilide may reduce, eliminate or prevent one or more symptoms of the inflammatory skin condition (e.g. dermatitis such as atopic dermatitis), including, but not limited to one or more of pruritus, erythema, induration, lichenification, scaling, oozing and crusting associated with the inflammatory skin condition.

[00168] The inflammatory skin condition may be any skin condition in a non-human subject which exhibits one or more symptoms of inflammation. Examples of inflammatory skin conditions include psoriasis, dermatitis, (e.g. atopic dermatitis, topic dermatitis, contact dermatitis, allergic contact dermatitis, irritant contact dermatitis, flea allergy dermatitis, malassezia dermatitis, intertrigo, pododermatitis, food allergic dermatitis, demodicosis ,seborrhoeic dermatitis, actinic dermatitis, pompholyx dermatitis, lichen simplex chronicus (including Canine acral lick dermatitis and neurodermatitis), digital dermatitis (including bovine digital dermatitis), exfoliative dermatitis (drythroderma), asteatotic dermatitis, carcinomatous dermatitis, nummular dermatitis, stasis dermatitis, perioral dermatitis, dermatomyositis, eczematous dermatitis, photoallergic dermatitis, phototoxic dermatitis, phytophotodermatitis and radiation-induced dermatitis); scleroderma, disorders of hair follicles and sebaceous glands rhinophyma, cutaneous lupus, and inflammatory reactions (e.g. drug eruptions, erythema multiforme, erythema nodosum, or granuloma annulare), otodectic acariasis, drug eruptions, psychogenic pruritus, self- induced psychogenic hair loss, dermatophytosis, pediculosis, skin inflammation associated with scabies, skin inflammation associated with otitis and skin inflammation associated with fungal or yeast infections (e.g. dermatophytosis).

Dermatitis

[00169] In an embodiment the dosage regimen of the invention is for use in the treatment or prevention of dermatitis. The dermatitis may be selected from, for example topic dermatitis, contact dermatitis, allergic contact dermatitis, irritant contact dermatitis, atopic dermatitis, seborrhoeic dermatitis, actinic dermatitis, pododermatitis, pompholyx dermatitis, lichen simplex chronicus (including Canine acral lick dermatitis and neurodermatitis), digital dermatitis (including bovine digital dermatitis), exfoliative dermatitis (drythroderma), carcinomatous dermatitis, nummular dermatitis, stasis dermatitis, flea allergy dermatitis, malassezia dermatitis, intertrigo, perioral dermatitis, dermatomyositis, eczematous dermatitis, photoallergic dermatitis, phototoxic dermatitis, phytophotodermatitis or radiation-induced dermatitis. In a particular embodiment the dermatitis is atopic dermatitis (AD).

[00170] Subjects with atopic dermatitis are prone to skin bacterial colonization by Gram- positive bacteria such as S. aureus because of the compromised physical epidermal barrier, coupled with diminished immune recognition and impaired antimicrobial peptide production.

[00171] Bacterial colonisation (e.g. with S. aureus) is a significant factor in the

pathogenesis of AD for recurrent complications that exacerbate the disorder. Its presence, even without overt infection, appears to trigger multiple inflammatory reactions, via toxins, that act as super-antigens and exogenous protease inhibitors that further damage the epidermal barrier and potentiate allergen penetration. Colonization with S. aureus is associated with cell death primarily mediated by secretion of alpha toxin (Wardenburg Infect Immun. 2007;75(2):1040-4, Taubler et al . J Bacteriol. 1963;86:51-7), a destructive pore-forming toxin often found in the lesions of human patients with severe AD (Travers et al. Pediatr. Dermatol. 2012;29, 289-296. Wichmann et al . Br J Dermatol. 2009;161 (2):300- 5). The increased cell death in AD skin correlates with increased exposure to Th2 cytokines, a landmark in the pathogenesis of the disease (Brauweiler et al J Invest Dermatol. 2014;134(8): 21 14-2121 ).

[00172] Accordingly reducing or eradicating Gram-positive bacteria from dermatitis lesions, especially AD lesions in a non-human subject may provide an anti-inflammatory effect on the dermatitis (e.g. AD). In an embodiment the dosage regimen of the invention is for use in reducing or preferably eliminating Gram-positive bacteria from a dermatitis lesion. The dermatitis lesion may be a lesion associated with any of the dermatitis forms listed herein, particularly an AD lesion. It may be that the lesion is infected with the Gram- positive bacteria. However, in particular embodiments the lesion is colonised with Gram- positive bacteria. Accordingly, in a particular embodiment the dosage regimen of the invention is for use in the decolonisation of a dermatitis lesion, particularly an AD lesion in a non-human subject.

[00173] In embodiments the dosage regimen of the invention is for use in the treatment of dermatitis (e.g. atopic dermatitis) in a non-human subject to reduce or eliminate one or more of pruritus, erythema, induration, excoriation, exfoliation, lichenification, scaling, oozing, crusting, xerosis, lesion nodules, prurigo nodules, lesion vesicles, lesion papules, lesion plaques or lesion swelling, associated with the dermatitis in the non-human subject. In embodiments the dermatitis is atopic dermatitis, for example canine atopic dermatitis.

[00174] In some embodiments the dosage regimen of the invention reduces or eliminates one or more of pruritus, erythema, induration, excoriation, lichenification, xerosis, lesion nodules, prurigo nodules, lesion vesicles, lesion papules, lesion swelling or exfoliation (e.g. self-induced alopecia) associated with the dermatitis (e.g. AD) in the non-human subject.

[00175] A particular problem associated with dermatitis, particularly AD, is pruritus

(itching). This symptom of the disease is unpleasant for subjects and often results in one or more of stress, anxiety, disturbed sleep, sleep deprivation and psychiatric effects including depression and anxiety, leading to impaired quality of life. Subjects are also prone to scratching lesions in an attempt to relieve the pruritus, however, this further damages the already compromised skin of the lesion leading to excoriation, increased erythema, induration and/or swelling. The additional damage to the barrier function of the skin associated with scratching the lesions also enhances exposure to allergens and irritants that can trigger an exacerbation of the dermatitis. Scratching of the lesions also increased the risk of infection of the dermatitis. Accordingly, in embodiments the dosage regimen of the invention is for use in reducing or eliminating pruritus associated with dermatitis (e.g. AD) in a non-human subject.

[00176] The pruritus in a subject may be assessed using a suitable scoring system for the pruritus associated with the dermatitis in a non-human subject. It may be that the topical treatment of the dermatitis using the halogenated salicylanilide results in a reduction in the pruritus score compared to the score immediately prior to treatment of the subject.

[00177] The dosage regimen of the invention may be for use in in the treatment of mild dermatitis (e.g. mild AD). The dosage regimen of the invention may be for use in the treatment of moderate dermatitis (e.g. moderate AD). The dosage regimen of the invention may be for use in the treatment of severe dermatitis (e.g. severe AD). The dosage regimen of the invention may be for use in the treatment of moderate to severe dermatitis (e.g. moderate to severe AD). The dosage regimen of the invention may be for use in the treatment of mild to moderate dermatitis (e.g. mild to moderate AD).

[00178] The severity of the dermatitis may be assessed using known methods. For example, a suitable scoring system that assesses the clinical signs of the dermatitis on the subject. One such scoring method suitable for determining the severity of AD is the Canine Atopic Dermatitis Extent and Severity Index (CADESI), for example CADESI-01 , CADESI-02 or CADESI-03 (Olivry et al. Validation of CADESi-03, a severity scale for clinical trials enrolling dogs with atopic dermatitis. Veterinary Dermatology, 18: 78-86), or CADESI-04 (Olivry T et al, Vet Dermatol. 2014 Apr;25(2):77-85). CADESI-4 is currently recommended by ICADA (International Committee on Allergic Diseases of Animals) and is a preferred scoring system for AD. These scoring systems may also be used to grade other, similar forms of dermatitis.

[00179] The CADESI scores quantitatively describe the dog’s skin condition, separately scoring areas of a dog’s body for erythema, lichenification, and / or excoriation as‘Normal or absent’ (0),‘Mild’ (1 ),‘Moderate’ (2), or‘Severe’ (3). CADESI-03 differs from CADESI- 02 in that it has an increased number of body sites assessed from 40 to 62, and includes another clinical sign (self-induced alopecia) and each sign is graded in a wider scale (scale of 0 to 5). CADESI-03. CADESI-04 requires only 20 defined body sites and takes approximately 33% of the time to conduct as compared to CADESI-03. Accordingly, a preferred dermatitis scoring system is CADESI-04.

[00180] It may be that the CADESI score (e.g. CADESI-03 or preferably CADESI-04 score) is reduced by 2, 4, 6 or 8 points compared to the score immediately before commencing treatment (the baseline score).

[00181] The dosage regimen of the invention may be for use in the treatment of acute AD. For example, the halogenated salicylanilide may be for use in the treatment or prevention of lesion redness (erythema, inflammation), induration, papulation, pruritus or excoriation in a non-human subject with acute AD. The acute AD may be mild, moderate or severe acute AD, for example moderate to severe acute AD or mild to moderate AD.

[00182] The dosage regimen of the invention may be for use in the treatment of a chronic form of dermatitis (e.g. chronic AD). For example, the halogenated salicylanilide may be for use in the treatment or prevention of lichenification (for example, lined skin or prurigo nodules), pruritus or excoriation in a subject with chronic AD. The chronic AD may be mild, moderate or severe chronic AD, for example moderate or severe chronic AD.

[00183] It may be that the dermatitis lesions are colonized by bacteria, for example the lesion may be colonized by Gram-positive bacteria. In certain embodiments the halogenated salicylanilide is for use in the treatment of a dermatitis lesion (e.g. an AD lesion) that is colonized by Gram-positive bacteria. The Gram-positive bacteria that may colonize the lesion include, but are not limited to Staphylococcus spp., Streptococcus spp. or Propionibacterium spp. The Gram-positive bacteria may be a Staphylococcus spp. or Streptococcus spp. The Gram-positive bacteria may be selected from Staphylococcus aureus or Streptococcus pyogenes. The bacteria may be resistant to conventional antibiotic agents. For example, the bacteria may be a MRSA, MRSP or MRSI strain.

[00184] In other embodiments the dermatitis lesion is not colonized by bacteria.

Reference to“not colonized” means that the lesion is substantially free from bacteria, for example the lesion to be treated in the subject carries less than 1000 CFU/cm². The CFU in a sample taken from the lesion may be determined using conventional cell culturing methods. The sample could be, for example, a swab or skin biopsy obtained from the lesion. Accordingly, it may be that the halogenated salicylanilide is for use in the treatment of dermatitis (e.g. AD) that is not colonized or infected by bacteria, for example the AD lesion is not colonized or infected with a Gram-positive bacteria.

[00185] Subjects with certain forms of dermatitis, including AD, are prone to exacerbation (flares) in their dermatitis. In the case of AD a flare could result from, for example, exposure to an irritant or allergen or a change in ambient conditions such as elevated temperature or humidity. Accordingly, the dosage regimen of the invention may be useful in the prevention or treatment of exacerbations of dermatitis (e.g. AD) in a subject. It may be that the dosage regimen of the invention is for use in reducing the frequency of exacerbations of dermatitis (e.g. AD) in a subject. It may be that the dosage regimen of the invention is for use in reducing the severity of an exacerbation of dermatitis (e.g. AD) in a subject. It may be that the dosage regimen of the invention is for use in reducing the duration of an exacerbation of dermatitis (e.g. AD) in a non-human subject.

[00186] Accordingly, in embodiments the dosage regimen of the invention is for use in the treatment of an exacerbation of dermatitis (e.g. AD). In embodiments the dosage regimen of the invention is for use in preventing or reducing the frequency of dermatitis (e.g. AD) exacerbations in a non-human subject. In embodiments the dosage regimen of the invention is for use in reducing the severity of exacerbations of dermatitis (e.g. AD) in a non-human subject.

[00187] In the embodiments described herein that refer to exacerbations of the dermatitis, the exacerbation may be an exacerbation of one or more of the symptoms of the dermatitis described herein (e.g. an exacerbation of one or more of pruritus, erythema, induration or excoriation).

Decolonisation [00188] In some embodiments, the dosage regimen of the invention is used to decolonise a non-human subject carrying Gram-positive bacteria (including any of the Gram-positive bacteria described herein, for example MRSA). Decolonisation may also be used to reduce or eliminate the risk of a non-human subject developing a skin infection. Decolonisation may also prevent or reduce the risk of surgical site infections resulting from surgical or medical procedures carried out on the non-human subject or at the site of medical devices such as catheters or IV lines or cannula. In some embodiments, the dosage regimen of the invention is for use in the decolonisation of a non-human subject prior to carrying out a surgical procedure on the subject, wherein the composition is applied topically to the subject. Such surgical procedures include, for example elective surgical procedures such as hip or knee replacement. In one embodiment, the dosage regimen of the invention is for use in the decolonisation of a subject prior to dialysis. Pre-dialysis decolonisation may prevent or reduce the risk of infection associated with dialysis such as vascular line infection or catheter related bloodstream infections (CRBSI) infections. Decolonisation may be achieved by topically administering the halogenated salicylanilide in accordance with the dosage regimen of the invention to sites on the subject which are colonised by the Gram-positive bacteria. It is known that a common site for bacterial colonisation such as MRSA is the nose in some animals, for example in horses. Accordingly, the halogenated salicylanilide may be applied topically to the nose, preferably to the anterior nares (the inner surface of the nostrils).

Mastitis

[00189] In some embodiments the skin condition is mastitis, particularly mastitis in large animals and especially mastitis in cattle. The halogenated salicylanilide is topically applied to the site of infection. Accordingly, there is provided a halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof, for use in the topical treatment or prevention of mastitis in a non-human subject. The halogenated salicylanilide (e.g.

oxyclozanaide or niclosamide) may be topically applied in accordance with the dosage regimen of the invention. Generally the halogenated salicylanilide will be topically applied to the teat canal. Suitably this is achieved by intramammary application of the

halogenated salicylanilide into the teat canal via the streak canal orifice using a suitable cannula, hypodermic needle or coated swab inserted into the teat canal so as to coat the inner surface of the teat canal. Once applied the halogenated salicylanilide can be worked into the teat by gently massaging the teat. The halogenated salicylanilide may also optionally be applied to the outer surface of the teat. Pyoderma

[00190] In some embodiments the skin condition is pyoderma. In embodiments the pyoderma is surface or superficial pyoderma which affects the surface or upper layers of the skin, especially the stratum corneum. In other embodiments the pyoderma is deep pyoderma affecting e.g. the dermis. In a preferred embodiment the pyoderma is canine or feline pyoderma. Preferably the pyoderma is canine pyoderma. It may be that the pyoderma is superficial canine pyoderma. It may be that the pyoderma is deep canine pyoderma.

Halogenated Salicylanilide

[00191] Halogenated salicylanilides are also known as 2-hydroxy-N-phenylbenzamides or 2-hydroxybenzanilides. Salicylanilides are weakly acidic phenolic compounds.

Halogenated salicylanilides are salicylanilides substituted by at least one halo group. A number of halogenated salicylanilide derivatives are known. Any halogenated salicylanilide possessing an effect on a skin infection (e.g. pyoderma (including canine pyoderma)) or inflammatory skin condition (e.g. dermatitis) may be used in the dosage regimen of the invention. For example, the halogenated salicylanilide may be any of the niclosamide analogues described in WO 2008/021088, which are incorporated herein by reference thereto.

[00192] It may be that the halogenated salicylanilide is a halogenated salicylanilide of the formula (I):

wherein

X is O or S;

R¹ and R² are at each occurrence independently selected from halo; R³ and R⁴ are at each occurrence independently selected from H, Ci-₆ alkyl, Ci-₆ haloalkyl, -OR^A1, -N0₂ and -CN;

R⁵ is H or -L¹-R⁷;

R⁶ is H or -C(0)R^A2;

L¹ is selected from a bond, O, S, or -(CR^A3R^B)₀-, wherein o is 1 or 2;

R⁷ is phenyl, unsubstituted or substituted with 1 , 2, or 3 groups selected from halo, Ci_-4 alkyl, Ci_-4 haloalkyl, -OR^M, -N0₂ and -CN;

R^A1, R^A2, R^A3 and R^M are at each occurrence independently selected from H and Ci_-4 alkyl; R^B is at each occurrence selected from H, Ci_-4 alkyl and -CN;

n and p are each independently selected from 0, 1 , 2, 3 or 4, with the proviso that n+p is at least 1 ;

t and v are independently selected from 0, 1 and 2;

or a pharmaceutically acceptable salt, or ester or hydrate thereof.

[00193] The following statements in the numbered paragraphs below apply to compounds of the formula (I). These statements are independent and interchangeable. In other words, any of the features described in any one of the following statements may (where chemically allowable) be combined with the features described in one or more other statements below. In particular, where a compound is exemplified or illustrated in this specification, any two or more of the statements below which describe a feature of that compound, expressed at any level of generality, may be combined so as to represent subject matter which is contemplated as forming part of the disclosure of this invention in this specification.

1. X is O.

2. R¹ and R² are at each occurrence independently selected from fluoro, chloro, bromo and iodo.

3. R¹ and R² are at each occurrence independently selected from chloro, bromo and iodo.

4. R¹ is chloro.

5. R¹ is bromo.

6. R¹ is iodo. 7. R² is chloro.

8. R² is bromo.

9. R² is iodo.

10. R³ and R⁴ are at each occurrence independently selected from H, Ci-4-alkyl, Ci_-4- haloalkyl, -OR^A1, -N0₂ and -CN.

11. R³ and R⁴ are at each occurrence independently selected from H, Ci-₄-alkyl, - OR^A1 and -N0₂.

12. R³ and R⁴ are at each occurrence independently selected from H, Ci_-4-alkyl, -CF₃, -OH, -OMe, -N0₂ and -CN, for example H, Ci_-4-alkyl, -OH or -N0₂.

13. R⁴ is at each occurrence independently selected from -CF3, -N0₂ and -CN.

14. R⁴ is at each occurrence independently selected from Ci_-4-haloalkyl, -N0₂ and -

CN.

15. R⁵ is H.

16. R⁵ is -L¹-R⁷.

17. L¹ is selected from -0-, -CH₂- and -CH(CN)-, for example -O- or -CH(CN)-.

18. R⁷ is phenyl, unsubstituted or substituted with 1 , 2, or 3 groups selected from halo, Ci-₄-alkyl, Ci_-4-haloalkyl and -CN.

19. R⁷ is phenyl unsubstituted or substituted with 1 , 2, or 3 groups (for example 1 or 2 groups) selected from halo.

20. R⁷ is unsubstituted phenyl.

21. L¹ is selected from -O- and -CH(CN)-; and R⁷ is phenyl unsubstituted or substituted with 1 , 2, or 3 groups selected from halo.

22. R⁶ is H.

23. R⁶ is -C(0)R^A2, for example -C(0)CH₃.

24. t = 0 or 1 .

25 t = 0.

26. v = 0 or 1 .

27. v = 0. 28. o is 1.

29. v = 1 and R⁴ is selected from -OH, Ci-₄-alkyl and -NO2.

30. v = 1 and R⁴ is selected from -CN, Ci-_4-haloalkyl (e.g. -CF3) and -NO2.

31. A compound of formula (I), or a pharmaceutically acceptable salt thereof.

[00194] Particular compounds of formula (I), or a pharmaceutically acceptable salt, hydrate or ester thereof are those wherein:

X is O;

R¹ and R² are at each occurrence independently selected from halo;

R³ and R⁴ are at each occurrence independently selected from H, Ci_-4 alkyl, -OR^A1, -NO2 and CN;

R⁵ is H or -L¹-R⁷;

R⁶ is H or -C(0)R^A2;

L¹ is selected from O and -CH(CN)-;

R⁷ is phenyl unsubstituted or substituted with 1 , 2, or 3 groups selected from halo;

R^A1 and R^A2 are at each occurrence independently selected from H and Ci_-4-alkyl;

t and v are independently selected from 0, 1 and 2;

or a pharmaceutically acceptable salt, or ester thereof.

It may be that the halogenated salicylanilide is selected from:

tetrachlorosalicylanilide closantel

oxyclosanide clioxanide

niclosamide

or a pharmaceutically acceptable salt or solvate (e.g. hydrate) thereof.

[00195] The halogenated salicylanilide may be a thioamide derivative, for example brotianide:

brotianide or a pharmaceutically acceptable salt, solvate (e.g. hydrate) thereof.

[00196] The halogenated salicylanilide may be selected from the group consisting of tetrachlorosalicylanilide, closantel, rafoxanide, oxyclozanide, resorantel, clioxanide, dibromosalan, tribromosalan, brotianide and niclosamide, or a pharmaceutically acceptable salt or prodrug or derivative thereof.

[00197] The halogenated salicylanilide may be selected from the group consisting of tetrachlorosalicylanilide, closantel, rafoxanide, oxyclozanide, resorantel, dibromosalan, tribromosalan and niclosamide, or a pharmaceutically acceptable salt or ester thereof.

[00198] The halogenated salicylanilide may be selected from the group consisting of clioxanide, closantel, oxyclozanide, rafoxanide, tribromosalan or a pharmaceutically acceptable salt or ester thereof.

[00199] The halogenated salicylanilide may be selected from the group consisting of tetrachlorosalicylanilide, closantel, rafoxanide, oxyclozanide, resorantel, clioxanide, dibromosalan, tribromosalan, brotianide and niclosamide, or a pharmaceutically acceptable salt or hydrate thereof.

[00200] The halogenated salicylanilide may be selected from the group consisting of tetrachlorosalicylanilide, closantel, rafoxanide, oxyclozanide, resorantel, clioxanide, dibromosalan, tribromosalan and niclosamide, or a pharmaceutically acceptable salt or hydrate thereof.

[00201] The halogenated salicylanilide may be selected from the group consisting of niclosamide, clioxanide, closantel, oxyclozanide, rafoxanide and tribromosalan, or a pharmaceutically acceptable salt or hydrate thereof.

[00202] The halogenated salicylanilide may be selected from the group consisting of clioxanide, closantel, oxyclozanide, rafoxanide and tribromosalan, or a pharmaceutically acceptable salt or hydrate thereof. [00203] The halogenated salicylanilide may be selected from the group consisting of clioxanide, closantel, rafoxanide and tribromosalan, or a pharmaceutically acceptable salt or hydrate thereof.

[00204] The halogenated salicylanilide may be selected from the group consisting of niclosamide and oxyclozanide, or a pharmaceutically acceptable salt or hydrate thereof

[00205] The halogenated salicylanilide may be selected from the group consisting of tetrachlorosalicylanilide, closantel, rafoxanide, oxyclozanide, resorantel, clioxanide, dibromosalan, tribromosalan, brotianide and niclosamide.

[00206] The halogenated salicylanilide may be selected from the group consisting of niclosamide, closantel, oxyclozanide and rafoxanide, or a pharmaceutically acceptable salt thereof.

[00207] The halogenated salicylanilide may be clioxanide, or a pharmaceutically acceptable salt or ester thereof, for example the halogenated salicylanilide is clioxanide or a pharmaceutically acceptable salt or hydrate thereof, suitably the halogenated

salicylanilide is clioxanide.

[00208] The halogenated salicylanilide may be closantel, or a pharmaceutically acceptable salt or hydrate thereof, for example the halogenated salicylanilide is closantel or a pharmaceutically acceptable salt thereof, suitably the halogenated salicylanilide is closantel.

[00209] The halogenated salicylanilide may be oxyclozanide, or a pharmaceutically acceptable salt or ester thereof, for example the halogenated salicylanilide is oxyclozanide or a pharmaceutically acceptable salt or hydrate thereof, suitably the halogenated salicylanilide is oxyclozanide.

[00210] The halogenated salicylanilide may be rafoxanide, or a pharmaceutically acceptable salt or hydrate thereof, for example the halogenated salicylanilide is rafoxanide or a pharmaceutically acceptable salt thereof, suitably the halogenated salicylanilide is rafoxanide.

[00211] The halogenated salicylanilide may be tribromosalan, or a pharmaceutically acceptable salt or hydrate thereof, for example the halogenated salicylanilide is

tribromosalan or a pharmaceutically acceptable salt thereof, suitably particularly the halogenated salicylanilide is tribromosalan. [00212] The halogenated salicylanilide may be niclosamide, or a pharmaceutically acceptable salt or hydrate thereof, for example the halogenated salicylanilide is niclosamide or a pharmaceutically acceptable salt thereof.

[00213] In certain embodiments the halogenated salicylanilide is niclosamide in the free acid form.

[00214] In certain embodiments the halogenated salicylanilide is a pharmaceutically acceptable salt of niclosamide, for example an ethanolamine salt, or piperazine salt.

[00215] The halogenated salicylanilide may be a hydrate of niclosamide or

pharmaceutically acceptable salt thereof. However, generally it is preferred that the niclosamide is not administered to the subject in the form of a hydrate. In certain embodiments the niclosamide is anhydrous niclosamide, or a pharmaceutically acceptable salt thereof. In a particular embodiment the niclosamide is anhydrous niclosamide.

PHARMACEUTICAL COMPOSITIONS

[00216] The halogenated salicylanilide is suitably administered to the subject in the form of a pharmaceutical composition comprising the halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable excipient. The halogenated salicylanilide is suitably compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 99 percent by weight of the composition. The compositions may be prepared using conventional procedures well known in the art. Conventional procedures for the selection and preparation of suitable pharmaceutical compositions are described in, for example, "Pharmaceuticals - The Science of Dosage Form Designs", M. E. Aulton, Churchill Livingstone, 1988.

