Halogenated salicylanilides for treating <i>Clostridium </i>infections

Abstract

The present invention relates to halogenated salicylanilides, or pharmaceutically acceptable salts or esters thereof, for use in the treatment of an infection in a subject caused by Clostridium bacteria, particularly a C. difficile infection. The halogenated salicylanilides are expected to be useful in the treatment of C. difficile associated diseases including C. difficile associated diarrhoea and C. difficile associated colitis.

Claims

1. A method of treating an infection caused by Clostridium difficile bacteria in a subject, the method comprising administering to said subject an effective amount of a halogenated salicylanilide, or a pharmaceutically acceptable salt or ester thereof, wherein the halogenated salicylanilide is of the formula: ##STR00009## wherein R.sup.1 and R.sup.2are each independently halo; L.sup.1 is selected from the group consisting of a bond and O; R.sup.7 is phenyl, unsubstituted or substituted with 1 or 2 groups each independently selected from the group consisting of halo and C.sub.1-4 alkyl; n is 1 or 2; p is 0 or 1.

2. The method of claim 1, wherein R.sup.1 is independently selected from I and Cl.

3. The method of claim 1, wherein n is 2 and R.sup.1 is independently selected from I and Cl.

4. The method of claim 1, wherein n is 2 and R.sup.1 is Cl.

5. The method of claim 1, wherein L.sup.1 is O and R.sup.7 is phenyl substituted with 1 halo.

6. The method of claim 1, wherein L.sup.1 is O and R.sup.7 is phenyl substituted with 1 Cl.

7. The method of claim 1, wherein p is 0.

8. The method of claim 1, wherein: R.sup.1 is Cl; L.sup.1 is O; R.sup.7 is phenyl substituted with 1 Cl; n is 2; and p is 0.

9. The method of claim 1, wherein the Clostridium difficile infection is associated with a disease selected from the group consisting of diarrhoea, colitis, pseudomembranous colitis and toxic megacolon.

10. The method of claim 1, wherein the Clostridium difficile infection is an antibiotic induced Clostridium difficile infection, wherein the antibiotic which induced the infection is other than the halogenated salicylanilide.

11. The method of claim 10, wherein the antibiotic which induced the infection is selected from clindamycin, a cephalosporin, cefotaxime, ceftazidime, ampicillin, amoxicillin, a quinolone, a fluoroquinolone, ciprofloxaxin and levofloxacin.

12. The method of claim 1, wherein the Clostridium difficile infection is induced by a gastric acid suppressive agent.

13. The method of claim 1, wherein the Clostridium difficile is resistant to an antibiotic agent other than the halogenated salicylanilide.

14. The method of claim 13, wherein the Clostridium difficile is a Clostridium difficile strain that is resistant to an antibiotic agent selected from metronidazole, vancomycin, fidaxomicin and a rifamycin.

15. The method of claim 1, wherein the Clostridium difficile infection has not been treated with an antibiotic prior to administration of the halogenated salicylanilide to the subject.

16. The method of claim 1, wherein the subject has a recurrent Clostridium difficile infection.

17. The method of claim 16, wherein the recurrent Clostridium difficile infection has recurred following prior treatment with an antibiotic other than the halogenated salicylanilide.

18. The method of claim 16, wherein the Clostridium difficile infection has recurred after being treated with an antibiotic selected from metronidazole, vancomycin, fidaxomicin and a rifamycin.

19. The method of claim 1, wherein treatment of the subject with the halogenated salicylanilide, or a pharmaceutically acceptable salt or ester thereof, prevents or inhibits sporulation of C. difficile in the subject.

20. The method of claim 1, wherein the halogenated salicylanilide, or a pharmaceutically acceptable salt or ester thereof, is orally administered to the subject.

21. The method of claim 1, wherein the subject is a human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the development of heat-resistant spore count over time for Clostridium difficile 7-6011209 in the presence of rafoxanide or fidaxomicin at a concentration of 8-fold above the MIC for rafoxanide and >8-fold above the MIC for fidaxomicin. The control shows the spore count over time in the Clospore medium used in the study.

DETAILED DESCRIPTION

Definitions

(2) Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

(3) It is to be appreciated that references to treating or treatment include prophylaxis as well as the alleviation of established symptoms of a condition. Treating or treatment of a state, disorder or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a subject, for example a human, that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms. Accordingly in the context of treating infections caused by a Clostridium bacteria includes:

(4) (i) the prevention of a disease caused by Clostridium species, particularly Clostridium difficile;

(5) (ii) the suppression of a disease caused by Clostridium species, particularly Clostridium difficile:

(6) (iii) the relief of symptoms of a disease caused by Clostridium species, particularly Clostridium difficile;

(7) iv) the eradication of a non-symptomatic colonization by Clostridium species, particularly Clostridium difficile from an area on or in the body;

(8) (v) the eradication of a Clostridium difficile symptomatic infection;

(9) (vi) the eradication a Clostridium species, particularly Clostridium dimffcile; from an area of the body affected by another disease that could enable establishment of an infection more readily, than in a non-disease affected areae.g. in the intestinal tract;
(vii) the suppression of a disease caused a Clostridium infection, particularly Clostridium difficile; from an area of the body affected by another noninfectious disease that enables establishment of an infection more readily, than in a non-disease affected area;
(viii) preventing or reducing the risk of transmission or spread of a Clostridium infection, particularly Clostridium difficile; or
(ix) preventing or reducing the risk of recurrence of a Clostridium infection, particularly Clostridium difficile.

(10) A therapeutically effective amount means the amount of a compound that, when administered to a subject, for example a human, for treating a disease, is sufficient to effect such treatment for the disease. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

(11) Minimum inhibitory concentration (MIC) is the lowest concentration of an antibacterial that will inhibit the visible growth of a microorganism after overnight incubation. Minimum inhibitory concentrations are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents. A MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism.

(12) The median lethal dose, LD50 (abbreviation for lethal dose, 50%) of a toxin, radiation, or pathogen is the dose required to kill half the members of a tested population after a specified test duration. LD50 figures are frequently used as a general indicator of a substance's acute toxicity.

(13) Therapeutic index (therapeutic ratio) is defined as the amount of a therapeutic agent causing the therapeutic effect measured as MIC to the amount that causes death in animal studies measured as LD50.

(14) The rate of resistance development is quantified as the frequency of spontaneous mutants in a population of bacteria that is able to resist a given concentration of an antibiotic. For example the rate of resistance development may be 10.sup.9 if on average 1 cell in 10.sup.9 cells is able to survive a concentration of antibiotic corresponding to 1MIC incubated at 37 C. for 48 hours using the method described in Drago et al. Journal of Antimicrobial Chemotherapy, 2005, 56(2), 353 to 359).

(15) In microbiology, colony-forming unit (CFU) is an