Topical Pharmaceutical Compositions

[00217] The halogenated salicylanilide is topically administered to the subject during the initial treatment period, preferably in the form of a topical pharmaceutical composition comprising the halogenated salicylanilide.

[00218] In some embodiments the halogenated salicylanilide is present in an amount of up to 15% by weight of the compositions described herein, for example from 0.01 % to 7.5% from 0.05% to 10% by weight of the composition, from 0.05% to 5% by weight, from 0.1 % to 4.5% by weight, from 1% to 3% by weight, from 1.5% to 4.5% by weight, from 2% to 12% by weight, or from 4 to 12% by weight. For example, at about 2% by weight of the composition, at about 4% by weight of the composition, at about 7% by weight of the composition or at about 10% by weight of the composition.

[00219] In some embodiments the halogenated salicylanilide is present in the composition at a concentration of up to 250 mg/ml, for example 200 mg/ml or less, 150 mg/ml or less, 100 mg/ml or less or 50 mg/ml or less. For example, the halogenated salicylanilide (e.g. niclosamide or oxyclozanide) is present in the composition at a concentration of from 0.5 mg/ml to 200 mg/ml, 1 mg/ml to 150 mg/ml_, 1 mg/ml_ to 120 mg/ml_, 1 mg/ml_ to 100 mg/ml_, 1 mg/ml_ to 50 mg/ml, 1 mg/ml_ to 20 mg/ml_ or 1 mg/ml_ to 10 mg/ml_. In some embodiments the halogenated salicylanilide (e.g. niclosamide or oxyclozanide) is present in the composition at a concentration of from 50mg/ml_ to 200 mg/ml_, from 50mg/ml_ to 200, or from 80 mg/ml_ to 120 mg/ml_, for example at about 100 mg/ml_.

[00220] In some embodiments the topical composition is an aqueous topical composition comprising the halogenated salicylanilide or pharmaceutically acceptable salt or hydrate thereof. The aqueous topical composition suitably comprises at least 5% by weight of water and one or more pharmaceutically acceptable excipients.

[00221] In other embodiments the topical composition is a non-aqueous topical composition comprising the halogenated salicylanilide or pharmaceutically acceptable salt or hydrate thereof.

[00222] The topical composition may be in any form suitable for topical administration, for example a cream, ointment, gel, foam, or aqueous, non-aqueous or oily solution or suspension comprising the halogenated salicylanilide. In some embodiments the topical composition may be in the form of an aqueous or non-aqueous gel comprising the halogenated salicylanilide and a gel forming agent. The gel forming agent may be any suitable gel-forming agent, including, but not limited to any of the gel forming agents described herein. In some embodiments the topical composition may be in the form of an aqueous cream or ointment comprising the halogenated salicylanilide and a suitable aqueous cream or non-aqueous ointment base. In some embodiments the topical composition may be in the form of a non-aqueous cream or ointment comprising the halogenated salicylanilide and a suitable non-aqueous cream or non-aqueous ointment base.

[00223] Suitably the topical composition is a spot-on, a pour-on or line-on topical composition. Spot-on compositions are applied to a single spot on the body of the animal suitably, between the animal's shoulders or neck. The active ingredients distribute through the epidermis to provide a therapeutically effective dose of the halogenated salicylanilide. [00224] Pour-on or line-on compositions are suitably applied to the non-human subject as a line or strip to the skin on the subject. Such compositions are particularly suitable for large animals such as horses or cattle as well as small non-human mammals (e.g. dogs or cats). Pour on and line on compositions are suitably applied against the grain of fur or hair of the non-human subject.

[00225] Reference to“Spot-on” compositions herein refers to a composition comprising the halogenated salicylanilide wherein the composition is topically applied (preferably as a single unit dose) to a single localized area (i.e. a spot) on the skin of the subject.

Reference to“line-on” compositions herein refers to a composition comprising the halogenated salicylanilide, wherein the composition is topically applied on the skin of the subject as a line or strip. Suitably line-on compositions are topically applied to the skin starting from the base of the tail along the spine to the shoulder blades, or from the middle of the back along the spine to the shoulder blades, or less of the subject (e.g. dog or cat). The length of the "line-on" application will depend on the subject being treated. For example, a line or strip about 30 cm, or 20 cm, or 15 cm, or 10 cm, or 5 cm long.

Preferably the length of the line or strip is about 10 cm. Line on compositions may also be applied specifically around a specific skin area to be treated (e.g. an area of infected skin or a dermatitis lesion). Spot on or line on composition are suitably formulated as a unit dose adapted to the weight and / or size of the animal, wherein the entire dose is applied to the animal in a single application.

[00226] The topical composition may be prepared using known carriers or“bases” in which the halogenated salicylanilide is dissolved or dispersed. For example, the topical composition may comprise the halogenated salicylanilide dissolved or dispersed in a suitable base formulation selected from an oleaginous base (e.g. petrolatum, white petrolatum, yellow ointment or white ointment), an absorption base (e.g. hydrophilic petrolatum or lanolin), a water-removable base (oil in water emulsion); a water-soluble base (e.g. a polyethylene glycol).

Non-aqueous topical compositions

[00227] In particular embodiments the halogenated salicylanilide is formulated as a non- aqueous pharmaceutical composition suitable for topical administration. For example, a non-aqueous cream, ointment, gel or foam comprising the halogenated salicylanilide (for example niclosamide or a pharmaceutically acceptable salt or hydrate thereof).

[00228] In certain embodiments the non-aqueous topical composition comprises: (i) a halogenated salicylanilide (for example selected from niclosamide, rafoxanide, oxyclozanide and closantel), or a pharmaceutically acceptable salt or hydrate thereof); and

(ii) polyethylene glycol (PEG), preferably a PEG with a melting point of less than 40°C.

[00229] In certain embodiments the non-aqueous composition comprises:

(i) a halogenated salicylanilide (for example selected from niclosamide, rafoxanide, oxyclozanide and closantel), or a pharmaceutically acceptable salt or hydrate thereof; and

(ii) greater than 60 % by weight of a PEG, preferably wherein the average molecular weight of the PEG is 800 or less and particularly 600 or less. For example, the average molecular weight of the PEG is less than 800. It may be that the average molecular weight of the PEG is less than 400.

[00230] In certain embodiments, the composition further comprises a non-polymeric glycol (for example an alkylene glycol, e.g. a C2-8 alkylene glycol, preferably a C2-6 alkylene glycol and especially propylene glycol).

[00231] In certain embodiments the non-aqueous topical composition comprises propylene glycol. Accordingly the composition may comprise:

(i) a halogenated salicylanilide (for example selected from niclosamide, rafoxanide, oxyclozanide and closantel), or a pharmaceutically acceptable salt or hydrate thereof;

(ii) polyethylene glycol (PEG), (preferably a PEG with a melting point of less than 40°C); and

(iii) a C2-8 alkylene glycol (preferably propylene glycol).

[00232] In certain embodiments the non-aqueous topical composition comprises:

(i) 0.1 to 5% by weight of a halogenated salicylanilide (e.g. selected from niclosamide, rafoxanide, oxyclozanide and closantel), or a pharmaceutically acceptable salt or hydrate thereof;

(ii) polyethylene glycol (PEG) with a melting point of less than 40°C; and

(iii) 0.5 to 30% (for example 5 to 25%) by weight of a non-polymeric glycol (preferably propylene glycol). [00233] Examples of PEG, preferably with an average molecular weight of less than 600, which may be used in the non-aqueous composition are described in more detail below under the section“Polyethylene Glycol (PEG)”

[00234] It may be that the non-aqueous composition comprises up to 10%, up to 20%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50% or up to 55% by weight of PEG. For example, wherein the lower limit of PEG is 1 % by weight and the upper limit is any of the values set out in this paragraph. For example, wherein the lower limit of PEG is 5% by weight and the upper limit is any of the values set out in this paragraph (e.g. a range of 5% to 20, 30, 40, 50, 60, 70, 80, 90 or 95% by weight PEG).

[00235] In some embodiments it has been found that a high concentration of PEG in the composition provides a non-aqueous topical composition with advantageous properties, for example one or more of improved dermal penetration and/or good tolerability when topically applied to the skin. Certain compositions described herein provide high concentration of the halogenated salicylanilide in skin tissues (e.g. the dermis and epidermis) and very low levels of systemic exposure (e.g. in the plasma) to the

halogenated salicylanilide. The compositions are therefore expected to provide an effective local topical treatment of, for example, a dermal condition, with little or no systemic side-effects, because the systemic exposure is low. Such compositions are expected to provide a wide therapeutic window between the beneficial therapeutic effects on the skin condition and the onset of undesirable systemic side effects (e.g. systemic toxicity) that may be associated with the halogenated salicylanilide.

[00236] It may be that the non-aqueous composition comprises more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% PEG (preferably with an average molecular weight of 600 or less, for example a PEG with an average molecular weight of 400 or less); and wherein the % is by weight of the composition.

Further amount of the PEG which may be present in the composition are described under the section“Polyethylene Glycol (PEG)”

[00237] It may be that the halogenated salicylanilide, or a pharmaceutically acceptable salt thereof is present in the non-aqueous composition in an amount of 0.01% to 10%, for example from 0.01% to 7.5%, from 0.01 % to 7%, from 0.01% to 6.5%, from 0.01% to 6%, from 0.01 % to 5.5%, 0.01% to 5%, from 0.01 % to 4.5%, from 0.01 % to 4%, from 0.01% to 3.5%, from 0.01 % to 3%, from 0.1 % to 5%, from 0.1% to 4.5%, from 0.1 % to 4%, from 0.1 % to 3.5%, from 0.1 to 3%, from 0.1 to 2.5%, from 0.1 to 2%, from 0.1 to 1.5%, from 0.1 to 1 %, from 0.5 to 3%, from 2% to 12%, from 4% to 12% or from 9% to about 11 %, for example about 1 %, about 2% about 2.5% about 3%, about 4%, about 4.5%, about 5%, about 6% about 7% or about 10% wherein the % are by weight based upon the weight of the composition. Suitable examples of halogenated salicylanilides which may be used are described herein, for example niclosamide, rafoxanide, oxyclozanide and closantel, or a pharmaceutically acceptable salt or hydrate thereof). The halogenated salicylanilides may be in the form of a hydrate, however, this is less preferred in the non-aqueous

compositions described herein. Accordingly, it is preferred that the halogenated salicylanilide is in a substantially anhydrous form.

[00238] It may be that the non-aqueous composition of the invention comprises:

(i) 0.01 to 7.5%, such as 0.01 to 4.5% (e.g. 0.1 to 4%, or 0.1 to 3.5, or 0.1 to 3% or about 2%, or about 4%) by weight of a halogenated salicylanilide, or a

pharmaceutically acceptable salt thereof; and

(ii) at least 70 % (for example at least 90%) by weight of a PEG, wherein the average molecular weight of the PEG is 600 or less (for example less than 600 or from about 200 to about 600 or about 400).

[00239] It may be that the non-aqueous compositions described herein further comprise a polar organic solvent for example a polar organic solvent selected from an alkylene glycol (e.g. propylene glycol), 2-(2-ethoxyethoxy)ethanol, glycerol, a macrogol stearyl ether (e.g. macrogol 15 stearyl ether) or a macrogol isostearate or a fatty alcohol, for example a C12- Cie-alcohol such as cetostearyl alcohol or a mixture two or more thereof. It may be that the polar organic is present in the composition in an amount of from about 5% to about 65%, about 10% to about 55% or about 25% to about 50% by weight of the composition.

[00240] It may be that the non-aqueous compositions described herein further comprise a glycol, for example an alkylene glycol (e.g. propylene glycol). It may be that the composition comprises from about 5% to about 30%, about 10% to about 30%, or about 14% to about 28% by weight of a glycol, particularly propylene glycol.

[00241] It may be that the non-aqueous compositions described herein further comprise 2- (2-ethoxyethoxy)ethanol. It may be that the composition comprises from about 1% to about 25%, about 5% to about 20% or about 10% to about 20% by weight of 2-(2- ethoxyethoxy)ethanol. [00242] It may be that the non-aqueous compositions described herein further comprise glycerol. It may be that the composition comprises from about 5% to about 30%, about 10% to about 30%, or about 15% to 25% by weight of glycerol.

[00243] It may be that the composition comprises one or more non-polar excipients, for example one or more non-polar oils, hydrocarbon solvents or waxes. It may be that the composition comprises one or more non-polar excipients selected from aromatic or aliphatic esters, a mineral oil, a vegetable oil and long-chain or medium chain triglycerides. For example, the non-polar excipients may be selected from one or more of a mineral oil, (e.g. liquid paraffin or a paraffin wax) and medium chain triglycerides. It may be that the non-polar excipients are present in the composition in an amount of from about 2% to about 50%, about 5% to about 40%, about 5% to about 30%, or about 5% to 25% by weight of the composition.

[00244] It may be that the non-aqueous compositions described herein further comprise one or more surfactant or emulsifiers, for example an ionic or non-ionic surfactant or emulsifiers. Representative examples of surfactants or emulsifiers include any of those described herein, for example a PEGylated fatty acid glyceride (labrasol), polyoxyethylene glycol sorbitan alkyl ester (polysorbate), a polyoxyethylene glycol alkyl ether (Brij), polyoxyethylene ethers of fatty alcohols (ceteareth), or a fatty acid ester of glycerol (e.g. glyceryl stearate). It may be that the surfactant or emulsifiers are present in the composition in an amount of from about 0.1 % to about 15%, about 0.2% to about 10%, or about 0.2% to about 5% by weight of the composition.

[00245] In certain embodiments the non-aqueous composition comprises a non-aqueous emulsion or microemulsion. Non-aqueous emulsion or microemulsion compositions are particularly suitable for providing compositions in the form of a non-aqueous topical cream composition. The non-aqueous emulsion comprise a non-aqueous hydrophilic phase (suitably comprising polar excipients) and a non-aqueous hydrophobic phase which is immiscible with the hydrophilic phase (suitably comprising non-polar excipients such as an oil). It may be that the hydrophilic phase comprises the continuous phase of the emulsion and the hydrophobic phase is dispersed within the hydrophilic phase as the discontinuous phase of the emulsion. In certain embodiments the non-aqueous hydrophobic phase comprises the continuous phase of the emulsion and the non-aqueous phase is dispersed within the non-aqueous hydrophobic phase as the discontinuous phase of the emulsion.

[00246] In certain embodiments the non-aqueous hydrophilic phase comprises the halogenated salicylanilide, PEG and optionally one or more of the polar solvents described herein. Accordingly it may be that the non-aqueous hydrophilic phase comprises niclosamide, PEG and optionally one or more polar solvents selected from propylene glycol, 2-(2-ethoxyethoxy)ethanol, glycerol, a macrogol stearyl ether (e.g. macrogol 15 stearyl ether) and a fatty alcohol, for example a Ci2-Cie-alcohol such as cetostearyl alcohol.

[00247] It may be that the non-aqueous hydrophobic phase of the emulsion or

microemulsion comprises one or more of the non-polar excipients described herein, for example, a mineral oil, a vegetable oil and long-chain or medium chain triglycerides.

[00248] In those embodiments where the composition is in the form of a non-aqueous emulsion or microemulsion the composition suitably comprises a surfactant or emulsifier, for example one or more of the surfactants or emulsifiers described herein.

[00249] Suitably the non-aqueous composition comprises a solution of the halogenated salicylanilide. Accordingly, it is preferred that the halogenated salicylanilide is completely dissolved in the non-aqueous composition. However, it is contemplated that the halogenated salicylanilide may present as a dispersion in the composition. Alternatively, in some embodiments at least a proportion of the halogenated salicylanilide is dissolved in the composition. In this embodiment it is preferred that at least 80%, preferably at least 90%, more preferably at least 95% by weight of the halogenated salicylanilide is dissolved in the composition.

Non-aqueous Gel Compositions

[00250] In certain embodiments the non-aqueous topical composition for use in the dosage regimen of the invention is in the form of a non-aqueous topical gel composition

[00251] In certain embodiments there is provided a non-aqueous topical gel composition comprising:

(ii) PEG with a melting point of less than 40°C; and

(iii) a gel forming agent.

[00252] In certain embodiments there is provided a non-aqueous topical gel composition comprising: (i) a halogenated salicylanilide (for example selected from niclosamide, rafoxanide, oxyclozanide and closantel), or a pharmaceutically acceptable salt or hydrate thereof;

(ii) greater than 60 % by weight of a PEG, preferably wherein the average molecular weight of the PEG is less than 600; and

(iii) a gel-forming agent.

[00253] Particular aspects of the non-aqueous gel compositions are described below.

Gel-forming agent

[00254] It may be that the gel-forming agent present in the compositions disclosed herein is an inorganic gel-forming agent. It may be that the gel-forming agent is a gel-forming polymer.

Inorganic gel forming agents

[00255] It may be that the gel-forming agent is an inorganic gel-forming agent, for example a bentonite or a silica. It may be that the gel-forming agent is magnesium aluminium silicate (Veegum®).

Gel-forming polymers

[00256] The gel-forming agent may be a gel-forming polymer. The gel-forming polymer may be a hydrophilic gel-forming polymer. The gel-forming polymer may be selected from the group consisting of: gelatin; agar; agarose; pectin; carrageenan; chitosan; alginate; starch; starch components (e.g. amylose or amylopectin); tragacanth gum; xanthan gum; gum Arabic (acacia gum); guar gum; gellan gum; locust bean gum; polyurethane;

polyether polyurethane; cellulose; cellulose ethers (for example methylcellulose, carboxymethyl cellulose, ethylcellulose, hydroxyethyl cellulose or hydroxypropyl cellulose), cellulose esters, cellulose acetates, cellulose triacetates; cross-bonded polyvinyl alcohol; polymers and copolymers of acrylic acid, hydroxyalkyl acrylates, hydroxyethyl acrylate, diethylene glycol monoacrylate, 2-hydroxypropylacrylate or 3-hydroxypropyl acrylate; carbomers (cross-linked poly(acrylic acids), for example carbomer 910, 934P, 940GE,

941 GE, 971 P, 974P; polymers and copolymers of methacrylic acid, hydroxyethyl methacrylate, diethyleneglycol monomethacrylate, 2-hydroxypropyl methacrylate, 3- hydroxypropyl methacrylate or dipropylene glycol monomethylacrylate; vinylpyrrolidone polymers; polymers and copolymers or acrylamide, N-methylacrylamide, N- propylacrylamide; methacrylamide, N-isopropylmethacrylamide, or N-2- hydroxyethylmethacrylamide; poloxamers (triblock copolymers comprising a central polyoxypropylene block flanked by two polyoxyethylene blocks, for example a Pluronic®); and gels comprising cross-linked polyalkylene glycols, for example gels comprising cross- linked polyethylene glycol or cross-linked polypropylene glycol. In specific embodiments binary or tertiary etc. combinations of any of the above gel-forming agents are foreseen. When the gel forming agent comprises a PEG, the PEG is suitably a higher molecular weight than the PEG used as a solvent to dissolve or disperse the halogenated salicylanilide in the gel composition. Accordingly it is to be understood that when the gel- forming agent is a PEG, the PEG of the gel-forming agent is different to the PEG present in component (ii) of the compositions of the invention. For example, where the gel forming agent comprises a PEG, the PEG suitably has a molecular weight greater than 600, for example greater than 1000, greater than 10000 or greater than 20000. Suitably, when the gel forming agent comprises a PEG it has an average molecular weight of from about 600 to about 35,000, for example from about 800 to about 25,000, or from about 1000 to about 20,000. Other gel-forming agents are also contemplated, for example as disclosed in Gels handbook Vols 1-4, Osada et al. 2001 Elsevier.

[00257] The gel-forming polymer may be a gum, for example a gum selected from tragacanth gum, xanthan gum; gum arabic (acacia gum); guar gum; gellan gum locust bean gum.

[00258] The gel-forming polymer may be a cellulose ether, for example methylcellulose, carboxymethyl cellulose, ethylcellulose, hydroxyethyl cellulose, hydroxy propyl methyl cellulose or hydroxypropyl cellulose.

Carbomer gel-forming polymers

[00259] In a particular embodiment the gel-forming agent is a carbomer. Carbomers are high molecular weight cross-linked poly(acrylic acid) polymers. The polymers may be cross-linked by polyalcohol allyl ethers, for example, allyl sucrose or allyl pentaerythritol The carbomer may be a homopolymer, for example 910, 934P, 940GE, 941 GE, 971 P, 974P, wherein“GE” refers to medical grade and“P” oral grade. Derivatives of Carbomer polymers may also be used, for example Carbopol interpolymers comprising a carbomer polymer comprising a block copolymer of polyethylene glycol and a long chain alkyl acid ester, such derivatives are commercially available as ETD 2020 NF and Ultrez 10 NF from Lubrizol.

[00260] Carbomers (also known as Carbopols) are well known and are characterised in the United States Pharmacopeia/National Formulary (USP/NF) monograph for Carbomers and the European Pharmacopeia (Ph. Eur.) monograph for Carbomers, reference to which is incorporated herein.

[00261] The carbomer may have a viscosity of from about 4,000 to about 70,000, for example about 10,000 to about 60,000, for about 20,000 to about 50,000, about 25,000 to about 45,000 or about 29,400 to about 39,400 cP, wherein the viscosity is that of a 0.5 wt% solution of the carbomer in water, neutralised to pH 7.3 - 7.8 at 25°C, measured using a Brookfield RVT, 20 rpm, spindle #6.

[00262] Suitably the carbomer comprises from about 56% to about 68.0 % by weight carboxylic acid (-COOH) groups. The proportion of carboxy groups present in the carbomer may be determined using known methods, for example by titrating an aqueous solution or dispersion of the polymer against NaOH.

[00263] Suitably the carbomer is substantially free of residual benzene (for example containing less than 0.5 parts per million). Accordingly, it is preferred that the carbomer is prepared without using benzene as a solvent during the polymerisation process. Preferred carbomers are those are prepared using ethyl acetate and optionally cyclohexane as the solvent during polymerisation.

[00264] A particular carbomer for use as a gelling agent in the present invention is Carbomer 974P. This carbomer suitably has a viscosity of 29400 to 39400 cP (0.5% solution in water neutralized to pH 7.3 - 7.8 and measured at 25°C using a Brookfield RVT, 20 rpm with spindle #6). The carbomer typically has a carboxylic acid content of from 56 to 68%.

[00265] Conventionally carbomer gels are formed by dispersing the carbomer in water, which results in ionisation of the carboxy groups present in the polymer. The resulting solution or dispersion is then neutralised using a base, resulting in an increase in viscosity and gel formation. However, in the present invention the gel is a non-aqueous gel and gel formation may be achieved by dissolving or dispersing the carbopol in the organic solvent together with the halogenated salicylanilides and heating the mixture to about 70°C.

[00266] The gel-forming polymer may also be referred to as a colloid i.e. a colloid system wherein the colloid particles are disperse in the organic solvent and the quantity of solvent available allows for the formation of a gel. In embodiments it is preferred to use reversible colloids preferably thermo-reversible colloids (e.g. agar, agarose and gelatin etc.) as opposed to irreversible (single-state) colloids. Thermo-reversible colloids can exist in a gel and sol state, and alternate between states with the addition or elimination of heat. Thermoreversible colloids which may be used according to the invention, whether individually or in combination, include for example, gelatin, carrageenan, gelatin, agar, agarose (a polysaccharide obtained from agar), pectin and cellulose derivatives for example methylcellulose, carboxymethyl cellulose, ethylcellulose, hydroxyethyl cellulose, hydroxy propyl methyl cellulose or hydroxypropyl cellulose. Another term which may be applied to gel forming polymers is“thermotropic”: a thermotropic gelling agent is one caused to gel by a change in temperature. In embodiments of the invention, therefore, the gel former is a thermotropic gel-forming polymer or a combination of such polymers.

[00267] The gel-forming polymer may be or comprise an ionotropic gel-forming polymer whose gelling is induced by ions. Suitable ionotrophic gel-forming agents are anionic or cationic polymers which can be cross-linked by multivalent counter ions to form a gel. The ionotrophic gel-forming polymers may be, for example chitosan, an alginate, carrageenan or pectin.

[00268] The gel-forming polymer may comprise or be a single gel-forming polymer or a mixture of two or more gel-forming polymers. For example, the gel-forming polymer may comprise a combination of two or more of the gel-forming polymers listed herein.

[00269] The amount of gel forming agent present in the composition should be selected so as to provide a gel composition having the required rheological properties, for example a viscosity suitable for topical application. Generally, the gel composition will be of a viscosity such that it can be readily dispensed and spread over and rubbed in the area of, for example, skin that is infected. The rheology of the gel composition will depend upon the particular gelling agent used, the molecular weight of the PEG, the particular halogenated salicylanilide and the amounts thereof in the composition. Generally, the gelling agent, for example a carbomer, will be present in the gel composition is an amount of up to about 10% by weight, for example up to about 1 %, 2%, 3%, 4%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%. 9% or 9.5% by weight of the gel composition. Suitably the gelling agent, for example a carbomer, may be present in an amount of from about 0.01% to about 10% by weight of the gel composition, for example about 0.01 % to about 8%, about 0.05% to about 7%, about 0.05% to about 6%, about 0.05% to about 5%, about 0.05% to about 4%, about 1 % to about 6%, about 1% to about 5% or about 1 % to about 4%, about 2% to about 5%, about 2% to about 4% or about 2% to about 3%, wherein the % is by weight based on the weight of the gel composition.

Polyethylene Glycol (PEG) [00270] In embodiments where PEG is present in the compositions comprising the halogenated salicylanilide described herein, the PEG suitably has one or more of the characteristics described in this section.

[00271] Suitably the PEG is liquid at ambient temperature (for example 20 to 25°C), accordingly the solvent may be a low molecular weight PEG. Particularly, the PEG has an average molecular weight of 600 or less, suitably less than about 600. For example, the PEG may have an average molecular weight of from about 200 to about 600, about 200 to about 500 or about 200 to about 400. A particular PEG is selected from PEG 200, PEG 300 and PEG 400. In one particular embodiment the PEG is PEG 400. Alternatively, the PEG may comprise a mixture of PEGs which together with the other components of the composition provide a composition which is suitable for e.g. topical application to the subject. Accordingly, the PEG may be a mixture of one or more low molecular weight PEGs with one or more higher molecular weight PEG, wherein the mixture of PEGs has a melting point below 40, or preferably below about 37°C.

[00272] Suitably the PEG is present in an amount at least sufficient to provide a solution of the halogenated salicylanilide in the composition. As will be realised the amount of PEG required to dissolve the halogenated salicylanilide will depend upon the particular halogenated salicylanilide used and the other components of the composition. In certain embodiments the PEG is present in the composition of the invention an amount of at least 60 %, suitably greater than 60% by weight of the composition. Non-aqueous compositions containing high amounts of PEG provide topical compositions which give high levels of the halogenated salicylanilide in skin tissues and only minimal systemic exposure to the halogenated salicylanilide. Such compositions have also been found to be well tolerated, despite containing high PEG concentrations. Suitably the PEG is present in an amount of greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% wherein the % is by weight based upon the weight of the composition. It may be that the PEG, preferably a PEG with an average molecular weight of 600 or less (particularly less than 600) is present in the non-aqueous composition of the invention in an amount of for example 65 to 98%, for example from 65% to 95%, 65% to 90%, 65% to 80%, 70% to 98%, 70% to 95%, 70% to 85%, 70% to 80%, 80% to 98%, 80% to 95%,

80% to 90%, 85% to 98% or 85% to 95%, wherein the % is by weight based upon the weight of the non-aqueous composition of the invention.

[00273] In certain embodiments the composition (e.g. a non-aqueous composition) comprise lower concentrations of PEG, for example 50% or less, 45% or less, 40% or less, 35% or less 30% or less, 25% or less, 20% or less, 15% or less, wherein the % is % by weight of the composition. It may be that the PEG is present from about 1 % to about 50%, from about 5% to about 40%, from about 5% to about 35%, or from about 5 to about 30% by weight of the composition.

Topical Foam Compositions

[00274] In certain embodiments the halogenated salicylanilide is formulated as a foam composition. The foam composition may be an aqueous foam composition such as an emulsion or nano-emulsion foams or a water-alcohol based foam (e.g. a water-ethanolic foam). Alternatively, the foam may be a non-aqueous (i.e. water-free) foam composition, including but not limited to oil-based foams, petrolatum-based foams, ointment foams; emollient foams and foams formed using non-aqueous hydrophilic excipients. When the foam is a foam formed from an emulsion, the emulsion may be a water-in-oil emulsion or an oil-in-water emulsion comprising the halogenated salicylanilide. Foams suitable for the delivery of pharmaceuticals are well-known and are described in for example Arzhavitina et al,“Foams for pharmaceutical and cosmetic application” Int. J. Pharm., 394, 1-17 (2010).

[00275] Suitably the foam is a breakable foam, i.e. a thermally stable foam which collapses (breaks) upon application of shear stress to the foam. Such breakable foams can be applied to the skin as a foam and then collapse when the foam is rubbed into the skin, thereby enabling the active to be applied to the skin in the area required.

[00276] In certain embodiments the foam is an emollient foam formed from an oil-in-water emulsion comprising the halogenated salicylanilide. The oil may be, for example a mineral oil, a plant derived oil (e.g. olive oil, soybean oil, coconut oil, or castor oil), medium or long-chain triglycerides and esters thereof, fatty acids, fatty acid esters, fatty acid alcohols and a wax. For example, the oil may comprise an alcohol selected from lauryl alcohol, myristyl alcohol, cetyl alcohol, stearyl alcohol, arachidyl alcohol, behenyl alcohol, tetracosanol, hexacosanol, octacosanol, triacontanol, and tetratriacontanol. The oil may comprise a fatty acid selected from dodecanoic acid, tetradecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid, tetracosanoic acid, hexacosanoic acid, heptacosanoic acid, octacosanoic acid,

triacontanoic acid, dotriacontanoic acid, tritriacontanoic acid, tetratriacontanoic acid and pentatriacontanoic acid. The oil may comprise a hydroxy fatty acid such a 12-hydroxy stearic acid. The oil may comprise a wax, for example carnauba wax, candelilla wax, ouricury wax, sugarcane wax, retamo wax, jojoba oil, an animal wax (e.g. beeswax) or a petroleum derived wax (e.g. paraffin wax).

[00277] The emulsion may include emulsifiers or surfactants to stabilise the emulsion, for example one or more non-ionic surfactant (including any of the surfactants described herein, particularly those in relation to the non-aqueous topical compositions described above). The foam may comprise further excipients, for example, solvents, gelling agents, humectants, preservatives, and absorption enhancers, including but not limited to those described herein.

[00278] In a particular embodiment the foam is a non-aqueous foam. Such foams can be prepared by forming one of the non-aqueous formulations described above, for example a non-aqueous gel composition, into a foam composition. Examples of non-aqueous foam compositions which may be suitable for the delivery of a halogenated salicylanilide are described in, for example WO2010/041141 , W02009/098595 and W02008/152444.

[00279] In certain embodiments the foams is a non-aqueous oil-based foam prepared using a suitable pharmaceutically acceptable oil, for example as discussed above in relation to emollient foams in which the halogenated salicylanilide is dispersed or dissolved. It may be that surfactants are used to stabilise the foams. It is also

contemplated that non-aqueous oil-based foams may be prepared which do not require a surfactant. Such foams include but are not limited to those described in WO2011/013008, WO2011/013009, WO201 1/064631 and WO201 1/039637.

[00280] Other examples of foam compositions that may be used to formulate the halogenated salicylanilide include analogous compositions to those described in, for example, WO2011/138678, WO2011/039638, WO/2010/125470, WO/2009/090558, W02009/090495, W02009/007785, W02008/038140, W02007/085902,

W02007/054818, W02007/039825, W02006/003481 , W02005/018530, W02005/01 1567 and W02004/037225.

[00281] Foam compositions comprising the halogenated salicylanilide are suitably formulated as a semi-solid or liquid composition packaged in a suitable aerosol pressurised container with a propellant. The foam is formed upon release of the composition from the pressurised container via a suitable aerosol nozzle in the outlet of the container. Suitable propellants include a hydrocarbon propellant such as propane or butane, or a halogenated fluorocarbon such as tetrafluoroethane. Suitable aerosol containers and nozzles are well-known. Spot-on/Line on Compositions

[00282] In some embodiments the composition is formulated as a spot-on or line-on composition comprising the halogenated salicylanilide. Examples of spot-on or line-on compositions comprise the halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof (e.g. niclosamide or oxyclozanide) and a solvent, preferably a non- aqueous solvent.

[00283] It may be that the spot-on or line on composition comprises the halogenated salicylanilide and one or more polar aprotic solvents. In some embodiments the polar aprotic solvent is selected from a ketone (e.g. acetone), N,N-dimethylformamide, acetonitrile, and dimethylsulfoxide (DMSO). Preferably the solvent is DMSO. In some embodiments the spot-on or line on compositions comprises one or more additional co- solvents. Suitably the co-solvent is a lipophilic solvent (for example an oil or, fat or lipid or any of the non-polar excipients described herein), a glycol described herein (e.g. PEG or propylene glycol), a glycol ether (e.g. 2-(2-ethoxyethoxy)ethanol), a protic polar solvent described herein, an alcohol (e.g. ethanol) and/or an alkanol amine (e.g. ethanolamine, diethanolamine, triethanolamine, isopropanolamine and diisopropanolamine). In some embodiments the spot-on or line-on composition comprises an absorption enhancer (e.g. one or more of the absorption enhancers described herein). It is to be understood that the solvent/co-solvent (e.g. Transcutol) may also act as an absorption enhancer in the composition. The presence of a solvent in the spot-on or line-on composition enables the composition to be formulated with a high concentration of the halogenated salicylanilide, thereby enabling the“spot-on” or“line-on” of a concentrated solution or dispersion of the halogenated salicylanilide to the subject.

[00284] In some embodiments the spot-on or line-on composition comprises 2 to 20 % wt/v, preferably 5 to 15 % wt/v, more preferably, 8 to 12 % wt/v of a halogenated salicylnalinide (e.g. niclosamide or oxyclozanide); and

35 to 55 % wt/v (preferably 30 to 50 % more preferably, about 45 %) of a polar aprotic solvent, for example dimethyl sulfoxide (DMSO).

[00285] Suitably the spot-on or line-on composition further comprises a glycol ether (e.g. 2-(2-ethoxyethoxy)ethanol) and optionally an alkanol amine (e.g. ethanolamine.

[00286] Accordingly, in a preferred embodiment the spot-on or line on composition comprises: 2 to 20 % wt/v, preferably 5 to 15 % wt/v, more preferably, 8 to 12 % wt/v of a halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof (e.g. niclosamide or oxyclozanide, more preferably oxyclozanide or a pharmaceutically acceptable salt or hydrate thereof);

35 to 55 % wt/v (preferably 30 to 50 % wt/v more preferably, about 45 % wt/v) of a polar aprotic solvent, for example dimethyl sulfoxide (DMSO);

25 to 55% w/v (preferably 30 to 55% wt/v, more preferably 35 to 50% w/v of a glycol ether (e.g. 2-(2-eth oxyeth oxy )etha nol );

and 0 to 10% w/v (preferably 0 to 5% w/v (e.g. 0, or 1 to 5% w/v) alkanol amine (e.g. ethanolamine.

[00287] In another embodiment the spot-on or line-on composition is a composition selected from formulation A’ to G shown in the Table below:

Table A

[00288] A further aspect of the invention provides a spot-on or line-on composition as described herein.

Optional Components for Topical Compositions

[00289] The following components and features may optionally be present in the halogenated salicylanilide compositions described herein, for example the non-aqueous topical compositions described herein.

Solvents

[00290] The topical composition may comprise one or more solvent(s). The presence of a further solvent may enhance the solubility of the halogenated salicylanilide and or help maintain the halogenated salicylanilide in solution during the preparation, storage and topical use of the non-aqueous composition. The additional solvent may be, for example, a polar organic solvent in which the halogenated salicylanilide is soluble, for example a polar organic solvent wherein the halogenated salicylanilides has a solubility of greater than 2% by weight in the additional solvent.

[00291] The polar organic solvent may be a protic polar organic solvent. In one embodiment the solvent is a protic polar organic solvent having a dielectric constant of from about 10 to about 45, for example a dielectric constant of from about 10 to about 25. Particular polar protic organic solvents are those which have a dielectric constant of from about 10 to about 20, wherein in each case the dielectric constant is measured at 20-25°C. The dielectric constant of organic solvents is well known or can be measured using well- known techniques

[00292] Representative protic polar organic solvents with a dielectric constant in the range of 10 to 45 include those set out in the table below:

[00293] Further polar organic solvents with a dielectric constant in the range are well known (see for example“Solubility and Solubilization in Aqueous Media” By Samuel H. Yalkowsky (University of Arizona). Oxford University Press: New York. 1999). For example, the polar organic solvent may be selected from ethyl acetate,

dimethylformamide, dichloromethane, glycerol, propylene glycol, or 2-(2- ethoxyethoxy)ethanol (Transcutol), propylene glycol stearyl ether and propylene glycol isostearate. [00294] In embodiments the polar organic solvent is an aprotic polar organic solvent having a dielectric constant of from about 10 to about 45, for example a dielectric constant of from about 10 to about 25 at 25°C.

[00295] When present the additional solvent(s) is suitably present in an amount of up to 35% by weight of the composition. For example, up to 30%, 25%, 20% 15% or 10% by weight of the composition. In particular embodiments the additional solvent(s) is present in an amount of less than 10%, for example less than 8%, less than 6%, less than 5% or less than 3%, wherein the % is by weight based upon the weight of the non-aqueous composition. It may be that the additional solvent is present in an amount of 1% to 30%, from 1 % to 25%, from 1 % to 20%, from 1 to 10%, from 3 to 30%, from 3 to 20%, from 3 to 15%, from 5 to 30%, from, 5 to 20% or from 5 to 10%, wherein the % is by weight based upon the weight of the composition.

Non-ethanolic compositions

[00296] The presence of ethanol in topical compositions can cause dryness and/or peeling of the skin, particularly in patients with sensitive skin. This can be a particular problem in subjects with dermal conditions such as dermatitis (e.g. AD). Accordingly, in certain embodiments the topical composition comprising the halogenated salicylanilide is ethanol free. Thus, in a preferred embodiment the topical halogenated salicylanilide composition comprises a non-aqueous, non-ethanol (ethanol free) composition, for example a non-aqueous, non-ethanol gel composition.

Absorption Enhancers

[00297] The topical composition may optionally comprise an absorption enhancer. The absorption may be any substance which acts to enhance the permeation of the

halogenated salicylanilide into the epidermis and epidermis. Suitable absorption enhancers include the transdermal absorption enhancers disclosed in for example Smith and Maibach (2005) Percutaneous Penetration Enhancers, Second Edition ISBN

9780849321528, incorporated herein by reference.

[00298] It may be that the absorption enhancer, when present in the topical composition is selected from, for example, a sulfoxide (for example dimethylsulfoxide);

dimethylacetamide; dimethylformamide; a urea; a fatty alcohol, for example a Cs-C-is fatty alcohol, which may be saturated or unsaturated (for example caprylic alcohol or cetostearyl alcohol); a polyol (for example glycerol; a glycol (for example propylene glycol or hexylene glycol); Azone ((1-dodecylazacycloheptan-2-one); an essential oil (for example a terpene or terpenoid); a pyrrolidone (for example N-methyl-2- pyrrolidone); an oxazolidinone (for example 4-decyloxazolidin-2-one) a surfactant (for example a non-ionic, anionic or cationic surfactant, particularly a non-ionic surfactant for example a

polyoxyethylene glycol sorbitan alkyl ester (for example polysorbates such as

Polysorbate 80 ((polyoxyethylene (20) sorbitan monooleate), Polysorbate 60

(polyoxyethylene (20) sorbitan monostearate), Polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate) or Polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate)), a polyoxyethylene glycol alkyl ether (Brij surfactants e.g. polyethoxylated stearyl ethers such as Brij S721 (a polyoxyethylene fatty ether derived from stearyl alcohols) or Brij S2 (Polyoxyethylene (2) stearyl ether)), a poloxamer or a PEGylated fatty acid glyceride such as caprylocaproyl polyoxyl-8 glycerides (e.g. Labrasol), a fatty acid ester of glycerol, for example glyceryl stearate, or polyoxyethylene ethers of fatty alcohols (for example cetyl alcohol and/or stearyl alcohol, particular examples include ceteareth-15, -16, -17, -18, -19, -20, -21 , -22, 23-, -24, or -25 and particularly ceteareth-20), a polyethoxylated sorbitan fatty acid ester, for example. The absorption enhancer may also be 2-(2- ethoxyethoxy)ethanol (Transcutol). Preferred absorption enhancers are those which have a minimal impact on the structure of the skin so as to minimise undesirable tolerability effects associated with the absorption enhancer, for example irritation, which could exacerbate the dermatitis (e.g. AD) in the subject. Particular absorption enhancers include polyols, for example propylene glycol or glycerol. Accordingly, the absorption enhancer may be propylene glycol. The absorption enhancer may be glycerol. It is to be understood that where the absorption enhancer may also act as an additional solvent in the composition, particularly when the halogenated salicylanilide is soluble in the absorption enhancer.

[00299] When present the absorption enhancer may be in an amount of up to 35% by weight of the topical composition (e.g. a gel composition), for example from 0.5% to 35%, from 1 % to 35%, from 5% to 30%, from 10% to 30%, from 5% to 35%, from 5% to 30% or from 10% to 30%, wherein the % is by weight of the composition.

Other Ingredients

[00300] The halogenated salicylanilide compositions described herein (e.g. a topical composition) may comprise one or more additional excipients in addition to the

halogenated salicylanilide and the other excipients described above (e.g. PEG in a non- aqueous topical composition). Additional excipients may be selected to provide compositions of the required form for topical administration. The additional excipients may be, for example one or more excipients selected from viscosity modifying agents, emulsifiers, surfactants, humectants, oils, waxes, solvents, preservatives, pH modifying agents (for example a suitable acid or base, for example an organic acid or organic amine base), buffers, antioxidants (for example butylated hydroxyanisol or butylated

hydroxytoluene), crystallisation inhibitors (for example a cellulose derivative such as hydroxypropyl methyl cellulose), colorants, fragrances,. Representative examples of such additional excipients are well known, for example as listed in the Handbook of

Pharmaceutical Excipients, 7^th Edition, Rowe et al. Further more specific excipients are set out in any of the compositions described in the Examples herein.

Local pH

[00301] WO 2016/038035 discloses that the antibacterial activity of halogenated salicylanilides, e.g. niclosamide, is higher at low pH than at neutral or basic pH, (see e.g. Figure 4 is WO 2016/038035).

[00302] In some embodiments the topical composition comprising the halogenated salicylanilide provides a local pH of less than 6 at the site of the skin where the

composition is topically applied (e.g. at the site of a skin infection) Thus, it may be that the topical composition does not comprise a buffer or pH modifier. Halogenated salicylanilides are weakly acidic by virtue of their phenolic groups. Thus, it may be that the halogenated salicylanilide, e.g. niclosamide, is in the free acid form (i.e. not in the form of a salt) in the composition. In other embodiments the topical composition comprises excipients to provide a local pH of less than 6 when the composition is topically applied to the subject, for example the composition may comprise pH modifier such as a suitable acid or base. The components of the topical composition may be selected such that it provides a local pH of greater than 4.5 (e.g. greater than 5) at the site of the skin condition to which is applied (e.g. the site of a skin infection infection). For example, the topical composition may provide a local pH of from about 4.5 to 6, suitably about 5.5. Reference to a“local pH” is to the pH at the site where the formulation is applied for example the pH on the surface of the skin after applying the composition comprising the halogenated

salicylanilide.

[00303] The desired local pH at the site of the skin to which the composition is topically applied (e.g. the site of a skin infection) can be obtained by routine methods, for example by adding a suitable acidic or basic material to the topical composition comprising the halogenated salicylanilide. For example, an inorganic or organic acid, base or buffer may be added to the composition comprising the halogenated salicylanilide in a sufficient amount to provide the desired local pH of less than 6 at the site of infection.

[00304] The topical gel compositions described herein wherein the gel forming agent comprises acidic moieties, for example carbomer polymers, may be particularly suitable to provide the desired low local pH following topical application without the need for additional agents to modify the pH.

[00305] The local pH following topical application, for example on the surface of the skin may be measured using known techniques. For example, the pH of the skin surface can be measured using a suitable flat glass electrode attached to a pH meter. Such apparatus are commercially available as for example, the Skin-pH-Meter PH 905 (Courage +

Khazaka electronic GmbH). Methods for measuring local skin pH are well known and include the ISO standard ISO 12505-1 :2014(en), section 4.4, incorporated herein by reference thereto.

Manufacture of Topical Compositions

[00306] The topical compositions described herein may be manufactured using well- known methods. For example, the non-aqueous gel compositions comprising PEG may be prepared by a process comprising the steps:

(i) dissolving the halogenated salicylanilide in the PEG;

(ii) combining the solution from step (i) with the gel-forming agent to form a mixture; and

(iii) causing the mixture to gel.

[00307] Suitably the halogenated salicylanilide is completely dissolved in the PEG in step (i) to form a solution. Dissolution may be aided by agitation of the mixture by stirring or by the application ultrasound. Optionally the mixture may be heated to facilitate dissolution. However, preferably the solution is prepared at ambient temperature. Optionally any halogenated salicylanilide that remains undissolved may be removed by a suitable filtration or other separation method prior to combining the solution with the gel-forming agent in step (ii) of the process.

[00308] The solution from step (i) may be added to the gel-forming agent or, alternatively, the gel-forming agent may be added to the solution. Optionally the gel-forming agent may be dissolved in some of the PEG to form a solution or dispersion prior to combining it with the solution from step (i). Suitably any additional optional components of the gel- composition, such as absorption enhancers, additional solvents etc. are added to the mixture prior to gelation of the composition. Alternatively, one or more of the optional components can be added after gel formation by mixing the additional component(s) with the gel.

[00309] Gel formation in step (iii) may be affected by various methods, depending on the nature of the gel-forming agent used. For example, where the gel-forming agent is thermotropic, the gel forming agent may be heated to form a liquid prior to adding the solution from step (i). Following mixing of the gel-forming agent with the solution, the resulting mixture may be cooled thereby causing the mixture to gel. Alternatively, where gelling is effected by ionic cross-linking, a suitable ionic agent is added to the mixture in step (iii), for example a suitable salt to thereby cause the mixture to gel. Gelling may also be induced by changing the pH of the mixture using a suitable acid or base to achieve the required pH for gelling to occur. The process is suitably carried out using anhydrous reagents under anhydrous conditions to ensure that the resulting gel composition is a non- aqueous gel composition.

[00310] When the gel-forming agent is a carbomer, a particular process for the preparation of the non-aqueous gel composition comprises:

(i) dissolving the halogenated salicylanilide in the PEG;

(ii) combining the solution from step (i) with a carbomer to form a mixture; and

(iii) heating the mixture to form a gel.

[00311] Step (i) of this process is suitably performed at room temperature. After combining the solution with the carbomer the mixture is mixed to provide a uniform dispersion. Mixing can be performed using any suitable method, for example stirring or, preferably, by homogenisation. The resulting dispersion is suitably de-gassed prior to gel formation in step (iii).

[00312] In step (iii) the mixture is suitably heated to a temperature of 60 to 80°C, for example at about 70°C, preferably under agitation. The mixture may be held at this temperature for a sufficient time to form a homogenous and transparent dispersion and to effect gel formation. Typically a holding time of about 30 minutes is sufficient to enable solvation of the carbomer and gel formation.

[00313] The process is suitably performed under anhydrous conditions using anhydrous reagents to ensure that the resulting gel composition is a non-aqueous gel. [00314] When the composition of the invention is in the form of a lotion, ointment or cream the composition may be prepared using known methods for the preparation of such compositions. For example, lotion or ointments may be prepared by simply blending the halogenated salicylanilide, and the other excipients comprising the formulation, for example viscosity modifiers, solvents and/or surfactants.

[00315] Non-aqueous topical compositions may also be prepared as non-aqueous emulsion or microemulsions to provide a composition in the form of, for example a non- aqueous cream. Non-aqueous emulsions and microemulsions may be prepared using well known methods. Non-aqueous emulsions and microemulsions may be prepared by mixing two immiscible non-aqueous phases. Suitably a non-aqueous hydrophilic phase (for example a hydrophilic phase comprising polar excipients and the halogenated

salicylanilide) is emulsified with an immiscible hydrophobic phase (e.g. comprising non- polar hydrophobic excipients). The non-aqueous emulsion may comprise a continuous hydrophobic phase and a discontinuous hydrophilic phase. Generally, however, the non- aqueous emulsion will comprise a continuous hydrophilic phase and a discontinuous hydrophobic phase. It may be that the non-aqueous hydrophilic phase comprises the halogenated salicylanilide and PEG and the non-aqueous hydrophobic phase comprises a non-polar liquid, which is immiscible with the hydrophobic phase, for example a medium chain triglyceride, a vegetable oil, a hydrocarbon oil or a mineral oil such as a paraffin. Generally the non-aqueous emulsion will be stabilised by one or more suitable surfactants or emulsifiers, for example one or more non-ionic surfactants (e.g. macrogol cetostearyl, cetostearyl alcohol, glyceryl stearate, polysorbate 80, Brij s721 , Brij S2, ceteareth-20 or macrogol stearyl ether). The emulsion or micro emulsion may be formed using well-known methods, for example by homogenisation of the hydrophilic phase with the hydrophobic phase together with the other components of the non-aqueous emulsion or microemulsion.

Embodiments comprising topical compositions

[00316] In some embodiments one or more of the following are excluded from the dosage regimen of the invention:

1. Compositions A, B, and/or C wherein the composition is topically administered to the subject for 1 day, 2 days or more than 3 days; and/or

2. Compositions A, B, and/or C wherein the composition is topically administered to the subject for 1 day, 2 days or more than 3 days, wherein the composition is topically administered to the subject at a frequency of once per day, twice per day, three times per day, four times per day, once every other day or once per week; and/or

3. Compositions A, B, and/or C wherein the composition is topically administered to the subject once per week;

wherein

Composition A is a non-aqueous topical composition comprising:

(i) a halogenated salicylanilide selected from niclosamide, rafoxanide, oxyclozanide and closantel, or a pharmaceutically acceptable salt or hydrate thereof; and

(ii) polyethylene glycol (PEG) with a melting point of less than 40°C;

with the proviso that when the composition comprises niclosamide or rafoxanide, or a pharmaceutically acceptable salt thereof, the composition further comprises a gel forming agent or a non-polymeric glycol;

Composition B is a non-aqueous topical composition comprising:

(ii) greater than 60 % by weight of a polyethylene glycol (PEG), wherein the average molecular weight of the PEG is 600 or less.

Composition C is a non-aqueous topical gel composition comprising:

(i) a halogenated salicylanilide selected from niclosamide, rafoxanide, oxyclozanide and closantel, or a pharmaceutically acceptable salt or hydrate thereof;

(ii) greater than 60 % by weight of a PEG, wherein the average molecular weight of the PEG is 600 or less; and

(iii) a gel-forming agent.

[00317] In some embodiments the topical composition is not a non-aqueous topical composition comprising a halogenated salicylanilide selected from niclosamide, rafoxanide, oxyclozanide and closantel, or a pharmaceutically acceptable salt or hydrate thereof; and polyethylene glycol.

[00318] In certain embodiments the topical composition comprising the halogenated salicylanilide (e.g. niclosamide or oxyclozanide) does not contain DMSO. [00319] In certain embodiments the composition comprising the halogenated salicylanilide (e.g. niclosamide or oxyclozanide) is not one of the compositions disclosed in WO

2019/053180.

[00320] In certain embodiments the topical composition comprising the halogenated salicylanilide (e.g. niclosamide or oxyclozanide) is not Composition W, Composition X or Composition Y:

Composition W: a topical veterinary spot on or line on composition comprising 2 to 20 wt/v % of at least one halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof, 35 to 55 wt/v % dimethyl sulfoxide, wherein the at least one halogenated salicylanilide is selected from niclosamide and/or oxyclozanide and wherein the composition is dissolved in diethylene glycol monomethyl ether.

Composition x: a topical veterinary spot on or line on composition comprising 2 to 20 wt/v % of at least one halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof, 35 to 55 wt/v % dimethyl sulfoxide, wherein the at least one halogenated salicylanilide is selected from niclosamide and/or oxyclozanide and wherein the composition is dissolved in diethylene glycol monoethyl ether (Transcutol).

Composition Y: a composition selected from Table A above.

[00321] In certain embodiments the topical composition is not Composition D, E, or F for use in the treatment or prevention of pyoderma or dermatitis in a non-human mammal.

[00322] In certain embodiments the topical composition is not Composition D, E, or F for use in the treatment or prevention of pyoderma or dermatitis in a non-human mammal wherein the composition is topically applied to the non-human mammal as a single application optionally repeated a number of times every 5 to 10 days, for example once every 5 to 10 days for 3 to 5 consecutive weeks.

Dosages

[00323] The halogenated salicylanilide is topically applied during the initial treatment period in an amount that is sufficient to provide an amount of the halogenated salicylanilide in the skin tissue which is sufficient to treat the skin condition, for example to relieve the subject of one or more of the symptoms of an inflammatory skin condition and/or to eradicate the bacteria responsible for the skin infection.

[00324] Generally the halogenated salicylanilide will be topically administered to the subject in the form of a topical composition. The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the subject being treated and the particular skin condition which is being topically treated with the halogenated salicylanilide. Generally, each dose of the topical composition comprising the halogenated salicylanilide administered to the subject during the initial treatment period will be in an amount sufficient to cover the areas of the skin affected by the skin condition. Suitably the composition is topically applied in an amount to such that each topical administration during the initial treatment period provides a dose of the halogenated salicylanilide of from about 0.001 to about 1 mg/cm²; about 0.01 to about 0.5mg/cm²; about 0.01 to about 0.5 mg/cm² or about 0.01 to about 0.3 mg/cm², for example about 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 1 , 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8,

1.9, 2.0, 2.1 , 2.2, 2.3, 2.4 or 2.5 mg/cm². In some embodiments the halogenated salicylanilide (e.g. oxyclozanide) is topically administered during the initial treatment period such that each topical administration of the halogenated salicylanilide is in a dose of from 0.1 mg. kg to 100 mg/kg (e.g. from 1 to 50 mg/kg, from 1 to 40 mg/kg, from 1 to 30 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg or about 25 mg/kg). Each application of the composition is generally topically applied in an amount sufficient to provide the desired dose of the halogenated salicylanilide. This will of course depend on the concentration of the halogenated salicylanilide in the composition. Typically the composition will be applied in an amount such that each topical administration during the initial treatment period in in an amount of about 0.1 to about 50 mg/cm²; about 1 to about 20 mg/cm²; about 1 to about 5 mg/cm² about 2 to 5 mg/cm²; about 2 to about 15 mg/cm² or about 4 to about 10 mg/cm².

[00325] In some embodiments the halogenated salicylanilide is topically administered to the subject is a dose of 0.5 to 5 ml per 10 kg of body weight, preferably, 1 to 3 ml per 10 Kg of body weight, even more preferably, about 2 ml per 10 Kg of body weight of the subject. For example, in this embodiment the halogenated salicylanilide is administered as a spot on / pour on / line on composition (for example a spot-on, line-on or pour-on composition described herein comprising the halogenated salicylanilide at a concentration of e.g. 2 to 20 % wt/v, preferably 5 to 15 % wt/v, more preferably, 8 to 12 % wt/v). The composition is suitably formulated as a unit dose adapted to the weight and / or size of the non-human mammal. Preferably the entire dose is topically applied to the animal as a single“spot” or“line”.

[00326] Suitably the topical composition and is gently rubbed into the skin at the sites affected by the skin condition to be treated to provide coverage of substantially all of the skin affected (e.g. to cover a dermatitis lesion or an area of infected skin). Optionally a composition comprising the halogenated salicylanilide may be topically applied to the subject using a suitable carrier substrate, for example a wound dressing or a patch impregnated with or carrying a composition comprising the halogenated salicylanilide. The carrier may be applied to a lesion such that the lesion is brought into contact with the halogenated salicylanilide present in or on the carrier substrate.

Subjects

[00327] the subject topically treated with the halogenated salicylanilide using the dosage regimen of the invention is a non-human subject. The subject may be a warm blooded non-human mammal. In some embodiments the subject is a commercial animal such as livestock (e.g. cows, sheep, chickens, pigs, geese, ducks, goats, etc.). In other embodiments subject is a companion animal such as a cat, dog or horse. In some embodiments the subject is a dog or a cat. In a particular embodiment the subject is a dog.

[00328] In some embodiments the subject is a dog and the skin condition is selected from canine atopic dermatitis, flea allergy dermatitis, scabies, malassezia dermatitis, intertrigo, pododermatitis, demodicosis, contact dermatitis and canine bacterial pyoderma.

[00329] In some embodiments the subject is a cat and the skin condition is selected from flea allergy dermatitis, atopic dermatitis, food allergic dermatitis otodectic acariasis and feline bacterial pyoderma.

EXAMPLES

Example 1 : Non Aqueous Topical Niclosamide Formulations

Non Aqueous Topical Niclosamide Gel Formulation

[00330] The topical gel compositions shown in Table 1 were prepared:

Table 1

[00331] The composition was prepared as follows. Niclosamide 200 mg, PEG 400 (9.56 g for Formulation A and 9.36 g for Formulation B) were weighed in blue cap bottles. The mixture was stirred at room temperature until a clear solution formed. 240 mg. Carbomer 974P was then dispersed in the niclosamide PEG 400 solution. The dispersion was homogenized and degassed. The suspension was then heated at 70 °C and stirred mechanically at 250 rpm until a homogeneous dispersion formed after about 30 minutes. The final solution was then cooled to give the title non-aqueous gel compositions.

[00332] The final formulations were protected from light prior to further use.

Further Non-Aqueous Topical compositions

[00333] The non-aqueous topical compositions shown in Tables 2 and 3 were prepared:

Table 2

Table 3

[00334] The ointment formulations D, E, F, G, H, I and J set out in Tables 2 and 3 were prepared as non-aqueous emulsions using the following general method.

[00335] The hydrophilic phase of the emulsion and the anhydrous niclosamide (see under heading“hydrophilic phase” in Tables 2 and 3) were mixed together with stirring in a vessel to form a solution of the niclosamide in the hydrophilic phase. Generally the hydrophilic phase was heated gently at a temperature of about 60 to 75°C (generally at about 70°C) to aid dissolution of the niclosamide.

[00336] A hydrophobic phase comprising the oils and emulsifiers under the heading “Hydrophobic phase and emulsifiers” were mixed together by stirring in a heated vessel. The temperature was about 60 to 75°C (generally at about 70°C).

[00337] The hydrophobic phase and the hydrophilic phases were mixed together with gentle stirring so as to avoid phase separation and the mixture was cooled to a temperature of about 40 to 50°C. The mixture was then homogenised to give the final composition.

[00338] The appearance and some of the properties of the resulting compositions is described in the row marked“Appearance” in Tables 1 to 3.

Spot-on or Line-on formulations

Compositions A to G shown in Table B below were prepared:

Table B

Transcutol P is diethylene glycol monomethyl

also known as 2-(2- ethoxyethoxy)ethanol.

[00339] The compositions may be prepared be dispersing the oxyclozanide into the

DMSO, Transcutol and optional monoethanolamine with stirring to form a solution.

Example 2: Dermal Tolerance and PK Study in Mini-pigs

Aim

[00340] The aim of the study was to assess the local tolerability of the non-aqueous dermal niclosamide formulations B, D, E, F, G, H, I and J in Tables 1 to 3. The study also assessed the distribution of niclosamide into the dermis and epidermis after 2 days and 28 days of topical application and also 14 days after the last topical application of niclosamide (i.e. 42 days after the start of the study).

Animals

[00341] The study was performed in 3 male and 3 female Gottingen SPF mini-pigs from

Ellegaard Gottingen Mini-pigs A/S, DK-4261 Dalmose, Denmark. At the start of the acclimatization period, the animals were approximately 6 months old and the body weight was 12.1 to 13.4 kg. An acclimatization period of at least 1 week was allowed, during which the animals were observed daily.

Dosing [00342] The compositions were applied dermally to a test area on the skin of the mini-pigs at an applied daily dose of 10 mg/m² niclosamide for 28 days. All test fields were semi- occluded with Mefix® (Molnlycke Health Care, Sweden), allowing the sites to“breathe” while keeping the test substance in place. The Mefix® was kept in place with adhesive tape (Tensoplast, BSN medical, Germany) for 6 hours ± 30 minutes after application to ensure that the compounds were absorbed into the skin.

Skin Biopsies and PK Analysis

[00343] Skin biopsies were taken from the mini-pigs on Day 28. Subdermal tissue was removed and the biopsies were placed in an Eppendorf tube and immersed in a 55 °C water bath for 5 minutes. After that, each biopsy was divided between the epidermal and dermal layer of the skin with tweezers. The weight of the dermal biopsy was recorded and the epidermal biopsy weight was estimated from weighing of epidermal slices from 6 mm biopsies of pig skin. The epidermal and the dermal biopsy samples were placed in separate Eppendorf tubes and snap-frozen in liquid nitrogen prior to analysis. The skin biopsies were analysed using a validated HPLC method to assess the concentration of niclosamide in the dermis and epidermis.

Blood Sampling

[00344] Blood samples were also taken at day 28 and plasma from the blood was analysed for niclosamide concentration.

Skin Reactions

[00345] Reactions at the application sites were observed daily after dosing and on the day of necropsy. Any dermal irritation was scored according to the OECD Guideline for Testing of Chemicals No. 404, adopted 24th April, 2002: "Acute Dermal Irritation/Corrosion" as follows:

Any other reaction was recorded and the area involved scored as follows;

Results

Skin Reactions

[00346] All dermal formulations tested were well tolerated in all mini-pigs following repeated dosing for 28 days.

[00347] In a comparison of the results of all the tested topical formulations, the following observations were made. [00348] The scoring of skin reactions revealed that formulation B (non-aqueous gel formulation) only caused slight erythema one day in one pig (Pig No. 6) indicating that Formulation B was extremely well tolerated.

[00349] Formulations F and G only revealed scores up to well defined in 3 pigs (Pig Nos. 1 , 2, and 3) and up to slightly in 3 pigs (Pig Nos. 4, 5, and 6), meaning that all pigs were affected to a minor degree by formulations F and G. Formulation I affected three pigs (Pig Nos. 1 , 4, and 5) up to slight erythema, while two pigs (Pig Nos. 2 and 3) were affected up to a moderate degree in erythema.

[00350] Formulations D, E, H, and J showed scores of up to moderate in all pigs, however the female pigs would never exceed scores of slight erythema. None of the formulations caused edema in any of the pigs throughout the study.

[00351] In conclusion, the anhydrous formulations dosed topically daily for 28 days in the local tolerance study in 3 female and 3 male mini-pigs revealed erythema macroscopically of slight to a moderate degree, however the erythema disappeared within 9 days of the last dosing. Additionally histopathology did not find any observable reactions caused by application of the formulations. Gel formula B was particularly well tolerated.

Bioanalysis in the Dermis, Epidermis and Plasma

[00352] Bioanalysis showed that niclosamide was mainly located in the epidermis, and the dermis, with only a slight concentration in the plasma (generally less than 0.651 ng/mL), indicting a highly local effect. The formulations that were observed to have the highest exposure in the epidermis and dermis, were formulations B (all pigs) and H (in four pigs). The mean concentration of niclosamide in the dermis and epidermis at day 2, day 28 and day 42 of the study are shown in Table 4:

Table 4

[00353] The data in Table 4 shows that very high concentrations of niclosamide were achieved in the skin after just 2 days of dosing. The concentration of niclosamide was similar at Day 2 and Day 28 during the period where the niclosamide was being topically applied to the pigs. The day 42 data shows that 14 days after the last dose of topical niclosamide was administered a high concentration of niclosamide was retained in the dermis and the epidermis.

[00354] The highest in-vitro MIC of niclosamide for S. aureus measured by the inventors is about 0.5 pg/mL, which corresponds to about 0.5 pg/g. Table 4 shows that the niclosamide concentration in the dermis and epidermis was well in excess of the MIC in both the dermis and the epidermis after just 2 days of topical administration of niclosamide. The data also shows that the concentration of niclosamide was higher than the MIC in the epidermis 14 days after administration of the last topical dose of niclosamide.

Example 3: Skin absorption of the 2% niclosamide gel composition (Formulation A) in human skin

[00355] This study assessed the absorption of niclosamide in human cadaver skin following application of the 2% niclosamide gel composition - Formulation A in Table 1.

Methods

Tissue source

[00356] Human cadaver skin tissue was purchased from a tissue bank in the U.S. These tissues are recovered within 24 hours of death. Three lots of the dermatomed skin tissue were received. Donor demographics: tissue 4838 (average thickness = 537 pm): sex = male, age = 31 , race = Caucasian and anatomical site = abdomen; tissue 4830 (average thickness = 610 pm): sex = male, age = 53, race = Caucasian and anatomical site = abdomen; tissue 4950 (average thickness = 633 pm): sex = male, age = 46, race =

Caucasian and anatomical site = abdomen). The tissue was received in dry ice packaging. Tissue 4830 and tissue 4848 were stored in the freezer (-20 °C) for about 4.5 months; tissue 4950 was stored in the freezer (-20 °C) for about 3.5 months. All tissues were wetted with a Zyleris' proprietary storage medium to preserve barrier integrity during the storage. It was stored at -20 °C until use.

Skin barrier integrity testing

[00357] After passing initial visual inspection, barrier integrity of the skin tissue was evaluated using trans-epidermal electrical impedance measurement (TEER measurement, Z value). [00358] Measurement was conducted using a LCR meter at frequency of 100 Hz at room temperature. The measurement medium was 0.9% NaCI solution using a pair of stainless steel electrodes.

[00359] Prior to the screening study, electrical impedance value for the lot of skin tissue was evaluated. Due to potential lot-to-lot variation in donor age, gender, race, anatomical site and tissue harvest time, objective of the measurement was to establish threshold value for intact cadaver tissue for the lot to be used in the present skin absorption and penetration study. Twelve (12) tissue samples from selected spots from the lot were taken. Electrical impedance value (Z value) was measured. All reported Z values are net values after subtraction of the measurement medium blank (1.0 KOhms). It was found that large majority of the tissues for # 4848 and 4950 had Z value 5.0 KOhms or greater; and for 4830 had Z value 7.0 KOhms or greater. For those tissues with visible defects, the Z value was found in the range of 0.1 -0.5 KOhms. Therefore, 5.0 and 7.0 KOhms was used as threshold value for the three lots.

[00360] After the tissues were mounted on High-Throughput Screening (HTS) cells, skin integrity was measured before incubation at 32 °C. Any tissue having Z value lower than 5.0 or 7.0 KOhms was discarded.

In vitro percutaneous absorption and penetration study

[00361] The study was carried out in Zyleris’ High-Throughput Screening (HTS) station. This is based on the Franz Cell principle. The station includes an 81 -cell screening station with the ability to conduct various types of skin absorption and penetration studies in each individual cell. It is fully validated against the standard Franz Cell technology. The skin samples after being washed with 1x phosphate buffer solution (PBS), pH 7.4 (10 mM phosphate, 137 mM NaCI, 2.7 mM KCI, pH 7.4) were mounted on diffusion cells in HTS station.

[00362] A total of 18 diffusion cells were used in the study. Each cell in the station has a diffusion area of 0.503 cm² (8 mm in diameter). Each individual cell is static Franz-Cell type. The receptor chamber was filled with 3.0 ml of 4% Bovine Serum Albumin (BSA) in water supplemented with 0.01% gentamicin sulfate. pH 7.1 , which was vigorously and continuously mixed. The rational for using this medium is that 4% BSA can mimic the protein level in the blood and can easily be removed before analysis. In addition, this medium with 0.01% gentamicin is best in maintaining the skin integrity through the experiments, compared with normal PBS buffer. The temperature was set at 32±0.1 °C. The tissue samples in the HTS cells were equilibrated at 32±0.1 °C for 1 hour before dosing. Each formulation was run in six replicates (N=6). All laboratory activities with niclosamide were performed under yellow lights (l=570 nm), because niclosamide is yellow and can absorb UV lights (l=290-450 nm).

[00363] At time points 2, 4, and 8 hours, the entire receptor fluid was collected and replaced with a fresh batch of receptor fluid (pre-incubated at 32°C). The samples collected from the receptor medium were stored in a freezer (-20 °C).

[00364] The dosing level was set at 5.0 mg per cell. The tested formulations were applied using a positive displacement pipette based on volume calculated from its density. The density of the 2% niclosamide gel (Formulation A in Table 1 ) had a density of 1 0154±0.1074. Total dosed weight for each the formulation was measured by weighing before and after dosing the six diffusion cells. The average dosing level was calculated by total measured weight divided by 6 (Table 5).

Table 5: Applied dose of Gel Formulation A

[00365] 24 hours, after removal of the skin tissue from the HT Franz Cell, the tissue surface and the donor chamber were carefully wiped with cotton swabs wetted by distilled water, followed by wiping with wet cotton swabs, then another cycle of wet cotton swabs to remove the“unabsorbed and unpenetrated” niclosamide. A standard tape-stripping method was used to remove the stratum corneum (SC) layer. The first two tape strips were also counted as“non-absorbed API. The collected cotton swabs and first two strips were combined and extracted with 5.0 ml of DMSO/acetonitrile (50/50 v/v) at room temperature overnight using an orbit shaker.

[00366] The tape-stripping cycle was continued for additional 15 times for each skin sample to remove SC layer completely. The tape strips were combined and extracted with 5.0 ml of DMSO/acetonitrile (50/50 v/v) at room temperature overnight using an orbital shaker. For each stripping cycle, all applied tapes/tissues were lined up on a Teflon plate. Then a hard rubber-lined roller was pressed against the tapes/tissues to apply equal pressure across all samples to ensure uniformity for each cycle and entire procedure. Then, the tape strip was removed in a quick motion from each tape/tissue pair. The tape strip was then collected. The extracts were collected and stored in a freezer (-20 °C). [00367] After removal of SC layer, the remaining tissue was cut into small pieces and extracted with 5.0 ml of DMSO/acetonitrile (50/50 v/v) at room temperature overnight using an orbital shaker. The supernatants were collected and stored in a freezer (-20 °C). The receptor fluid was collected and stored in a freezer (-20 °C).

Drug penetration profile after 24 hour application

[00368] The distribution of niclosamide in the skin samples after 24 hours, i.e. the amount expressed as ng and % of the applied dose present in receptor fluid, viable epidermis and dermis and stratum corneum was determined. The amount of niclosamide on skin surfaces and on the walls of the donor chambers was also determined, and was considered to be non-penetrated drug.

[00369] The analysis was performed using an LC-MS-MS method. The LC system consisted of an advance liquid chromatograph (Bruker, Fremont, CA, USA) equipped with a solvent delivery compartment with high-pressure mixing, a column compartment and a CTC an autosampler. The injection volume was 1 pi. Separation of compounds was performed on a Zic-cHILIC (Merck sequant) 2.1^*100 mm; particle size 3 pm (Merck sequant, Umea, Sweden). In front of the separation column was a Phenomena krudkatcher filter, 0.5 pm. The total flow rate of eluent A (20 mM ammonium formate adjusted to pH 3.5 (with formic acid) and B (acetonitrile) was 0.4 ml/min. The initial gradient was 95% B and held for 1 min, decreasing to 50% B after 3 min, then back to 95% B after 0.1 min and held at 95% B 1.4 min. The total run time was 4.5 min.

The column temperature was set at 40°C.

[00370] The MS-MS detection was performed on an EVOQ triple quadrupole instrument (Bruker, Fremont, CA, USA) equipped with an atmospheric pressure ionization (API) interface. The mass spectrometer was operated with electrospray both in the negative-ion mode (ESI-).

[00371] The average of the results for Formulation A are shown in Tables 6 and 7

Table 6. Average drug penetration profile of niclosamide through human skin after applying Formulation A for 24 hr (n=6)

Table 7 Average niclosamide concentration in the stratum corneum, and the dermis and epidermis

Active drug exposure in the human skin tissue

[00372] The average thickness of stratum corneum layer of adult abdomen is about 22.38 pm (Holbrook, K. A.; Odland, G. F., Regional differences in the thickness (cell layers) of the human stratum corneum: an ultrastructural analysis. Journal of Investigative

Dermatology 1974, 62 (4), 415-422.). The thickness of the epidermis and dermis was calculated by subtracting the stratum corneum layer from the thickness of the whole piece of skin (Table 8). Thus, the volume of stratum corneum and epidermis plus dermis can be calculated. The concentrations of niclosamide in different skin layers can be calculated by using the deposited niclosamide amount and the calculated volume. As shown in Table 8, approximately 10,000 times the MIC of niclosamide was present in the stratum corneum layer after applying the 2% niclosamide gel Formulation A (220.17 pg/cm²) for 24 hours.

[00373] 688 times the MIC of niclosamide was present in the epidermis and dermis after applying Formulation A (220.17 pg/cm²). These results suggest that gel Formulation A will provide a highly potent topical antibacterial composition.

Table 8 Average values of active drug exposure in human skin tissue after applying Formulation A for 24 hr (n=6)

1 , The MIC mentioned above was for a methicillin-resistant staphylococcus aureus (MRSA) strain which had a niclosamide MIC of 250 ng/ml_.

Example 4: Clinical Trial to Assess the Safety Topically Applied Niclosamide in Healthy Volunteers Study design

[00374] A prospective, single centre, randomized, double-blind, Placebo controlled study.

Primary Objective of the Trial

[00375] The primary objective of the study is to demonstrate the safety and tolerability of topical niclosamide formulations in healthy volunteers.

Secondary Objectives:

• To determine the local and systemic exposure of the topical niclosamide

composition.

Exploratory Objective:

• To collect illustrative information on local tolerability of the topical niclosamide composition.

• To determine the best tolerated formulation to advance into further development.

[00376] Randomization ratio 1 :1 ; randomized niclosamide composition or Placebo application on right or left arm.

Inclusion criteria:

• Signed and dated informed consent has been obtained.

• Age 18 - 70 years.

• Male or female.

• Female subjects of childbearing potential must be confirmed not pregnant by a negative urine pregnancy test prior trial treatment.

• Female subjects of childbearing potential must be willing to use effective

contraceptive at trial entry until completion.

• Male subjects must agree to use adequate contraception for the duration of the trial.

Exclusion criteria:

• Regular use of medications unless considered clinically irrelevant by the

Investigator.

• Use of any dermatological drug therapy on the arms within 14 days before day 1 of this study.

Protocol

[00377] The study comprised a group with 30 healthy volunteers. Each of these volunteers were treated in four separate areas two times daily with the niclosamide topical formulations or the vehicle controls during a seven-day period. [00378] The following topical niclosamide formulations were tested:

2% niclosamide non-aqueous dermal gel: Formulation A described in Table 1 above 2% niclosamide non-aqueous dermal cream: Formulation G described in Table 2 above

[00379] For each arm of the trial a placebo formulation comprising the vehicle only (i.e. without the niclosamide) was also tested.

Dosage and Administration

Route of administration: topical.

Duration of treatment: 7 days.

[00380] Each volunteer had 4 formulations (2 active formulations and their respective Placebos) applied to defined skin areas in the dorsal arms. The body area to be treated was a circle marked by a skin marker with a diameter of 5 cm (approx. 20 cm²). The healthy volunteers had the body areas treated two times per day, at 08:00 (+/- 2 hours) and 20:00 (+/- 2 hours), respectively for 7 days. The expected dose of each formulation was 2 to 5 mg of product/cm²/day (corresponding to 0.04-0.1 mg niclosamide/cm²). The dermal formulation was left to dry for 10 minutes after application.

[00381] A screening visit was performed at Day 0. At Day 1 the patients were

randomized, and this was also be the first day of treatment. On days 1-7 the healthy volunteers were treated twice daily at the study site. On Day 8 a final dose was applied in conjunction with the PK analysis. A final examination (end of study) was made on Day 15.

[00382] Six additional healthy volunteers were also enrolled for method testing.

Treatments were blinded to both volunteers and doctors. The body area to be treated was be a circle with a diameter of approximately 5 cm and the expected dose of 2-5 mg of product/cm² (0.04-0.1 mg active substance per cm²).

[00383] The healthy volunteers in the trial were also subjected to a PK analysis after the last dose. The PK analysis involved sampling of blood after the final exposure to assess systemic exposure to niclosamide and skin biopsy sampling to assess local exposure to niclosamide in the skin. The 30 healthy volunteers were randomized for single punch biopsies to collect 10 biopsy samples from each active formulation. This meant that 1 active treatment area for each healthy volunteer had to be unblinded prior to biopsy sampling. To ensure that this did not interfere with the blind assessment of the safety of the formulations, safety was assessed in the morning of day 8, then on day 8 a 15th dose was given in conjunction with the bioanalysis. Biopsies were taken 1 h (+/- 10 min) after application of the respective formulation. Punch biopsies

[00384] The skin biopsies were taken using sterile single use disposable biopsy punches (BP40F, Kai Europe GmbH, Solingen, Germany). For the 6 non-treated healthy volunteers biopsies were taken on Day 1. 10 mL of blood was collected at day 1 for the method validation group to determine niclosamide concentration in the blood.

[00385] For the 30 treated healthy volunteers biopsies were taken on day 8 one hour after the 15^th application (+/- 10 minutes).

[00386] The concentration of niclosamide in the skin biopsy samples was determined using validated bioanalytical UPLC-MS/MS methods.

[00387] The following chromatographic conditions were used:

[00388] Mass spectrometry was performed using a Shimadzu 8050 mass spectrometer operating in Electrospray negative mode (ESI ^ve).

Preparation of skin biopsy samples containing niclosamide

[00389] Extraction of skin biopsy samples was performed as follows:

1. Cut the tissue into small pieces and extract with 5.0 ml of DMSO/acetonitrile (50/50 v/v) at room temperature overnight using a shaker. 2. Spin down the tissue at 3700 g, collect supernatant and store it in a freezer (-20°C). Determination of niclosamide concentration in skin biopsies

[00390] 50 pi of untreated human skin extract was spiked with 10 mI of working standard solution (concentration of standard will be provided for each parameter). Samples were vortexed, then 200 mI of methanol/water solution 1 ;1 (v:v) was added. Finally, samples were centrifuged for10 minutes at 4°C at 2000 g. The supernatant was transferred into HPLC plate and analysed using UPLC-MS/MS.

Assessment of local tolerability

[00391] Local dermal tolerability at the sites of application of the topical formulations was assessed by the investigator at all treatment visits using an 8-point dermal assessment score, in accordance with the FDA guideline on Skin Irritation and Sensitization Testing (1999). A dermal assessment score of 0 to 7 was defined as follows:

0 = no evidence of irritation,

1 = minimal erythema, barely perceptible,

2 = definite erythema, readily visible; minimal edema or minimal papular response,

3 = erythema and papule,

4 = definite edema,

5 = erythema, edema, and papule,

6 = vesicular eruption,

7 = strong reaction spreading beyond test site.

Results

[00392] All of the topical dermal niclosamide formulations and placebo formulations were well tolerated with no signs of adverse reactions at the sites of administration. All 6 investigational medicinal products were scored at 0 in all subjects at all time points, see Table 9.

Table 9: Mean Local Tolerability Scores at Treatment Visits

[00393] Systemic exposure to niclosamide was minimal with mean serum concentration of niclosamide 0.24 ng/mL, while local exposure to the skin was substantial (see Table 10). Table 10: Niclosamide Concentration in Skin Biopsies

[00394] The results of the study showed that the dermal niclosamide and placebo formulations tested were well tolerated locally with no signs of adverse reactions at the sites of administration. No safety concerns were identified and formulations delivered therapeutically relevant concentrations to the skin with minimal systemic exposure.

[00395] The data in Table 10 shows that high concentrations of niclosamide were observed in human skin which are well in excess of the in-vitro MIC of niclosamide against S. aureus.

Example 5: A Double-blind, Randomized. Intraindividual Vehicle-Controlled, Phase

Study to Evaluate Efficacy and Safety of Topically Applied Niclosamide in Human Patients with Moderate Atopic Dermatitis

The topical niclosamide Formulation G, as described in Table 2 above, was tested in the following clinical trial.

Rationale for the study Wu et al (2014, Ibid) reports that niclosamide exhibited anti-inflammatory properties in vitro by modulating the activation of dendritic cells and repressing the expression of proinflammatory cytokines. This study will investigate whether niclosamide possesses anti- inflammatory properties capable of translating into a therapeutic effect on the signs and symptoms of atopic dermatitis.

Study Design

31 patients with moderate atopic dermatitis (Investigator Global Assessment [IGA] of 3) were included in this double-blind, randomized, intraindividual vehicle-controlled, Phase 2 study to evaluate the efficacy and safety of topically applied niclosamide. Patients had at least 2 areas of at least 3 x 3-cm of atopic dermatitis with a Total Sign Score (TSS) of >5. 2 patients discontinued the study before Day 22.

Patients received topical applications of a niclosamide 2% composition and vehicle once daily for 3 weeks, followed by a 1-week follow up period. Topical niclosamide 2% and vehicle was applied on two separate target lesions of atopic dermatitis (lesions of at least 3 x 3-cm that are at least 2 cm apart, excluding the face, scalp, genitals, hands, and feet). The application areas (5 ^c 5-cm) were randomized (1 :1 ) to once daily application of niclosamide 2 % or vehicle at 5mg/cm² without occlusion 6 days per week. Patients came to the study site for all study product application for a total of 3 weeks.

Efficacy was assessed using Total Sign score (TSS) and Treatment Areas Assessment (TAA). Safety was assessed with vital signs, physical examination, clinical laboratory tests (haematology; biochemistry; urinalysis), and by collecting adverse events (AEs).

Three skin biopsies were collected in all patients (one from lesional skin at baseline, pre- dosing at Day 1 , and two from lesional skin at Day 22 (one where topical niclosamide 2% had been applied and one where the vehicle had been applied). The lesional skin biopsies were analysed for skin thickness and inflammation biomarkers.

STUDY ENDPOINTS Primary endpoint:

• Number of local and systemic treatment-emergent adverse events (AEs) in each treatment group (during 34 days).

Secondary endpoints:

• Change from baseline (pre-dosing at Day 1 ) in lesional TSS at Days 8, 15 and 22.

• Change from baseline (pre-dosing at Day 1 ) in lesional T reatment Areas Assessment (TAA) at Days 8, 15 and 22, for area randomized to topically applied niclosamide 2%, as compared with vehicle.

• Change from baseline in skin barrier and biomarker levels at Day 22, for area randomized to topically applied niclosamide 2%, as compared with vehicle.

Inclusion criteria

Patients will be eligible for participation in the study if they meet all the following inclusion criteria at the screening and baseline (pre-dosing at Day 1 ) visits, unless specified otherwise:

1. Man or woman 18 years of age or older at the time of consent.

2. Patient has clinically confirmed diagnosis of active atopic dermatitis, according to Hanifin and Rajka criteria (Hanifin et al.“Diagnostic feature of atopic dermatitis", Acta. Derm. Ven. vol 92, (suppl):44-47, 1980).

3. Patient has at least a 6-month history of atopic dermatitis and had no significant flares in atopic dermatitis for at least 4 weeks before screening (information obtained from medical chart or patient’s physician, or directly from the patient).

4. Patient has moderate atopic dermatitis at baseline (pre-dosing at Day 1 ), as defined by an IGA of 3.

5. Patient has at least two areas of atopic dermatitis (excluding face, scalp, genitals, hands, and feet) of at least 3 x 3 cm; with a TSS of at least 5 at baseline (Day 1 ). These areas should be at least 2 cm apart.

6. For patient (man and woman) involved in any sexual intercourse that could lead to pregnancy, patient agrees that an effective contraceptive method will be used, from at least 4 weeks before baseline (Day 1 ) until at least 4 weeks after the last study product administration. Effective contraceptive methods include hormonal contraceptives (combined oral contraceptive, patch, vaginal ring, injectable, or implant), intrauterine devices or intrauterine systems, vasectomy, tubal ligation, or a barrier method of contraception (male condom, female condom, cervical cap, diaphragm, contraceptive sponge) in conjunction with spermicide. Note: Hormonal contraceptives must have been on a stable dose for at least 4 weeks before baseline (Day 1 ).

Note: Woman of nonchildbearing potential is as follows: Woman who has had surgical sterilization (hysterectomy, bilateral oophorectomy, or bilateral salpingectomy)

Woman >40 years of age who has had a cessation of menses for at least 12 months and a follicle-stimulating hormone (FSH) test confirming nonchildbearing potential (refer to laboratory reference ranges for confirmatory levels) or cessation of menses for at least 24 months without FSH levels confirmed

7. For woman of childbearing potential, has had a negative serum pregnancy test at screening and negative urine pregnancy test at baseline (Day 1 ).

8. Patient is willing to participate and is capable of giving informed consent. Note:

Consent must be obtained prior to any study-related procedures.

Exclusion criteria

Patients will not be eligible for participation in the study if they meet any of the following criteria at the screening and baseline (Day 1 ) visits, unless specified otherwise:

1. Patient is a woman who is breastfeeding, pregnant, or who is planning to become pregnant during the study.

2. Patient has clinically infected atopic dermatitis.

3. Patient has a Fitzpatrick’s Skin Phototype >5.

4. Presence of any tattoos, scratches, open sores, excessive hair, or skin damages in the target lesion areas that in the opinion of the investigator may interfere with study evaluations.

5. Patient is known to have immune deficiency or is immunocompromised.

6. Patient has a history of cancer or lymphoproliferative disease within 5 years prior to baseline (Day 1 ). Patients with successfully treated nonmetastatic cutaneous squamous cell or basal cell carcinoma and/or localized carcinoma in situ of the cervix are not to be excluded.

7. Patient had a major surgery within 8 weeks prior to baseline (Day 1 ) or has a major surgery planned during the study.

8. Patient has any clinically significant medical condition or physical/laboratory/vital signs abnormality that would, in the opinion of the investigator, put the patient at undue risk or interfere with interpretation of study results. 9. Patient has a known history of chronic infectious disease (e.g., hepatitis B, hepatitis C, or infection with human immunodeficiency virus).

10. Patient has used hydroxyzine or diphenhydramine within 1 week prior to Day 1.

1 1. Patient has used dupilumab within 12 weeks prior to Day 1.

12. Patient has received any nonbiological investigational product or device within 4 weeks prior to Day 1

13. Patient has used crisaborole and any other topical PDE-4 inhibitor within 4 weeks prior to Day 1.

14. Patient has used doxepin within 1 week prior to Day 1.

15. Patient has used topical products containing urea on target areas within 1 week prior to baseline (Day 1 ).

16. Patient used nonurea-containing emollient anywhere on the body from 1 day before Day 1.

17. Patient has used systemic antibiotics within 2 weeks or topical antibiotics on target areas within 1 week prior to baseline (Day 1 ).

18. Patient has used any topical medicated treatment for atopic dermatitis within 1 week prior to baseline (Day 1 ), including, but not limited to, topical corticosteroids, calcineurin inhibitors, tars, bleach, antimicrobials, medical devices, and bleach baths.

19. Patient has used systemic treatments (other than biologies) that could affect atopic dermatitis less than 4 weeks prior to baseline (Day 1 ) (e.g., retinoids, calcineurin inhibitors, methotrexate, cyclosporine, hydroxycarbamide [hydroxyurea], azathioprine, oral/injectable corticosteroids). Note: Intranasal corticosteroids and inhaled corticosteroids for stable medical conditions are allowed if patient has been on a stable dose for at least 4 weeks prior to baseline (Day 1 ) and will continue usage at the same dose for the duration of the study. Eye drops containing corticosteroids are allowed.

20. Patient has received any marketed or investigational biological agent within 12 weeks or 5 half-lives (whichever is longer) prior to baseline (Day 1 ).

21. Patient has excessive sun exposure, is planning a trip to a sunny climate, or has used tanning booths within 4 weeks prior to baseline (Day 1 ), or is not willing to minimize natural and artificial sunlight exposure during the study. Use of sunscreen products and protective apparel are recommended when exposure cannot be avoided. 22. Patient has a known or suspected allergy to niclosamide or any component of the formulation to be tested.

23. Patient has a known history of clinically significant drug or alcohol abuse in the last year prior to baseline (Day 0).

24. Patient has a history of an allergic reaction or significant sensitivity to lidocaine or other local anaesthetics.

25. Patient has a history of hypertrophic scarring or keloid formation in scars or suture sites.

26. Patient is taking anticoagulant medication, such as heparin, low molecular weight (LMW)-heparin, warfarin, antiplatelets (nonsteroidal anti-inflammatory drugs [NSAIDs] and low-dose aspirin <81 mg will not be considered antiplatelets), or has a contraindication to skin biopsies.

Diagnosis of AD

Diagnosis of AD in a subject will use the criteria according to Hanifin et al, ibid and set out in the Description of the present application. To be diagnosed with AD the subject should have at least three of the Major Criteria and at least three of the Minor Criteria

Treatment

The study involved a comparison of the niclosamide topical composition with a matching vehicle, administered topically once daily for 3 weeks, without occlusion, at 5 mg/cm²/day (application areas 5 x 5 cm). The niclosamide formulation and placebo vehicle will be applied on two separate target lesions of atopic dermatitis (lesions of at least 3 ^c 3 cm that are at least 2 cm apart, excluding the face, scalp, genitals, hands, and feet). As the chosen target lesion areas are expected to have a significant effect on outcomes, it is important to make a considerable effort to ensure select treatment areas with similar severity to reduce bias. Subjects came to the study site for all study product (active or vehicle) applications.

Efficacy Assessments

Clinical evaluations of atopic dermatitis were performed by an experienced and qualified dermatologist (board certified or equivalent) or other suitably qualified and experienced designee. To assure consistency and reduce variability, the same assessor performed all assessments on a given subject whenever possible.

Eczema Area and Severity Index The Eczema Area and Severity Index (EASI) were assessed pre-dosing (Day 1 ). It quantifies the severity of the atopic dermatitis based on both lesion severity and the percentage of body surface area (BSA) affected. The EASI is a composite score ranging from 0 to 72 that takes into account the degree of erythema, induration/infiltration

(papules), excoriation, and lichenification (each scored from 0 to 3 separately) for each of four body regions, with adjustment for the percentage of BSA involved for each body region and for the proportion of the body region to the whole body. The EASI score calculation is set out in the description.

Body Surface Area

The overall BSA affected by atopic dermatitis was evaluated (from 0% to 100%) -pre- dosing (Day 1 ). For example, one subject’s palm represents 1 % of total BSA.

Total Sign Score (TSS)

The lesional TSS on each of the two treatment areas was assessed pre-dosing (Day 1 ). It quantifies the severity of a subject’s atopic dermatitis based on severity of erythema, edema/papulation, oozing/crusting, excoriation, lichenification, and dryness (each scored from 0 to 3, separately). The lesional TSS is a composite score ranging from 0 to 18. A detailed procedure of lesional TSS score calculation is set out in the description. To be eligible for this study, subjects had a TSS score of >5 pre-dosing (Day 1 ) for each treatment area.

Treatment Areas Assessment (TAA)

The lesional TAA on each of the two treatment areas was assessed at the visits. The lesional TAA grades the severity of disease (each area scored from 0 to 5, separately).

Skin biopsies

Skin barrier and inflammation biomarker levels were determined from lesional skin biopsies from application areas. All subjects had a total of three skin biopsies: one biopsy at Day 1 and 2 biopsies at Day 22 (one where niclosamide was applied and one where the vehicle was applied).

Subjects who discontinued from the study but had completed at least the Day 15 visit, received treatment applications on Days 13 and 14, and received at least 12 applications up to Day 14, inclusively, had a biopsy taken as was planned for Day 22.

The skin biopsy samples were analysed by immunohistochemistry (IHC), and by gene expression studies by RT-PCR using TaqMan Low Density Array (TLDA), and by microarray using Affymetrix U133A Plus 2. The immunohistochemistry (IHC) was used to analyse cell biomarkers. The methodologies as disclosed by Guttman-Yassky et al,“Major differences in inflammatory dendritic cells and their products distinguish atopic dermatitis from psoriasis”, The Journal of Allergy and Clinical Immunology, vol. 1 19, issue 5, pages 1210-1217, 2007, were followed except for that U133A Plus 2-set Gene Chip probe arrays was used instead of U95A-set Gene Chip probe arrays.

TLDA Data analysis

Expression values (threshold cycle [Ct]) were normalized to RplpO by negatively transforming the Ct values to -dCt (IL17A was normalized to hARP, as analysed by qPCR). The undetected -dCt values were estimated for each gene as the 20% of the minimum across all samples. qRT-PCR expression data were modelled using a mixed effect model with Visit and Treatment Area as a fixed effect and a random intercept for each patient. This formulation intrinsically models the within patient correlation structure as in the case of a paired t-test. This approach introduces less bias than restricting the analysis for those patients who completed the study. Contrasts were used to estimate the fold changes with treatment within each treatment group and conduct hypothesis testing.

Microarray Data Analysis

Experimental design: The hybridization strategy was in concordance with experimental design principles, by for example keeping all samples from the same patient in the same date, and always include samples from every treatment arm/group.

Quality Control and Pre-processing : Quality control of microarray chips were carried out using standard QC metrics and R package microarray Quality Control. Expression measures were obtained using GCRMA algorithm (Wu & Irizarry, 2004). Several visual and modelling techniques were used to elucidate if batch effect existed. Principal

Component analysis plots were used to detect if any evident batch effect existed. If such batch effects were found, they were adjusted using Combat, an empirical Bayes method for adjusting data for batch effects that is robust to outliers in small sample sizes (Johnson, Li, & Rabinovic, 2007). The implementation of Combat by package sva was used.

Probe-sets with at least 5% samples with expression larger than 3 (in log2-scale) were kept for further analysis. Expression values were modelled using mixed-effect models with fixed factors Visit and Treatment Area and a random effect for each patient. Fold changes for the comparisons of interest were estimated and hypothesis testing was conducted on such comparisons using contrasts under the general framework for linear models in limma package. The inter-replicate correlation was computed by Duplicate Correlation function and the linear model was estimated by ImFit. P-values from the moderated (paired) t-test were adjusted for multiple hypotheses using the Benjamini-Hochberg procedure, which controls for FDR.

Statistical analysis - TSS and TAA

Continuous variables were summarized in tables and included the number of patients, mean, standard deviation, median, minimum, and maximum. Categorical variables were presented in tables as frequencies and percentages.

The comparison between the treatment groups change from baseline in TSS at Day 22, was done using a paired Student t-test. The difference between treatments was estimated and presented along with a 95% confidence interval.

The other endpoints involving change from baseline were analysed using the same approach as described for the primary endpoint.

Analysis Sets

Data from subjects who were randomized were included in the Intent to Treat (ITT) analysis set. Data from subjects who received at least one administration of study treatment on each lesion were included in the modified ITT (MITT) analysis set. Data were analyzed according to the treatment group to which the subject was randomized.

The Per Protocol (PP) analysis set included data from subjects who were randomized, had no significant protocol deviations effecting the efficacy assessment, and have evaluable data for the primary endpoint.

The Safety analysis set (SAF) was defined as data from subjects who received at least one administration of the study product. Analysis was performed according to the actual treatment subjects received.

Efficacy Analysis - lesional TSS at Day 22

The comparison between the treatment groups for change from baseline in lesional TSS at Day 22, was done using a paired Student t-test. The difference between treatments was estimated and presented along with a 95% confidence interval. Descriptive statistics for the baseline, Day 22 and change from baseline to Day 22 lesional TSS were presented for lesions treated with niclosamide and vehicle in the MITT population. Ninety-five percent confidence intervals (Cls) using a t-distribution were determined for the point estimates for change from baseline in each treatment group. Descriptive statistics and a 95% Cl using a t-distribution will also be provided for the difference between the change from baseline in lesional TSS for the niclosamide and placebo lesions. Subjects with missing TSS at Day 22 were included in the analysis using a last observation carried forward imputation for the missing data. Analyses of the primary efficacy endpoint was repeated in the PP

population.

Efficacy endpoints include TSS at Days 1 (pre-dosing), 8,15 and 22, and TAA at Days 1 (pre-dosing), 8, 15 and 22. Analyses of endpoints were conducted in the same manner as described for the other efficacy endpoint.

Efficacy Analysis - Biomarker/clinical score correlation analysis

The variables that were used for the correlation analysis were the clinical score (Total Sign Score (TSS) and Target Area Assessment (TAA)) of Day 22 and Baseline (Day 1 ) and the normalized biomarker expression values that were analysed with qRT-PCR (TLDA) and for the same days. The absolute change with treatment at Day 22 were calculated for each patient and each treatment. For the assessment of pairwise correlation the Spearman correlation coefficient was used. It is a non-parametric measure of rank correlation. The significant correlations were plotted with the respective linear regression line, a confidential interval of 95% and its respective rho (spearman coefficient, R) and p value. For this correlation analysis biomarkers were selected that showed significant changes in qRT- PCR and/or microarray. The correlation analysis was made on qRT-PCR data only except for the immune cells were IHC data was taken.

The same procedure was applied when analysing the correlation of individual scores and biomarker expression values. For this analysis biomarkers were taken that showed significant correlation to TSS or/and TAA. The correlation analysis was made on qRT-PCR data only.

Biomarkers

Immune effectors (herein also referred to as biomarkers) included in the

immunohistochemistry (IHC) and in the gene expression analysis using qRT-PCR were grouped as shown in Table 1 1 :

Thymic stromal lymphopoietin protein receptor (TSLP-R) is the receptor for the proinflammatory cytokine thymic stromal lymphopoietin (TSLP).

CD3 (cluster of differentiation 3) is a biomarker for T cells. FOXP3 (also known as scurfin) is a biomarker for a subpopulation of T cells called regulatory T cells (also known as suppressor T cells).

As mentioned above, the biomarkers that showed significant changes in qRT-PCR (TLDA) expression analysis were selected for correlation analysis with TSS and TAA.

Further biomarkers were included in the microarray analysis, see Tables 18 to 20.

Results

Skin thickness at Day 22

No differences in skin thickness were found following treatment with 2% niclosamide compared to baseline and compared to vehicle.

Expression levels of biomarkers at Day 22 and correlation versus Total Severity Score (TSS) and Target Area Assessment (TAA)

Biomarkers were analysed by qRT-PCR or microarray in the skin biopsies taken at Day 1 and at Day 22 as described hereinbefore.

The results for all biomarkers analysed by qRT-PCR are presented in Tables 12-17.

The results for all biomarkers analysed by microarray are presented in Tables 18-20. Table 12: qRT-PCR - all Biomarkers results

^***(p<0.001 ) ^**(p<0.01 ) ^*(p<0.05) +(p<0.1 )

Table 13: Biomarkers that changed significant with treatment at Day 22 compared to Baseline (qRT-PCR)

^***(p<0.001) ^**(p<0.01) ^*(p<0.05) +(p<0.1)

Table 14: Biomarkers that are significant changed with treatment compared to vehicle (qRT-PCR)

^***(p<0.001) ^**(p<0.01) ^*(p<0.05) +(p<0.1)

Table 15: Biomarkers that are significant changed to Baseline and vehicle with Niclosamide (qRT-PCR)

^***(p<0.001 ) ^**(p<0.01 ) ^*(p<0.05) +(p<0.1 )

Table 16: Significant correlations of biomarker expression (based on qRT-PCR/IHC data) to TSS at Day 22

Table 17: Significant correlation of biomarker expression (based on qRT-PCR data) to TAA at Day 22

Table 18: Biomarker expression levels that changed significant with treatment compared to Baseline (Microarray)

Table 19: Biomarker expression (microarray) that changed significant with treatment compared to vehicle

Table 20: Biomarkers that are significant changed compared to Baseline and vehicle with treatment (Microarray)

Conclusions

As evident from the above presented results, significant changes from baseline (pre- dosing at Day 1 ) were found for certain immune effectors in the biopsies taken at Day 22.

S100A12 was found to be significantly downregulated at Day 22 following topical administration of 2% niclosamide compared to baseline (-3.62) and compared to vehicle (- 2.30), p<0.05). S100A12 was found to be significantly correlated with TSS and TAA. Results are shown in Figures 1a and 1 h, respectively. The graphs show the correlation of change in biomarker expression at Day 22 compared to baseline to change in TSS at Day 22.

S100A9 was found to be significantly downregulated at Day 22 following topical administration of 2% niclosamide compared to baseline (-2.81 ) and compared to vehicle (- 1.88) (p<0.05). S100A9 was found to be significantly correlated with TSS and TAA.

Results are shown in Figures 1 b and 1f respectively. The graphs show the correlation change in biomarker expression at Day 22 compared to baseline to change in TSS at Day 22.

PI3 was found to be significantly downregulated at Day 22 following topical administration of 2% niclosamide compared to baseline (-3.13) and compared to vehicle (-1.87) (p<0.05). PI3 was found to be significantly correlated to TSS and TAA. Results are shown in Figures 1 c and 1 g respectively. The graphs show the correlation of change in biomarker expression at Day 22 compared to baseline to change in TSS at Day 22.

CXCL1 was found to be significantly downregulated at Day 22 following topical administration of 2% niclosamide compared to baseline (-2.83) and compared to vehicle (- 2.10) (p<0.05). CXCL1 was found to be significantly correlated to TSS. Results are shown in Figure 1 d. The graphs show the correlation of change in biomarker expression at Day 22 compared to baseline to change in TSS at Day 22.

S100A7 was found to be significantly downregulated at Day 22 following topical administration of 2% niclosamide compared to baseline (-3.04) and compared to vehicle (- 2.20) (p<0.05). S100A7 was found to be significantly correlated to TSS and TAA. Results are shown in Figures 1 e and 1 i, respectively. The graphs show the correlation of change in biomarker expression at Day 22 compared to baseline to change in TSS at Day 22.

Thus, S100A12, S100A9, PI3, S100A7 and CXCL1 were all shown to be significantly downregulated in expression compared to baseline as well as vehicle and were all found to be clinically correlated to TSS.

Among these biomarkers that showed significant change compared to vehicle and baseline, S100A7 and S100A9 were found to have the highest correlations to TSS and S100A7 and S100A9 to were found to have the highest correlations to TAA.

The levels of the biomarkers listed in Table 17 above and analysed by qRT-PCR were also found to have changed significantly at Day 22 compared to baseline following topical administration of 2% niclosamide. Results are shown in Figures 16 - 25, where A denotes vehicle and B denotes

niclosamide at Day 22 compared to baseline.

Figure 16 shows changes in biomarkers (IL6, IL8, IL17C, IL1 B) associated with innate immunity.

Figure 17 shows changes in biomarkers (I L15, IL15RA, IL2, CCL5) associated with T cell activation.

Figure 18 shows changes in biomarkers (IFNG, CXCL9, I L12A/IL12p35, CXCL10) associated with Th1 related genes.

Figure 19 shows changes in biomarkers (I L13, I L10, IL33, TSLP-R, IL31 , IL5) associated with Th2 related genes.

Figure 20 shows changes in biomarkers (CCL17, CCL18, CCL22, CCL26) associated with Th2 related chemokines.

Figure 21 shows changes in biomarkers (IL17A, IL17F, IL23A/I L23p19, CAMP/LL37, I L19, IL12B/IL23p40) associated with Th17 cytokine related genes.

Figure 22 shows changes in biomarkers (DEFB4A/DEFB4B, CXCL1 , CXCL2, CCL20, PI3) associated with Th17 chemokine related genes.

Figure 23 shows changes in biomarkers (IL22, S100A7, S100A8, S100A9, S100A12) associated with Th17/Th22 related genes.

Figure 24 shows changes in biomarkers (FLG, PPL, LOR) associated with terminal differentiation.

Figure 25 shows changes in biomarkers (KRT16) associated with proliferation, general inflammation (MMP12), Th9 (IL9) and T regulatory cells (FOXP3).

Correlations between change in biomarker expression versus TSS are shown in Figures 2- 5. The graphs show the correlation of biomarker change at Day 22 compared to baseline to change in TSS at Day 22.

Figures 2a and 2b show biomarkers (KRT16, MMP12) associated with proliferation / general inflammation.

Figures 2c, 2d and 2e show biomarkers (IL13, CCL17, CCL22) associated with Th2 related chemokines and cytokines.

Figure 3a show biomarkers (IL8) associated with innate immunity. Figures 3b and 3c show biomarkers (LOR, FLG) associated with skin barrier / terminal differentiation.

Figure 3d show biomarkers (CD11 c Dermis) associated with dendritic cells.

Figures 4a-4e show biomarkers (S100A8, S100A12, S100A7, S100A9, IL22) associated with Th17/Th22 related chemokines and cytokines.

Figures 5a-5f show biomarkers (PI3, CXCL1 , IL17A, IL19, CAMP, DEFB4A/DEFB4B) associated with Th17 related chemokines and cytokines.

Correlations between change in biomarker expression versus TAA are shown in Figures 12-15. The graphs show the biomarker change at Day 22 compared to baseline.

Figures 12a and 12b show biomarkers (KRT16, MMP12) associated with proliferation / general inflammation.

Figures 12c, 12d and 12e show biomarkers (IL13, CCL17, CCL22) associated with Th2 related chemokines and cytokines.

Figure 13a show biomarkers (IL8) associated with innate immunity.

Figures 13b and 13c show biomarkers (LOR, FLG) associated with skin barrier / terminal differentiation.

Figures 14a-14e show biomarkers (S100A8, S100A12, S100A7, S100A9, IL22) associated with Th17/Th22 related chemokines and cytokines.

Figures 15a-15c show biomarkers (PI3, DEFB4A/DEFB4B, IL19) associated with Th17 related chemokines and cytokines.

All these biomarkers analyzed with qRT-PCR except for LOR and FLG were found to have decreased significantly at Day 22 following topical administration of 2% niclosamide compared to baseline (see Tables 12-17).

LOR and FLG were found to have increased significantly at Day 22 following topical administration of 2% niclosamide compared to baseline, see Figures 13b and 13c. LOR and FLG are involved in terminal differentiation of epidermal cells and an increased expression of any one of these proteins is associated with a better skin barrier. Increased expression of LOR induced by topical niclosamide was shown to be associated with an improvement of signs and symptoms of AD.

Also, some skin barrier proteins and lipids analyzed with microarray (see Tables 18-20) were found to have increased significantly at Day 22 following topical administration of 2% niclosamide compared to baseline and vehicle. Skin barrier lipids that were found to have increased compared to baseline and vehicle, by using the microarray analysis, were ACOX2, EVOLV3, FA2H, FAR2, KRT79, PNPLA3. Skin barrier proteins that were found to have increased compared to baseline and vehicle, by using the microarray analysis, were DGAT2 and FAXDC2.

The increased expression of structural skin barrier proteins and lipids indicate that niclosamide are useful for treatment of an inflammatory skin condition associated with skin barrier dysfunction, e.g. an inflammatory skin condition associated with skin barrier deficiency in one or more skin barrier molecules, such as AD, by improving the skin barrier function.

Treatment with 2% niclosamide was shown to decrease inflammation and immune cell infiltrates compared to baseline (pre-dosing at Day 1 ). Significant reductions in

inflammatory cells (dendritic cells: CD1 1c, FceR1 in epidermis, and Langerhans cells: langerin/CD207) compared to baseline (pre-dosing at Day 1 ) in patients topically treated with 2% niclosamide were found (Figures 27-29). CD11 c Dermis was significantly changed in expression level compared to baseline and clinically correlated to TSS (see Figure 28).

No significant change of the total amount of T cells (i.e. T cells expressing CD3D and CD3G) was found (in dermis and epidermis) compared to baseline (pre-dosing at Day 1 ) in patients topically treated with 2% niclosamide, see Figure 26.

In patients treated with 2% niclosamide, there were significant changes from baseline in certain inflammatory markers including those of general inflammation (MMP12), proliferation (KRT16), innate immunity (IL6, IL17C, IL8, IL1 B), terminal differentiation (FLG, LOR), T-Cell/NK cell activation (IL15, IL15RA), Th1 pathway (CXCL10), Th2 pathway (CCL17, CCL18, CCL22, IL10, IL13, IL5, TSLPR), Th17 pathway (IL17A, IL23p19, IL23A, CCL20, CXCL1 , CXCL2, PI3, DEFB4A/DEFB4B, PI3, IL12B), general inflammation (MMP12), T regulatory cells (FOXP3), Th17/TH22 pathway (S100A7, IL22, S100A8, S100A9, S100A12).

The results show that topical administration of niclosamide significantly downregulates expression of immune effectors associated with the Th1 , Th2, Th17 and Th22-type immune responses, including innate immune effectors. Th2, Th17, Th22 responses are crucial in the inflammatory loop of AD. The reduced expression of these key biomarkers and the direct correlation of these biomarkers to clinical signs and symptoms strongly support use of niclosamide for treatment of AD.

Brunner et al (The Journal of Allergy and Clinical Immunology, Volume 139, Issue 4, Supplement, Pages S65-S76, 2017) discloses the effects of dupilumab on lesional AD skin, such as reduction in expression of Th2-associated molecules, such as CCL17, CCL18, and CCL26, and decrease in mediators associated with TH17 and TH22

responses. The biomarker profile in lesional AD skin treated with dupilumab is shown in Figure 4 of the Brunner reference.

Reference is here also made to Hamilton, Jennifer D., et al.“Dupilumab improves the molecular signature in skin of patients with moderate-to-severe atopic dermatitis.” Journal of Allergy and Clinical Immunology 134.6 (2014): 1293-1300; and Brunner, Patrick M., et al.“A mild topical steroid leads to progressive anti-inflammatory effects in the skin of patients with moderate-to-severe atopic dermatitis.” Journal of Allergy and Clinical Immunology 138.1 (2016): 169-178.

Example 6: Exploratory pharmacokinetic study in dogs

A preliminary PK study was carried out in dogs in which oxyclozanide was orally administered in the following treatment arms: 5 mg/kg orally once per day for 4

consecutive days; topically administered as a single 20 mg/kg (using Composition K described in Example 7 below); and intravenously as a single 2.5 mg/kg dose.

Oxyclozanide was distributed according to the two-compartment model following intravenous administration. Terminal half-life was 38 h and Vss was about 0.60 L/kg suggesting a low to moderate volume of distribution. Bioavailability was 48% following oral administration and 12% after topical application suggesting a small systemic exposure. Oxyclozanide accumulated well in stratum corneum following topical application for a long period of time when tape stripping (adhesive films) were collected at about 8 cm of the application site (along the backbone).

Figure 30 illustrates the arithmetic profile of oxyclozanide (pg/g) in stratum corneum of skin flank following the oral or topical administration in dogs.

Example 7: Dermatopharmacokinetic study oxyclozanide in plasma and skin following topical administration to dogs

Study objective The aim of this study was to determine on dog (i) the tolerance and pharmacokinetic profile in plasma and skin of oxyclozanide after topical application of Composition K, and (ii) the spread of oxyclozanide on several areas of the skin 10-40 cm away from the application site following topical application of composition K for 4 weeks.

Inclusion criteria

12 adult beagle dogs having a body weight of more than 1 1 kg were used in the study. An inclusion clinical examination was performed on D-3_a (i.e. 3 days before first

administration) and D-4_b (i.e. 4 days before first administration) of each phase. Twelve healthy dogs with haematological-biochemical parameters within the supplier’s normal range were included in the study.

Management of test system

During the 28-days animal study phase (from DO to D28), the animals were housed individually in order to guarantee the quality of the results by avoiding the dogs licking each other after the treatment is applied. No treatment, apart from those treatments included under the study, was administered without the approval of the study director.

Treatment

The cutaneous route (topical administration) was the route of administration for composition K on dogs. Composition K was applied evenly along the backbone starting from the base of the tail to the back of the neck. The administered dosage was 20 mg oxyclozanide/kg (0.20 ml/kg of body weight).

The 12 dogs were divided into 2 groups of 6 animals, with homogeneous averages of age and weight. They were treated with the same composition but the activities following administration were different:

Group A: dogs 1 to 6

Blood samplings Skin biopsies, preceded by cutaneous cells (stratum corneum) samplings at the belly zone

Cosmetic and dermal assessments

Group B: dogs 7 to 12

- Cutaneous cells (stratum corneum) samplings at several sites (ear, shoulder, belly, fore leg, hock joint and chest zones).

The volumes administered for each dog are described in the table below:

Clinical follow-up

On D-3a or D-4b, a physical examination, including a weigh-in, was performed on the 6 dogs of each group by a qualified person.

On DO of each phase, in the hour following treatment administration, a clinical observation was performed. Special attention was given to the following clinical signs: constitutional symptoms (such as fever, fatigue, shivering), neurological and vascular, ptyalism, vomiting, pruritus, pain, unusual behaviour (e.g. rolling on back).

All dogs in group A were shaved at D-3a in the belly zone (lower part of the abdomen to the right or left of the animal) on sufficient areas for biopsies. Shaving was repeated at D4a, D11a, D18a and D25a alternating the right and left side of the animal for each sampling time. All dogs of group B were shaved on D-4b at the following areas, alternating the right and left side of the animal for each sampling time: ear, belly, chest, hock (tarsal) joint, fore leg, and shoulder. Each area was shaved on a zone of around 5 cm x 5 cm.

Dermal tolerance assessment Dermal tolerance was assessed for the 6 dogs of group A on: D0a+1 h, D0a+3h, D0a+24h, D3a, D7a, D14a, D21 a and D28a.

Upon each assessment, dermal tolerance in the treatment area was assessed according to the following scoring system:

Erythema:

- No erythema

- Very mild erythema (barely visible)

- Clearly-defined erythema

- Moderate erythema

- Severe erythema (beetroot red) with slight sores (deep lesions)

Oedema (Yes/No)

Excoriation (Yes/No)

Scabs (Yes/No)

Sampling

Dry tube blood sampling

Before the inclusion examination and on D28 of both phases, blood samples were taken from the jugular vein of each dog with a 4 ml dry tube and a 3 ml EDTA K3 tube. These samplings were done for hemato-biochemical analysis.

After centrifugation (around 3500 revolutions/min, for 15 minutes at +4°C), the serum was collected and then divided into two equal aliquots in tubes (Nunc 1.8 ml type). Each aliquot was identified with the study code; the identification number of animal and its case number; the type, date and time of sampling. The aliquots were stored at 5°C +/-3°C until their shipping. The aliquots were shipped on the day of sampling, in cold packaging, to the analytical laboratory.

The laboratory analysed the following: Biochemical parameters: total bilirubin, total protein, glucose, alkaline phosphatase (ALP), alanine aminotransferase (ALAT), aspartate aminotransferase (AST), creatine kinase (CK) and gamma glutamyl transferase (GGT).

Haematological parameters: haemoglobin, haematocrit, RBC, MCV, MCH, MCHC and reticulocytes.

Lithium heparin blood sampling

For all animals of group A, a blood sample of around 4 ml was collected from the jugular vein using tubes containing lithium heparin at the following times (with tolerance of 10%): D-3a, D0a+1 h, D0a+6h, D0a+12h, D0a+16h, D0a+24h, D0a+32h, D0a+48h, D3a, D5a et D7a.

Centrifugation (around 3500 revolutions/min, for 15 minutes at +4°C) was performed a maximum of 30 minutes after sampling. The plasma was collected and then divided into two aliquots in tubes (Nunc 1.8 ml type) as follows:

51 aliquot: around 0.5 ml

52 aliquot: the remainder of the plasma (more than 0.5 ml)

The aliquots were stored at -70°C +/-5°C until their shipping. The aliquots were shipped at the end of each phase packaged in dry ice to the analytical laboratory.

Skin biopsies

For all dogs of group A, a skin biopsy was performed under anaesthesia in the belly zone (, alternating the right and left side of the animal for each sampling time) with a 4 mm ‘Biopsy-Punch’ at the following times: D1 a, D7a, D14a, D21 a and D28a. Prior to each biopsy a cutaneous cell sampling was performed on the area of the biopsy. The vials were frozen at -70°C +/- 5°C until shipping.

Stratum corneum sampling (D-Squame discs)

Tape-stripping (adhesive films) was carried out in an analogous method to that described in Emilie Videmont, et al., (“Characterization of the canine skin barrier restoration following acute disruption by tape stripping,” Veterinary Dermatology, vol. 23, pp. 103-123, 201 1 ) and Lionel Trottet (Dermal Drug Selection and Development, An Industrial Perspective. Springer 2017. Page 57). Tape was pressed onto the surface of the skin with a fixed amount of pressure before removal. The superficial layers of the SC which adhere to the film were stripped from the Stratum corneum which was then accessible for further investigation.

For all animals of group B, cutaneous cell sampling was performed on fasted animal on the six shaved zones using discs (D-Squame DISCS), at the following times: D-4b, D0b+24h, D0b+24h, D3b, D7b, D14b, D21 b and D28b alternating the right and left side of the animal. 20 discs (D-Squame Discs) of 22 mm-diameters were applied in succession on the target area, in the same zone defined with a marker.

The discs were applied as follow:

Tweezers were used to carefully remove the discs from its backing using the edge provided.

The disc was applied to the defined area.

The disc was pressed for 1 second with D-Squame Pressure Instrument

150 g/cm² and 1 mg of stratum corneum was removed after each disc application. 20 discs were pressed per sample corresponding to removal of 20 mg of stratum corneum per sampling time.

For each cycle, the 20 discs removed from the skin were divided into 2 samples (S1 and S2) as follows:

51 : the first 10 discs were placed in a scintillation vial

52 : the last 10 discs were placed in a scintillation vial

The pigmentation of the skin of the sampled area was recorded in raw data. The vials were frozen at -70°C +/-5°C until shipping to the analytical laboratory.

PK Analysis

The pharmacokinetic analyses was performed using Phoenix software (version 6.3, Pharsight, USA). Data points indicated as“missing” were systematically ignored during the calculation and therefore have no effect on the results. A missing status is assigned and no flag symbol is used. Values were excluded if, in the judgement of the

pharmacokineticist, they were deemed not to be“pharmacodynamically relevant.” If outliers were suspected, they were identified using the Outlier identification procedure of STATGRAPHICS.

The following pharmacokinetic parameters were evaluated:

Statistical analysis

Mean (arithmetic average), standard deviation (SD), standard error of the mean, coefficient of variation (CV%, SD/mean^*100), maximum value and minimum value were calculated per day and treatment for each concentration parameter:. The mean concentrations and the standard deviations were calculated and the mean concentration time curves were plotted. This descriptive statistics of the pharmacokinetic parameters were calculated per treatment.

Analytical methods

The oxyclozanide concentration in samples was quantified using an LC-MS-MS method.

Results

The dogs’ general health condition was satisfactory and comparable all along the study. Their results of haematological-biochemical analysis were acceptable without any indication of possible negative effect of the active substance on the parameters examined for the animals of group A. For the animals of group B, there were some anomalies in the haematological-biochemical parameters such as an increase of alanine aminotransferase. This is due to close and repeated anaesthesia for more than 4 weeks by tiletamine metabolized in the liver.

No symptoms or abnormalities were observed after treatment on DOa. After treatment on DOb, two dogs (dogs 9 and 12) licked the base of their tail and they shook themselves. Some slight scabs were observed on the left shoulder (area of skin sampling) on one dog (dog 12) on DOb.

After the cutaneous treatment, no significant sign appeared for dermal tolerance all along the phase A. On the six dogs of the group A, the fur was greasy appearance at D0a+1 h and D0a+3h with an orange coloration on light skin for 3 dogs.

Plasma samples

Figure 31 illustrates the mean plasma concentration-time of oxyclozanide (pg/L) obtained following topical administration in dogs.

Oxyclozanide presents a PK profile in plasma similar to those obtained in the early PK study of Example 4 and at same systemic exposition. Bioavailability was not calculated because there are not intravenous route in this study. However, bioavailability appeared to be similar (about 12%) to that the exploratory pharmacokinetics study of Example 4, when comparing AUCinf (60604 h^*pg/L against 55712 h^*pg/L). Low concentrations of oxyclozanide in plasma were observed after topical administration suggesting a low systemic exposure..

Epidermis/dermis (biopsies) + stratum corneum (strip samples)

Figure 32 illustrates the mean (pg/g) skin biopsies concentration-time of oxyclozanide obtained following topical administration in dogs.

Oxyclozanide accumulates about 10 fold less in deep skin (epidermis and dermis) than in the stratum corneum. Oxyclozanide concentrations were above MIC (staphylococcus spp) for 1-2 weeks after treatment. The inter-variability between animals was low after topical application.

Table 21 below presents the mean exposure (AUC) of each collected site. Results show that exposure of oxyclozanide in the superficial skin and deep skin for several period of time after administration. Table 21

Stratum corneum (strip samples)

Oxyclozanide accumulates well in stratum corneum following topical application for a long period of time as observed in the previous study of Example 6. Oxyclozanide diffuse well on all the skin area zones of the body and stay significantly above the MICs although it is difficult to compare concentrations of oxyclozanide solubilized in skin lipids and measurement of the MIC determined in a buffered aqueous medium.

Figure 33 illustrates mean (pg/g) stratum corneum (strips) concentration-time of oxyclozanide obtained following topical administration in dogs on 6 zones.

Results show that concentrations of oxyclozanide were quite correlated with the distance between the site of administration and the sampling site of skin collection.

Oxyclozanide is slowly eliminated from the superficial skin (stratum corneum) and elimination rate seems correlated to the process of cell migration through the layers of the epidermis (epidermal renewal) takes approximately 22-28 days.

Table 22 below presents the mean exposure (AUC) of each collected site. Results show that exposure of oxyclozanide in the superficial skin was quite correlated with the distance between the site of administration and the sampling site of skin collection for several period of time after administration.

Conclusions

All twelve dogs remained in good general condition during the study and no significant signs of dermal intolerance were observed.

High concentrations of oxyclozanide were observed in the stratum corneum for a prolonged period in all the skin area zones of the body. The distribution of oxyclozanide between the superficial and deep skin was about 10:1. Low systemic concentrations were observed as in the earlier pharmacokinetic study of Example 4.

Example 8: Efficacy and safety of oxyclozanide in the treatment of superficial and deep pyoderma due to Staphylococcus SOD in dogs

Study objective

The objective of this concept study was to assess the clinical field safety and efficacy of a line-on solution containing 10% (100 mg/ml) oxyclozanide in the treatment of superficial and deep pyoderma due to Staphylococcus spp. in dog. Composition K (see Example 4) was used in the study.

Superficial pyoderma is defined as skin bacterial infection restricted to the epidermis that do not penetrate below the basement membrane. Superficial pyodermas are typically exudative and lesions include papules, pustules, epidermal collarettes, scales and crusts. Pruritus is often present.

Deep pyoderma is defined as serious skin bacterial infection below the basement membrane into the dermis and deeper tissues. The study was an open single arm multi-site field study.

Inclusion criteria

Dogs eligible for enrolment in this study needed to meet all of the following criteria:

Owner older than 18 years and who signed the owner's consent prior to any study specific physical examination is performed.

Dog in general good health based on physical examination (except for pyoderma).

Dog aged at least of 12 months at inclusion.

Dog shows clinical evidence of superficial or deep pyoderma:

o based on suggestive clinical signs (e.g. pustules, papules, scaling/crusts, erythema, pruritus, furunculosis, cellulitis), and

o the presence of neutrophils (with or without intracellular cocci) in cytological samples

A systemic antibiotherapy or anti-infective shampoo was required.

Suspicion of Staphylococcus spp. infection (e.g. S. pseudintermedius, S. aureus, S. hyicus, S. schleifen).

If Malassezia were observed, they were not suspected to be the primary cause of the skin lesions.

Dog with no flea infestation or fleas controlled with oral insecticide.

Exclusion criteria

A dog was not enrolled if one or more of the following exclusion criteria apply:

Dog intended for breeding, or known to be pregnant or lactating.

Dog with localized pyoderma that would require a local treatment only.

Dog with surface pyoderma (absence of neutrophils in cytology).

Dog that has been treated with anti-infective medicines, or with corticosteroids within the last 4 weeks.

Suspicion of fungal or parasitological disease (flea infestation, skin scrapings positive for sarcoptic mange or demodicosis).

Dog that has been washed with a shampoo within the last 3 days. Dog with any severe or uncontrolled concomitant disorder or disease, that might have interfered with the evaluation of response to composition K.

Dog with known or suspected hypersensitivity to the IVP ingredients.

Exclusion diet initiated less than 6 weeks ago.

Dog receiving immunomodulating drugs or hormones for which the treatment has been modified within the last 8 weeks prior to inclusion.

Treatment

Composition K was administered at the dose of 20 mg/kg, corresponding to 0.2 ml/kg body weight (BW) at each administration, as following:

Superficial pyoderma: 3 consecutive weekly treatments starting on Day O.

Deep pyoderma: a minimum of 3 up to a maximum of 8 consecutive weekly treatments starting on Day O. If the clinical cure was observed before achieving the 8 weekly treatments, the dog received 2 additional weekly treatments after the visit when first occurrence of clinical cure (first occurrence of clinical cure was upon evaluation of the investigator based on his/her assessment of skin lesions, pruritus intensity and cytology observations). For example, if clinical cure was observed on Day 28, the dog was treated again on Day 28 and Day 35.

Composition K was applied topically directly to the dog's skin by emptying the whole content of the syringe as a line starting from the base of the tail to the back of the neck. The hair could be parted so the composition was applied as close as possible to the skin.

The study calendar after enrolment varied based on the type of pyoderma:

Superficial pyoderma: 3 consecutive weekly treatments + 3 follow-up visits at 7, 14 and 28 days after last administration

Deep pyoderma: 3 to 8 consecutive weekly treatments + 3 follow-up visits at 7, 14 and 28 days after last administration.

The three follow-up visits at 7, 14 and 28 days after the last administration were also named Follow-up 1 , Follow-up 2 and Follow-up 3 or Study end, respectively.

Evaluated parameters

Haematology/Biochemistry

On day 0 and study end: Blood sample collected for complete haemato / biochemical profiles

Bacteriology

On day 0, then on Day 21 and study end if skin lesions were still present:

Swab collected for culture and identification of bacteria and Malassezia and antimicrobial susceptibility testing.

Cytology

On day 0, then at each visit if skin lesions were still present:

On Day 0, superficial pyoderma was identified by the presence of neutrophils (with or without intracellular cocci). Deep pyoderma was identified by the presence of neutrophils (often degenerate) and macrophages.

Presence/absence of neutrophils, intracellular cocci, macrophages, Malassezia

Examination of skin lesions

At each visit:

Scoring of severity of each type of lesions, by body area:

o Lesion types (9): erythema, lichenification/hyperpigmentation, excoriation, alopecia, keratoseborrheic condition, papules, pustules, macules and epidermal collarettes.

o Body areas (14 ): face, ears, neck, axilliae {L +R), thorax, abdomen & groin, lumber & flanks, front leg & paw (L +R), hind leg & paw (L +R), perineum, tail

o Severity score: none (0); mild (1 ); moderate (2); severe (3)

A total skin score per visit was obtained by summing the score from each lesion and each body area.

Post-administration observations

Approx. 1 and 6 hours after each treatment:

Assessment of general condition and application site (normal/abnormal)

Owner's assessments of pruritus and application site

At each visit: Assessment of pruritus severity on PVAS with clinical descriptors (10 cm scale; see below).

- Assessment of coat colour and aspect (e.g. moist, greasy, clumps of fur)

Extremely severe itching/almost continuous: Itching doesn’t stop whatever is happening, even in the consulting room (needs to be physically restrained from itching)

Severe itching/prolonged episodes: Itching might occur at night (if observed) and also when eating, playing, exercising or being distracted.

Moderate itching / regular episodes: Itching might occur at night (if observed) but not when eating, playing, exercising or being distracted.

Mild itching / a bit more frequent: Wouldn’t itch when sleeping, eating, playing, exercising or being distracted.

Very mild itching / only occasional episodes: The dog is slightly more itchy than it was before the skin problem started.

Normal dog: I don’t think itching is a problem.

Investigator's clinical improvement assessment

At each visit after Day 0, the investigator made his/her assessment of the clinical improvement compared to the previous visit, as follows:

The dog is clinically cured, i.e. disappearance of skin lesions associated to pyoderma.

Significant clinical improvement but the dog is not yet clinically cured. No significant clinical improvement but I consider the dog can continue the study per protocol schedule.

Treatment failure. Response to treatment is not satisfactory.

Relapse. The dog was clinically cured at previous visit(s) but skin lesions reappeared.

The dog is withdrawn from the study for other reasons.

Investigator's overall assessment

At the end of the study i.e. 28 days after the last treatment or at the time of premature withdrawal when applicable, the investigator made his/her assessment on the overall efficacy of the product, the treatment outcome and gave his feed-back on the ease of product administration and cosmetic effects at application site.

Evaluation criteria

Study populations

Safety population (SAF)

All dogs having received at least one dose of treatment. This population was used for safety analyses.

Full Analysis Set (FAS)

Dogs having actually presented the studied disease, having received at least one dose of study treatment and having been assessed at least once for efficacy parameters.

Per Protocol (PP)

Dogs from the Full Analysis Set without any major deviation which could affect the evaluation of the efficacy. This population was used for efficacy analyses.

Data collected from animals withdrawn from the study after inclusion up to and including the date of removal were included in the final statistical evaluation.

Efficacy analysis

The efficacy analyses were performed on the Per Protocol (PP) population. Results are displayed separately for superficial and deep pyoderma.

Skin lesions % reduction of the total skin score at each visit compared to Day 0.

Dogs having skin lesions associated to pyoderma (papules, pustules, macules and epidermal collarettes) at score O (absent) at the end of the study.

Owner's pruritus assessment

- % reduction of the pruritus score at each visit compared to Day 0.

Investigator's clinical improvement assessment

clinically cured / significant clinical improvement/ no significant clinical

improvement/ treatment failure/ relapse at each visit after Day 0.

Investigator's overall efficacy assessment

- very good / good / poor / very poor) at the end of study.

Treatment outcome as assessed by the investigator

clinical cure without relapse / clinical improvement/ treatment failure/ relapse at the end of study.

Clinical success

Clinical success is defined as the percentage of dogs having skin lesions associated to pyoderma (papules, pustules, macules, epidermal collarettes) at score 0 (absent) at the end of study i.e. 28 days after last treatment or at premature withdrawal.

Safety analysis

Description of Adverse Events (number, type, severity).

- Changes from baseline of haematology and biochemistry parameters.

Additional analysis

Feedback from investigator on ease of product administration.

Cosmetic aspects at administration site.

Performance of study

A total of 30 dogs were enrolled in the study, including 23 dogs with superficial pyoderma (77%) and 7 dogs with deep pyoderma.

Four dogs, all having superficial pyoderma, were excluded from the Per Protocol population. Three dogs with superficial pyoderma and four dogs with deep pyoderma from the Per Protocol population were prematurely withdrawn from the study due to treatment failure or relapse.

Six dogs out of 30 (2 with superficial and 4 with deep pyoderma) had a known medical history of skin inflammation or infection. In addition, one of these dogs (deep pyoderma) was under treatment with oclacitinib for several months before inclusion in the study, to control atopic dermatitis. The treatment was continued during the study.

Bacteriology at inclusion

The bacteria isolated from the swab samples collected at the time of inclusion (Day 0) in the Per-Protocol population (N=26) are listed below:

Staphylococcus was isolated on an additional swab sample on Day 7.

^** Staph intermedius then isolated on an additional swab sample on Day 7.

Cytology at inclusion

Superficial pyoderma:

All the dogs had neutrophils in cytology at inclusion, including 74% with intracellular cocci.

One dog had also macrophages but was diagnosed as superficial pyoderma by the investigator based on the skin lesions. Deep pyoderma:

All the dogs had neutrophils in cytology at inclusion, including 86% with intracellular cocci.

Two dogs had not macrophages but were diagnosed as deep pyoderma by the investigator based on the skin lesions.

Administration

87% of cases with superficial pyoderma (20/23) received the recommended treatment duration of 3 weeks. Three dogs received shorter treatment duration due to premature withdrawal.

Most of the dogs with deep pyoderma (57%, 4/7) received the maximum recommended treatment duration of 8 weeks. The other dogs received 3 (ending the study as treatment failure), 5 (clinical cure) or 7 (relapse) administrations.

The average volume of composition K administered per application was 4.5 ml (min-max: 0.7-13.2 ml).

Results

Efficacy results

The efficacy results described apply to the Per Protocol (PP) population representing 19 cases with superficial pyoderma and 7 cases with deep pyoderma.

Figure 34 shows the distribution of total skin score in dogs with superficial pyoderma treated with composition K. These results showed that the mean total skin score decreased after treatment initiation starting from Day 7 until follow-up visit 2 ( 14 days after last treatment). A slight increase of skin score was observed between follow-up visit 2 and Study end, exacerbated by an increased skin score for two dogs withdrawn prematurely from the_study because of treatment failure or relapse.

Figure 35 shows the distribution of total skin score in dogs with deep pyoderma treated with composition K. These results showed that the mean total skin score decreased after treatment initiation starting from Day 7 until follow-up visit 1 (7 days after last treatment), with a slight and temporary increase observed at Day 21 (3/7 dogs). A slight increase of mean total skin score was observed between follow-up visit 1 and Study end due to 2 out of 7 dogs with an increased skin score at study end, including one dog withdrawn prematurely from the study on Day 21 for treatment failure. The most severely affected body areas on Day 0 were abdomen and groin, lumbar and flanks, and axillae. Mean severity at study end was reduced in all body areas.

Figure 36 shows evolution of mean pruritus score in dogs with superficial pyoderma treated with composition K.

The results show that in dogs having superficial pyoderma, the pruritus severity decreased from Day 7 until Day 28 (Follow-up 2), then increased on Day 42 (study end) due to four dogs.

Figure 37 shows evolution of mean pruritus score in dogs with deep pyoderma treated with composition K.

The results show that in dogs having deep pyoderma, the pruritus severity decreased from Day 7 until study end, with a slight "rebound" on Day 21 as also observed for skin lesions.

In the investigator’s clinical improvement assessment of the 19 dogs having superficial pyoderma, 63% of dogs showed significant clinical improvement by Day 7 after treatment initiation. First clinical cure was observed in about 16% of dogs at 14 days after treatment initiation (Day 14). Then the proportion of dogs considered clinically cured by the investigator increased up to 63% of dogs at 14 days after last treatment (Follow-up 2) and 52.6% at study end (i.e. 28 days after last treatment or premature withdrawal).

In the investigator’s clinical improvement assessment of the 7 dogs having deep pyoderma, 57% (4/7) of dogs exerted significant clinical improvement by 7 days after treatment initiation. First clinical cure was observed from 21 days after treatment initiation (Day 21 ). Then, the proportion of dogs considered clinically cured by the investigator increased to about 43% at 7 days after last treatment (Follow-up 1 ) and was maintained until the study end i.e. 28 days after last treatment. Treatment failure in 43% of dogs (3/7) on Day 21 , due to worsening or re-occurrence of skin lesions. Out of these dogs, two dogs continued the study until receiving the eight maximum administrations; one dog ended the study as clinically cured and one dog as treatment failure but with a good overall efficacy per investigator's assessment because of the cure of skin lesions in some of the body areas but not in others.

The distribution of the overall efficacy and treatment outcome assessed by the

investigator, and the percentage of dogs free of primary lesions associated to pyoderma (i.e. papules, pustules, macules and epidermal collarettes) at the end of the study (i.e. 28 days after last treatment or at premature withdrawal) is given in Table 23 below. Table 23

Primary lesions associated to pyoderma include papules, pustules, macules and epidermal collarettes.

Percentage of clinical cure assessed by investigator was slightly lower than % dogs free of primary skin lesions for both superficial and deep pyoderma, because of remaining secondary lesions (e.g. erythema, excoriation, alopecia) at study end for some cases not considered as clinically cured by the investigator.

Relapses occurred 7 days (1 dog with superficial, 1 dog with deep pyoderma) or 28 days (1 dog with superficial) after end of treatment.

Clinical success based on the percentage of dogs without primary lesions associated to pyoderma at study end:

Superficial pyoderma: 63.2 %

Deep pyoderma: 57.1 %

Effect of topical oxyclozanide on lesion type

The clinical data was analysed to determine the effect of topical oxyclozanide treatment on each of the skin lesions assessed in the clinical trial. Table 24 shows the mean % reduction in severity score compared to the mean severity score prior to treatment (Day 0) for each lesion assessed. Table 24 also shows the % of dogs with a 100% reduction at the end of the study for each lesion.

These results are also shown in Figure 38. A reduction in mean severity score was observed for all lesions and was particularly high for pustules and papules.

Safety results

17 non-serious adverse events (AE) were reported, among 10 dogs out of 30 (33%). No serious AE occurred. The adverse events consisted of:

Skin desquamation at application site (3 cases)

Hair coat coloration (1 case). Actually this cosmetic effect was observed for more dogs (10/30, 33%) but was not reported as adverse event

A diffuse hair loss (without alopecia) (1 case)

Re-occurrence of pyoderma (4 cases)

Pruritus (3 cases)

Others: Corneal ulcer (1 ), Otitis (1 ), T remors (1 ), Vocalization (1 ), Vomiting (1 No significant changes were observed in the haematological and biochemical profiles.

Conclusions

Mean total skin lesion score and pruritus scores decreased starting by 7 days following treatment initiation until 14 days (superficial) or 7 days (deep) after last treatment.

Mean total skin lesion score slightly increased at study end, mainly due to treatment failures and relapses.

In deep pyoderma, a slight and temporary rebound in skin lesions and pruritus was observed at 21 days after treatment initiation, then followed by second phase of improvement.

Clinical success based on the percentage of dogs free of primary lesions at study end (28 days after last treatment or at premature withdrawal) was: superficial pyoderma: 63.2 %; deep pyoderma: 57.1 %. (Note in deep pyoderma the number of dogs that were free of lesions following treatment was only 7 and the statistical significance of this result could not be determined).

The tested product was well tolerated. No adverse event of concern was reported.

Further Embodiments

Further embodiments of the invention are set out in the following numbered clauses

1 . A halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof, for use in the topical treatment or prevention of a skin condition in a non-human subject; wherein the skin condition is selected from (i) a skin condition or skin infection caused by or associated with Gram-positive bacteria; and (ii) an inflammatory skin condition;

2. The halogenated salicylanilide for the use of clause 1 , wherein the halogenated salicylanilide is topically administered to the subject once or twice per day during the initial treatment period. 3. The halogenated salicylanilide for the use of any of clause 1 or clause 2, wherein the treatment-free period is at least 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, 2 months, 3 months, 4 months, 6 months, 9 months or 1 year.

4. The halogenated salicylanilide for the use of any of clauses 1 to 3, wherein further halogenated salicylanilide is topically administered to the subject at the end of the treatment-free period.

5. The halogenated salicylanilide for the use of clause 4, wherein the further halogenated salicylanilide is topically administered to the subject for a second initial period (e.g. 1 , 2, 3, 4 or 5 days) followed by a second treatment-free period of at least three days during which no further halogenated salicylanilide is topically administered to the subject.

6. The halogenated salicylanilide for the use of any of clauses 1 to 3, wherein no further halogenated salicylanilide is topically administered to the subject following completion of the initial treatment period.

7. The halogenated salicylanilide for the use of any of clauses 1 to 6, wherein the topical application of the halogenated salicylanilide provides a therapeutically effective concentration of the halogenated salicylanilide in the epidermis (e.g. stratum corneum) and/or the dermis at the site of administration following completion of the initial treatment period.

8. The halogenated salicylanilide for the use of clause 7, wherein the concentration of the halogenated salicylanilide is measured in a sample of the dermis or the epidermis taken from the site of topical administration of the halogenated salicylanilide within 12 hours after completion of the initial treatment period.

9. The halogenated salicylanilide for the use of any of clauses 1 to 8, wherein the skin condition affects one or more of the, the epidermis (e.g. the stratum corneum) or the dermis.

10. The halogenated salicylanilide for the use of any of clauses 1 to 9, wherein the skin condition is a skin infection and the concentration of the halogenated salicylanilide in, the epidermis (e.g. the stratum corneum) and/or the dermis at the site of administration upon completion of the initial treatment period is at least 5 (e.g. 5 to at least 10) times the minimum inhibitory concentration of the Gram-positive bacteria responsible for or associated with the skin infection.

1 1. A halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof, for use in the topical treatment of a skin infection caused by or associated with Gram-positive bacteria in a non-human subject, wherein the halogenated salicylanilide is topically administered to the subject at the site of infection for an initial treatment period of 5 days or less to provide a concentration of the halogenated salicylanilide in the epidermis (e.g. the stratum corneum) and/or the dermis at the site of infection upon completion of the first treatment period of at least 5 times the minimum inhibitory concentration of the Gram- positive bacteria responsible for or associated with the skin infection;

and wherein the bacteria responsible for the infection are eradicated from the site of infection and/or the infection resolves without further topical administration of the halogenated salicylanilide to the subject after the initial treatment period; or

wherein no further halogenated salicylanilide is topically administered after the initial treatment period until the concentration of the halogenated salicylanilide in the epidermis (e.g. the stratum corneum) and/or the dermis at the site of administration has reduced to about 3 times the minimum inhibitory concentration of the Gram-positive bacteria responsible for or associated with the skin infection.

12. A method for the topical treatment or prevention of a skin condition in a non- human subject wherein the skin condition is selected from (i) a skin condition or skin infection caused by or associated with Gram-positive bacteria; and (ii) an inflammatory skin condition; the method comprising topically administering to the subject an effective amount of a halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof, to the subject;

13. The method of clause 13, wherein the halogenated salicylanilide is topically administered to the subject once or twice per day during the initial treatment period.

14. The method of clause 12 or clause 13, wherein the treatment-free period is at least 3 day, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 28 days, 2 months, 3 months, 4 months, 6 months, 9 months or 1 year.

15. The method of any of clauses 12 to 14, wherein further halogenated salicylanilide is topically administered to the subject at the end of the treatment-free period.

16. The method of clause 15, wherein the further halogenated salicylanilide is topically administered to the subject for a second initial period (e.g. 1 , 2, 3, 4 or 5 days) followed by a second treatment-free period of at least three days during which no further halogenated salicylanilide is topically administered to the subject.

17. The method of any of clauses 12 to 14, wherein no further halogenated salicylanilide is topically administered to the subject following completion of the initial treatment period.

18. The method of any of clauses 12 to 17, wherein the topical application of the halogenated salicylanilide provides a therapeutically effective concentration of the halogenated salicylanilide in the epidermis (e.g. stratum corneum) and/or the dermis at the site of administration following completion of the initial treatment period.

19. The method of clause 18, wherein the concentration of the halogenated salicylanilide is measured in a sample of the dermis or the epidermis taken from the site of topical administration of the halogenated salicylanilide within 12 hours after completion of the initial treatment period.

20. The method of any of clauses 12 to 19, wherein the skin condition affects one or more of the, the epidermis (e.g. the stratum corneum) or the dermis.

21. The method of any of clauses 12 to 20, wherein the skin condition is a skin infection and the concentration of the halogenated salicylanilide in, the epidermis (e.g. the stratum corneum) and/or the dermis at the site of administration upon completion of the initial treatment period is at least 5 (e.g. 5 to at least 10) times the minimum inhibitory concentration of the Gram-positive bacteria responsible for or associated with the skin infection.

22. A method for the topical treatment or prevention of a skin infection caused by or associated with Gram-positive bacteria in a non-human subject, the method comprising topically administering an effective amount of a halogenated salicylanilide, or a

pharmaceutically acceptable salt or hydrate thereof;

wherein the halogenated salicylanilide is topically administered to the subject at the site of infection for an initial treatment period of 5 days or less to provide a

concentration of the halogenated salicylanilide in the epidermis (e.g. the stratum corneum) and/or the dermis at the site of infection upon completion of the first treatment period of at least 5 times the minimum inhibitory concentration of the Gram-positive bacteria responsible for or associated with the skin infection; and wherein the bacteria responsible for the infection are eradicated from the site of infection and/or the infection resolves without further topical administration of the halogenated salicylanilide to the subject after the initial treatment period; or

23. The halogenated salicylanilide for the use of any of clauses 1 to 1 1 , or the method of any of clauses 12 to 22, wherein the concentration of the halogenated salicylanilide in a sample of the epidermis (e.g. stratum corneum) or the dermis taken from the site of topical administration of the halogenated salicylanilide within 12 hours after completion of the initial treatment period is greater than the mean plasma C_max of the halogenated

salicylanilide measured during the initial treatment period.

24. The halogenated salicylanilide for the use or the method of any of clauses 1 to 23, wherein the halogenated salicylanilide is topically applied to the site of the skin condition.

25. The halogenated salicylanilide for the use or method of any of clauses 1 to 24, wherein the skin condition is a skin infection caused by or associated with Gram-positive bacteria selected from Staphylococcus spp. or Streptococcus spp.

26. The halogenated salicylanilide for the use or method according to clause 25, wherein the Gram-positive bacteria are selected from Streptococcus uberis,

Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus intermedius and Staphylococcus schleiferi, preferably Staphylococcus pseudintermedius or

Staphylococcus aureus.

27. The halogenated salicylanilide for the use or method according to clause 25, wherein the Gram-positive bacteria are selected from Staphylococcus aureus and Staphylococcus pseudintermedius.

28. The halogenated salicylanilide for the use or method of any of clauses 1 to 27, wherein the Gram-positive bacteria is not an antibiotic resistant strain.

29. The halogenated salicylanilide for the use or method of any of clauses 1 to 27, wherein the Gram-positive bacteria is an antibiotic resistant strain. 30. The halogenated salicylanilide for the use or method of clause 29, wherein the Gram-positive bacteria is an antibiotic strain resistant to a drug selected from fusidic acid, mupirocin, retapamulin, erythromycin, clindamycin, lincomycin, a tetracycline, a b-lactam (e.g. methicillin, amoxicillin, oxacillin, dicloxacillin, flucloxacillin or a cephem (e.g. cefazolin, cefalexin or cefixime)), a cephalosprin (e.g. cephalexin or cefovecin), a fluoroquinolone (e.g. enrofloxacin, orbifloxacin, marbofloxacin or ciprofloxacin), trimethoprim, sulfamethoxazole and sulfadiazine or a combination of two or more thereof, for example wherein the bacteria are selected from methicillin resistant Staphylococcus aureus, methicillin resistant Staphylococcus pseudintermedius and methicillin resistant Staphylococcus intermedius.

31. The halogenated salicylanilide for the use or method of clause 29 or clause 30, wherein the Gram-positive bacteria are resistant to a drug selected from fusidic acid, mupirocin and retapamulin.

32. The halogenated salicylanilide for the use or method of any of clauses 1 to 31 , wherein the skin condition is a superficial skin infection caused by or associated with Gram-positive bacteria.

33. The halogenated salicylanilide for the use or method of any of clauses 1 to 31 , wherein the skin condition is a skin condition caused by or associated with Gram-positive bacteria and is selected from the group consisting of pyoderma (e.g. canine or feline pyoderma), otitis, bacterial conjunctivitis, dermatitis (including topic dermatitis, contact dermatitis, allergic contact dermatitis, irritant contact dermatitis, atopic dermatitis, seborrhoeic dermatitis, actinic dermatitis, pododermatitis, lichen simplex chronicus

(including Canine acral lick dermatitis and neurodermatitis), digital dermatitis (including bovine digital dermatitis), exfoliative dermatitis (erythroderma), carcinomatous dermatitis, nummular dermatitis, stasis dermatitis, perioral dermatitis, dermatomyositis, eczematous dermatitis, photoallergic dermatitis, phototoxic dermatitis, phytophotodermatitis and radiation-induced dermatitis, flea allergy dermatitis, malassezia dermatitis, intertrigo and food allergic dermatitis), folliculitis, superficial folliculitis, erythrasma, dermatoses, carbuncles, abscesses, cysts (including pilonidal and sebaceous cysts), furunculosis, ecthyma, cellulitis, paronychia, erysipelas, necrotising fasciitis, secondary skin infections, scabies, chronic ulcers (including varicose ulcers and decubitus ulcers), vesicular eruptions, bullous eruptions, mastitis, chronic rhinosinusitis, traumatic skin lesions

(including infected surgical wounds, burns, cuts, abrasions, animal bites or insect bites) and Staphylococcal scalded skin syndrome (SSSS or Ritter’s disease) 34. The halogenated salicylanilide for the use or method of any of clauses 1 to 31 , wherein the skin condition is a skin infection caused by or associated with Gram-positive bacteria selected from the group consisting of infected dermatitis (e.g. infected atopic dermatitis, infected canine atopic dermatitis, infected flea allergy dermatitis, infected malassezia dermatitis, infected intertrigo, infected pododermatitis, infected demodicosis, infected food allergic dermatitis or infected contact dermatitis), pyoderma (preferably canine pyoderma), otitis, infected ulcers, infected traumatic skin lesions (e.g. infected surgical wounds, burns, cuts, abrasions, animal bites or insect bites) and folliculitis.

35. The halogenated salicylanilide for the use or method of any of clauses 1 to 31 , wherein the skin condition is pyoderma, preferably canine pyoderma, for example pyotraumatic dermatitis, impetigo (superficial pustular dermatitis), superficial bacterial folliculitis, chin pyoderma (canine acne), skin fold dermatitis (intertrigo, skin fold pyoderma), mucocutaneous pyoderma, nasal pyoderma, bacterial pododermatitis, canine pedal furunculosis (interdigital bullae, interdigital pyogranuloma), more preferably the skin condition is surface or superficial pyoderma.

36. The halogenated salicylanilide for the use or method of clause 35, wherein the treatment reduces or eliminates one or more of: blistering, exudate/pus, crusting, itching, pain, erythema, or inflammation associated with the pyoderma.

37. The halogenated salicylanilide for the use or method of any of clauses 1 to 36, wherein the skin condition is a skin infection caused by or associated with Gram-positive bacteria and wherein the bacteria causing or associated with the infection are tested for susceptibility to the halogenated salicylanilide prior to or during topical treatment of the subject; and continuing the treatment if the subject is susceptible to the halogenated salicylanilide or discontinuing the treatment if the subject is resistant to the halogenated salicylanilide.

38. The halogenated salicylanilide for the use or method of any of clauses 1 to 37, wherein the Gram-positive bacteria causing or associated with the skin infection are eradicated from the site of infection within 7 days after completion of the initial treatment period (e.g. within 3 days).

39. The halogenated salicylanilide for the use or method of any of clauses 1 to 24, wherein the skin condition is an inflammatory skin condition.

40. The halogenated salicylanilide for the use or method of clause 39, wherein the skin condition is an inflammatory skin condition selected from psoriasis, dermatitis, (e.g. atopic dermatitis, contact dermatitis, allergic contact dermatitis, irritant contact dermatitis, flea allergy dermatitis, malassezia dermatitis, intertrigo, pododermatitis, food allergic dermatitis, demodicosis, seborrhoeic dermatitis, actinic dermatitis, pompholyx dermatitis, lichen simplex chronicus (including Canine acral lick dermatitis and neurodermatitis), digital dermatitis (including bovine digital dermatitis), exfoliative dermatitis (drythroderma), asteatotic dermatitis, carcinomatous dermatitis, nummular dermatitis, stasis dermatitis, perioral dermatitis, dermatomyositis, eczematous dermatitis, photoallergic dermatitis, phototoxic dermatitis, phytophotodermatitis and radiation-induced dermatitis); scleroderma, disorders of hair follicles and sebaceous glands, rhinophyma, cutaneous lupus, and inflammatory reactions (e.g. drug eruptions, erythema multiforme, erythema nodosum, or granuloma annulare), otodectic acariasis, drug eruptions, psychogenic pruritus, self- induced psychogenic hair loss, dermatophytosis, pediculosis, skin inflammation associated with scabies, skin inflammation associated with otitis, skin inflammation associated with fungal or yeast infections (e.g. dermatophytosis)

41. The halogenated salicylanilide for the use or method of clause 39, wherein the skin condition is a dermatitis (e.g. atopic dermatitis).

42. The halogenated salicylanilide for the use or method of clause 39 or clause 40, wherein the treatment reduces or eliminates one or more of pruritus, erythema, induration, lichenification, scaling, oozing and crusting associated with the inflammatory skin condition (e.g. atopic dermatitis).

43. The halogenated salicylanilide for the use or method of any of clauses 1 to 42, wherein the initial treatment period is 1 day, 2 days, 3 days or 4 days, e.g. wherein the initial treatment period is 1 day or 2 days.

44. The halogenated salicylanilide for the use or method of any of clauses 1 to 43, wherein the subject is selected from livestock or a companion animal.

45. The halogenated salicylanilide for the use or method of any of clauses 1 to 43, wherein the subject is a companion animal, preferably a dog or cat, more preferably a dog.

46. The halogenated salicylanilide for the use or method of any of clauses 1 to 45, wherein the halogenated salicylanilide is selected from rafoxanide, oxyclozanide, closantel and niclosamide or a pharmaceutically acceptable salt, solvate or ester thereof.

47. The halogenated salicylanilide for the use or method of any of clauses 1 to 45, wherein the halogenated salicylanilide is niclosamide or a pharmaceutically acceptable salt or hydrate thereof, optionally niclosamide or a pharmaceutically acceptable salt thereof, e.g. wherein the halogenated salicylanilide is niclosamide.

48. The halogenated salicylanilide for the use or method of any of clauses 1 to 45, wherein the halogenated salicylanilide is oxyclozanide, or a pharmaceutically acceptable salt or hydrate thereof, optionally oxyclozanide or a pharmaceutically acceptable salt thereof, e.g. wherein the halogenated salicylanilide is oxyclozanide.

49. The halogenated salicylanilide for the use or method of any of clauses 1 to 48, wherein the halogenated salicylanilide is topically administered in the form of a topical composition.

50. The halogenated salicylanilide for the use or method of clause 49, wherein the topical composition is selected from a topical cream, ointment, gel, paste, foam, solution, suspension, pour-on, spot-on or line-on composition.

51. The halogenated salicylanilide for the use or method of clause 49 or 50, wherein the topical composition comprises the halogenated salicylanilide and a base selected from an oleaginous base (e.g. petrolatum, white petrolatum, yellow ointment or white ointment), an absorption base (e.g. hydrophilic petrolatum or lanolin), a water-removable base (oil in water emulsion); a water-soluble base (e.g. a polyethylene glycol).

52. The halogenated salicylanilide for the use or method of any of clauses 49 to 51 , wherein the topical composition is a non-aqueous composition.

53. The halogenated salicylanilide for the use or method of any of clauses 49 to 51 , wherein the topical composition is an aqueous composition.

54. The halogenated salicylanilide for the use or method of clause 49 or clause 50, wherein the topical composition is a non-aqueous topical composition comprising:

(i) the halogenated salicylanilide (e.g. selected from niclosamide, rafoxanide, oxyclozanide and closantel), or a pharmaceutically acceptable salt or hydrate thereof; and

55. The halogenated salicylanilide for the use or method of clause 56, wherein the topical composition is a non-aqueous topical composition comprising:

(i) 0.01 to 4.5% (e.g. 0.1 to 3% or about 2%) by weight of the halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof; and (ii) at least 70 % (e.g. at least 90%) by weight of a PEG, wherein the average molecular weight of the PEG is 600 or less (e.g. less than 600 or from about 200 to about 600 or about 400).

56. The halogenated salicylanilide for the use or method of clause 54 or clause 55, wherein the topical composition further comprises a non-polymeric glycol (e.g. propylene glycol).

57. The halogenated salicylanilide for the use or method of any of clauses 49 to 56, wherein the halogenated salicylanilide is dissolved or partially dissolved in the composition.

58. The halogenated salicylanilide for the use or method of any of clauses 1 to 57, wherein compositions A, B and/or C are excluded when:

(a) the composition is topically administered to the subject for 1 day, 2 days or more than 3 days; and/or

(b) the composition is topically administered to the subject for 1 day, 2 days or more than 3 days, wherein the composition is topically administered to the subject at a frequency of once per day, twice per day, three times per day, four times per day, once every other day or once per week; and/or

(c) the composition is topically administered to the subject once per week; wherein

Composition A is a non-aqueous topical composition comprising:

(ii) polyethylene glycol (PEG) with a melting point of less than 40°C;

Composition B is a non-aqueous topical composition comprising:

(i) a halogenated salicylanilide selected from niclosamide, rafoxanide, oxyclozanide and closantel, or a pharmaceutically acceptable salt or hydrate thereof; and (ii) greater than 60 % by weight of a polyethylene glycol (PEG), wherein the average molecular weight of the PEG is 600 or less.

Composition C is a non-aqueous topical gel composition comprising:

(iii) a gel-forming agent.

59. A topical composition comprising 2 to 20 % wt/v, preferably 5 to 15 % wt/v, more preferably, 8 to 12 % wt/v of a halogenated salicylanilide (e.g. niclosamide or oxyclozanide or a pharmaceutically acceptable salt or hydrate thereof);

25 to 55% w/v (preferably 30 to 55% wt/v, more preferably 35 to 50% w/v of a glycol ether (e.g. 2-(2-eth oxyeth oxy )eth an ol ) ;

and 0 to 10% w/v (preferably 0 to 5% w/v (e.g. 0%, or 1 to 5% w/v) alkanol amine (e.g. ethanolamine.

60. The topical composition of clause 59 selected from composition is selected from Formulation A’ to G shown in the table below:

61. The topical composition of clause 59 or clause 60 wherein the halogenated salicylanilide is oxyclozanide or a pharmaceutically acceptable salt or hydrate thereof. 62. The topical composition of any of clauses 59 to 61 , wherein composition is in the form of a spot-on or line-on composition

Claims

1. A halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof, for use in the topical treatment or prevention of a skin condition in a non-human subject; wherein the skin condition is selected from (i) a skin condition or skin infection caused by or associated with Gram-positive bacteria; and (ii) an inflammatory skin condition;

wherein the halogenated salicylanilide is topically administered to the subject for an initial treatment period of 5 days or less followed by a treatment-free period of at least 2 days (preferably at least 3 days) during which no further halogenated salicylanilide is topically administered to the subject;

with the proviso that Compositions W and X for use in the treatment or prevention of pyoderma or dermatitis in a non-human mammal are excluded, wherein:

Composition W is a topical veterinary spot on or line on composition comprising 2 to 20 wt/v % of at least one halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof, 35 to 55 wt/v % dimethyl sulfoxide, wherein the at least one halogenated salicylanilide is selected from niclosamide and/or oxyclozanide and wherein the composition is dissolved in diethylene glycol monomethyl ether; and

Composition X is a topical veterinary spot on or line on composition comprising 2 to 20 wt/v % of at least one halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof, 35 to 55 wt/v % dimethyl sulfoxide, wherein the at least one halogenated salicylanilide is selected from niclosamide and/or oxyclozanide and wherein the composition is dissolved in diethylene glycol monoethyl ether.

2. The halogenated salicylanilide for the use of claim 1 , wherein the halogenated salicylanilide is topically administered to the subject once or twice per day during the initial treatment period.

3. The halogenated salicylanilide for the use of claim 1 or claim 2, wherein the initial treatment period is one day.

4. The halogenated salicylanilide for the use of any of claims 1 to 3, wherein the halogenated salicylanilide is topically administered to the subject once per day during the initial treatment period.

5. The halogenated salicylanilide for the use of any of claims 1 to 4, wherein the treatment-free period is at least 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, 2 months, 3 months, 4 months, 6 months, 9 months or 1 year.

6. The halogenated salicylanilide for the use of any of claims 1 to 4, wherein the treatment free period is at least 5 days, for example 5 days to 10 days, preferably 7 days.

7. The halogenated salicylanilide for the use of any of claims 1 to 6, wherein further halogenated salicylanilide is topically administered to the subject at the end of the treatment-free period.

8. The halogenated salicylanilide for the use of claim 7, wherein the further halogenated salicylanilide is topically administered in accordance with the dosage regimen defined in any one of claims 1 to 6, optionally wherein said dosage regimen is repeated 2 or more times.

9. The halogenated salicylanilide for the use of claim 7, wherein the further halogenated salicylanilide is topically administered to the subject for a second initial period (e.g. 1 , 2, 3, 4 or 5 days) followed by a second treatment-free period of at least three days during which no further halogenated salicylanilide is topically administered to the subject.

10. The halogenated salicylanilide for the use of any of claims 1 to 6, wherein no further halogenated salicylanilide is topically administered to the subject following completion of the initial treatment period.

1 1. The halogenated salicylanilide for the use of any of claims 1 to 6, wherein the topical application of the halogenated salicylanilide provides a therapeutically effective concentration of the halogenated salicylanilide in the epidermis (e.g. stratum corneum) and/or the dermis at the site of administration following completion of the initial treatment period.

12. The halogenated salicylanilide for the use of claim 1 1 , wherein the concentration of the halogenated salicylanilide is measured in a sample of the dermis or the epidermis taken from the site of topical administration of the halogenated salicylanilide within 12 hours after completion of the initial treatment period.

13. The halogenated salicylanilide for the use of any of claims 1 to 12, wherein the skin condition affects one or more of the, the epidermis (e.g. the stratum corneum) or the dermis.

14. The halogenated salicylanilide for the use of any of claims 1 to 13, wherein the skin condition is a skin infection and the concentration of the halogenated salicylanilide in, the epidermis (e.g. the stratum corneum) and/or the dermis at the site of administration upon completion of the initial treatment period is at least 5 (e.g. 5 to at least 10) times the minimum inhibitory concentration of the Gram-positive bacteria responsible for or associated with the skin infection.

15. A halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof, for use in the topical treatment of a skin infection caused by or associated with Gram-positive bacteria in a non-human subject, wherein the halogenated salicylanilide is topically administered to the subject at the site of infection for an initial treatment period of 5 days or less to provide a concentration of the halogenated salicylanilide in the epidermis (e.g. the stratum corneum) and/or the dermis at the site of infection upon completion of the first treatment period of at least 5 times the minimum inhibitory concentration of the Gram- positive bacteria responsible for or associated with the skin infection;

16. A method for the topical treatment or prevention of a skin condition in a non- human subject wherein the skin condition is selected from (i) a skin condition or skin infection caused by or associated with Gram-positive bacteria; and (ii) an inflammatory skin condition; the method comprising topically administering to the subject an effective amount of a halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof, to the subject;

wherein the halogenated salicylanilide is topically administered to the subject for an initial treatment period of 5 days or less followed by a treatment-free period of at least 2 days (preferably at least 3 days) during which no further halogenated salicylanilide is topically administered to the subject; with the proviso that Compositions W and X for use in the treatment or prevention of pyoderma or dermatitis in a non-human mammal are excluded, wherein:

17. The method of claim 16, wherein the halogenated salicylanilide is topically administered to the subject once or twice per day during the initial treatment period.

18. The halogenated salicylanilide for the use of claim 16 or claim 17, wherein the initial treatment period is one day.

19. The halogenated salicylanilide for the use of any of claims 16 to 18, wherein the halogenated salicylanilide is topically administered to the subject once per day during the initial treatment period.

20. The method of any one of claims 16 to 19, , wherein the treatment-free period is at least 3 day, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 28 days, 2 months, 3 months, 4 months, 6 months, 9 months or 1 year; optionally wherein the treatment free period is at least 5 days, for example 5 days to 10 days, preferably 7 days.

21. The method of any of claims 16 to 20, wherein further halogenated salicylanilide is topically administered to the subject at the end of the treatment-free period.

22. The method of any of claims 16 to 20, wherein no further halogenated

salicylanilide is topically administered to the subject following completion of the initial treatment period.

23. The method of any of claims 16 to 22, wherein the topical application of the halogenated salicylanilide provides a therapeutically effective concentration of the halogenated salicylanilide in the epidermis (e.g. stratum corneum) and/or the dermis at the site of administration following completion of the initial treatment period.

24. The method of claim 23, wherein the concentration of the halogenated

salicylanilide is measured in a sample of the dermis or the epidermis taken from the site of topical administration of the halogenated salicylanilide within 12 hours after completion of the initial treatment period.

25. The method of any of claims 16 to 24, wherein the skin condition affects one or more of the, the epidermis (e.g. the stratum corneum) or the dermis.

26. The method of any of claims 16 to 25, wherein the skin condition is a skin infection and the concentration of the halogenated salicylanilide in, the epidermis (e.g. the stratum corneum) and/or the dermis at the site of administration upon completion of the initial treatment period is at least 5 (e.g. 5 to at least 10) times the minimum inhibitory concentration of the Gram-positive bacteria responsible for or associated with the skin infection.

27. A method for the topical treatment or prevention of a skin infection caused by or associated with Gram-positive bacteria in a non-human subject, the method comprising topically administering an effective amount of a halogenated salicylanilide, or a

pharmaceutically acceptable salt or hydrate thereof;

concentration of the halogenated salicylanilide in the epidermis (e.g. the stratum corneum) and/or the dermis at the site of infection upon completion of the first treatment period of at least 5 times the minimum inhibitory concentration of the Gram-positive bacteria responsible for or associated with the skin infection;

28. The halogenated salicylanilide for the use of any of claims 1 to 15, or the method of any of claims 16 to 27, wherein the concentration of the halogenated salicylanilide in a sample of the epidermis (e.g. stratum corneum) or the dermis taken from the site of topical administration of the halogenated salicylanilide within 12 hours after completion of the initial treatment period is greater than the mean plasma C_max of the halogenated

salicylanilide measured during the initial treatment period.

29. The halogenated salicylanilide for the use or the method of any of claims 1 to 28, wherein the halogenated salicylanilide is topically applied to the site of the skin condition.

30. The halogenated salicylanilide for the use or method of any of claims 1 to 29, wherein the skin condition is a skin infection caused by or associated with Gram-positive bacteria selected from Staphylococcus spp. or Streptococcus spp.

31. The halogenated salicylanilide for the use or method according of any of claims 1 to 29, wherein the Gram-positive bacteria are selected from Streptococcus uberis, Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus intermedius and Staphylococcus schleiferi, preferably Staphylococcus pseudintermedius or

Staphylococcus aureus.

32. The halogenated salicylanilide for the use or method according to of any of claims 1 to 29, wherein the Gram-positive bacteria are selected from Staphylococcus aureus and Staphylococcus pseudintermedius, preferably wherein the Gram-positive bacteria are Staphylococcus pseudintermedius.

33. The halogenated salicylanilide for the use or method of any of claims 1 to 32, wherein the Gram-positive bacteria is not an antibiotic resistant strain.

34. The halogenated salicylanilide for the use or method of any of claims 1 to 32, wherein the Gram-positive bacteria is an antibiotic resistant strain.

35. The halogenated salicylanilide for the use or method of claim 34, wherein the Gram-positive bacteria areresistant to a drug selected from fusidic acid, mupirocin, retapamulin, erythromycin, clindamycin, lincomycin, a tetracycline, a b-lactam (e.g. methicillin, amoxicillin, oxacillin, dicloxacillin, flucloxacillin or a cephem (e.g. cefazolin, cefalexin or cefixime)), a cephalosprin (e.g. cephalexin or cefovecin), a fluoroquinolone (e.g. enrofloxacin, orbifloxacin, marbofloxacin or ciprofloxacin), trimethoprim, sulfamethoxazole and sulfadiazine or a combination of two or more thereof, for example wherein the bacteria are selected from methicillin resistant Staphylococcus aureus, methicillin resistant Staphylococcus pseudintermedius and methicillin resistant Staphylococcus intermedius.

36. The halogenated salicylanilide for the use or method of claim 34 or claim 35, wherein the Gram-positive bacteria are resistant to a drug selected from fusidic acid, mupirocin and retapamulin.

37. The halogenated salicylanilide for the use or method of any of claims 1 to 36, wherein the skin condition is a superficial skin infection caused by or associated with Gram-positive bacteria.

38. The halogenated salicylanilide for the use or method of ony of claims 30 to 37, wherein the skin infection is in skin that has a compromised barrier function, for example in skin affected by dermatitis (e.g. atopic dermatitis).

39. The halogenated salicylanilide for the use or method of any of claims 1 to 38, wherein the skin condition is a skin condition caused by or associated with Gram-positive bacteria and is selected from the group consisting of pyoderma (e.g. canine or feline pyoderma), otitis, bacterial conjunctivitis, dermatitis (including topic dermatitis, contact dermatitis, allergic contact dermatitis, irritant contact dermatitis, atopic dermatitis, seborrhoeic dermatitis, actinic dermatitis, pododermatitis, lichen simplex chronicus

(including infected surgical wounds, burns, cuts, abrasions, animal bites or insect bites) and Staphylococcal scalded skin syndrome (SSSS or Ritter’s disease)

40. The halogenated salicylanilide for the use or method of any of claims 1 to 36, wherein the skin condition is a skin infection caused by or associated with Gram-positive bacteria selected from the group consisting of infected dermatitis (e.g. infected atopic dermatitis, infected canine atopic dermatitis, infected flea allergy dermatitis, infected malassezia dermatitis, infected intertrigo, infected pododermatitis, infected demodicosis, infected food allergic dermatitis or infected contact dermatitis), pyoderma (preferably canine pyoderma), otitis, infected ulcers, infected traumatic skin lesions (e.g. infected surgical wounds, burns, cuts, abrasions, animal bites or insect bites) and folliculitis.

41. The halogenated salicylanilide for the use or method of any of claims 1 to 36, wherein the skin condition is pyoderma, preferably canine pyoderma, for example pyotraumatic dermatitis, impetigo (superficial pustular dermatitis), superficial bacterial folliculitis, chin pyoderma (canine acne), skin fold dermatitis (intertrigo, skin fold pyoderma), mucocutaneous pyoderma, nasal pyoderma, bacterial pododermatitis, canine pedal furunculosis (interdigital bullae, interdigital pyogranuloma), more preferably the skin condition is surface or superficial pyoderma.

42. The halogenated salicylanilide for the use or method of any of claims 1 to 36, wherein the skin condition is superficial pyoderma.

43. The halogenated salicylanilide for the use or method of any of claims 1 to 36, wherein the skin condition is deep pyoderma.

44. The halogenated salicylanilide for the use or method of any of claims 41 to 43, , wherein the treatment reduces or eliminates one or more of: blistering, exudate/pus, crusting, itching, pain, erythema, or inflammation caused by or associated with the pyoderma, for example wherein the treatment reduces or eliminates one or more of papules and pustules caused by or associated with the pyoderma.

45. The halogenated salicylanilide for the use or method of any of claims 1 to 44, wherein the skin condition is a skin infection caused by or associated with Gram-positive bacteria and wherein the bacteria causing or associated with the infection are tested for susceptibility to the halogenated salicylanilide prior to or during topical treatment of the subject; and continuing the treatment if the subject is susceptible to the halogenated salicylanilide or discontinuing the treatment if the subject is resistant to the halogenated salicylanilide.

46. The halogenated salicylanilide for the use or method of any of claims 1 to 45, wherein the Gram-positive bacteria causing or associated with the skin infection are eradicated from the site of infection within 7 days after completion of the initial treatment period (e.g. within 3 days).

47. The halogenated salicylanilide for the use or method of any of claims 1 to 29, wherein the skin condition is an inflammatory skin condition.

48. The halogenated salicylanilide for the use or method of claim 47, wherein the skin condition is an inflammatory skin condition selected from psoriasis, dermatitis, (e.g. atopic dermatitis, contact dermatitis, allergic contact dermatitis, irritant contact dermatitis, flea allergy dermatitis, malassezia dermatitis, intertrigo, pododermatitis, food allergic dermatitis, demodicosis, seborrhoeic dermatitis, actinic dermatitis, pompholyx dermatitis, lichen simplex chronicus (including Canine acral lick dermatitis and neurodermatitis), digital dermatitis (including bovine digital dermatitis), exfoliative dermatitis (drythroderma), asteatotic dermatitis, carcinomatous dermatitis, nummular dermatitis, stasis dermatitis, perioral dermatitis, dermatomyositis, eczematous dermatitis, photoallergic dermatitis, phototoxic dermatitis, phytophotodermatitis and radiation-induced dermatitis); scleroderma, disorders of hair follicles and sebaceous glands, rhinophyma, cutaneous lupus, and inflammatory reactions (e.g. drug eruptions, erythema multiforme, erythema nodosum, or granuloma annulare), otodectic acariasis, drug eruptions, psychogenic pruritus, self- induced psychogenic hair loss, dermatophytosis, pediculosis, skin inflammation associated with scabies, skin inflammation associated with otitis, skin inflammation associated with fungal or yeast infections (e.g. dermatophytosis)

49. The halogenated salicylanilide for the use or method of claim 47, wherein the skin condition is a dermatitis (e.g. atopic dermatitis).

50. The halogenated salicylanilide for the use or method of any of claims 47 to 49, wherein the treatment reduces or eliminates one or more of pruritus, erythema, induration, lichenification, scaling, oozing and crusting caused by or associated with the inflammatory skin condition (e.g. atopic dermatitis).

51 . The halogenated salicylanilide for the use or method of any of claims 1 to 50, wherein the initial treatment period is 1 day, 2 days, 3 days or 4 days, e.g. wherein the initial treatment period is 1 day or 2 days.

52. The halogenated salicylanilide for the use or method of any of claims 1 to 51 , wherein the subject is selected from livestock or a companion animal.

53. The halogenated salicylanilide for the use or method of any of claims 1 to 51 , wherein the subject is a companion animal, preferably a dog or cat, more preferably a dog.

54. The halogenated salicylanilide for the use or method of any of claims 1 to 53, wherein the halogenated salicylanilide is selected from rafoxanide, oxyclozanide, closantel and niclosamide or a pharmaceutically acceptable salt, solvate or ester thereof.

55. The halogenated salicylanilide for the use or method of any of claims 1 to 53, wherein the halogenated salicylanilide is niclosamide or a pharmaceutically acceptable salt or hydrate thereof, optionally niclosamide or a pharmaceutically acceptable salt thereof, e.g. wherein the halogenated salicylanilide is niclosamide.

56. The halogenated salicylanilide for the use or method of any of claims 1 to 53, wherein the halogenated salicylanilide is oxyclozanide, or a pharmaceutically acceptable salt or hydrate thereof, optionally oxyclozanide or a pharmaceutically acceptable salt thereof, e.g. wherein the halogenated salicylanilide is oxyclozanide.

57. The halogenated salicylanilide for the use or method of any of claims 1 to 56, wherein the halogenated salicylanilide is topically administered in the form of a topical composition.

58. The halogenated salicylanilide for the use or method of claim 57, wherein the topical composition is selected from a topical cream, ointment, gel, paste, foam, solution, suspension, pour-on, spot-on or line-on composition.

59. The halogenated salicylanilide for the use or method of claim 57 or 58, wherein the topical composition comprises the halogenated salicylanilide and a base selected from an oleaginous base (e.g. petrolatum, white petrolatum, yellow ointment or white ointment), an absorption base (e.g. hydrophilic petrolatum or lanolin), a water-removable base (oil in water emulsion); a water-soluble base (e.g. a polyethylene glycol).

60. The halogenated salicylanilide for the use or method of any of claims 57 to 59, wherein the topical composition is a non-aqueous composition.

61. The halogenated salicylanilide for the use or method of any of claims 57 to 59, wherein the topical composition is an aqueous composition.

62. The halogenated salicylanilide for the use or method of claim 57 or claim 58, wherein the topical composition is a non-aqueous topical composition comprising:

63. The halogenated salicylanilide for the use or method of claim 62, wherein the topical composition is a non-aqueous topical composition comprising: (i) 0.01 to 7.5 % (e.g. 0.01 to 4.5 %, or 0.1 to 3%, or about 2 %) by weight of the halogenated salicylanilide, or a pharmaceutically acceptable salt or hydrate thereof; and

(ii) at least 70 % (e.g. at least 90%) by weight of a PEG, wherein the average molecular weight of the PEG is 600 or less (e.g. less than 600 or from about 200 to about 600 or about 400).

64. The halogenated salicylanilide for the use or method of claim 62 or claim 63, wherein the topical composition further comprises a non-polymeric glycol (e.g. propylene glycol).

65. The halogenated salicylanilide for the use or method of any of claims 57 to 64, wherein the halogenated salicylanilide is dissolved or partially dissolved in the topical composition.

66. The halogenated salicylanilide for the use or method of any of claims 57 to 64, wherein the halogenated salicylanilide is present in the composition at a concentration of from 0.5 mg/ml to 200 mg/ml, optionally at a concentration of from 50 mg/ml_ to 100 mg/ml_, for example 100 mg/ml_.

67. The halogenated salicylanilide for the use or method of any of claims 1 to 66, wherein compositions A, B and/or C are excluded when:

(d) the composition is topically administered to the subject for 1 day, 2 days or more than 3 days; and/or

(e) the composition is topically administered to the subject for 1 day, 2 days or more than 3 days, wherein the composition is topically administered to the subject at a frequency of once per day, twice per day, three times per day, four times per day, once every other day or once per week; and/or

(f) the composition is topically administered to the subject once per week; wherein

Composition A is a non-aqueous topical composition comprising:

(ii) polyethylene glycol (PEG) with a melting point of less than 40°C; with the proviso that when the composition comprises niclosamide or rafoxanide, or a pharmaceutically acceptable salt thereof, the composition further comprises a gel forming agent or a non-polymeric glycol;

Composition B is a non-aqueous topical composition comprising:

Composition C is a non-aqueous topical gel composition comprising:

(iii) a gel-forming agent.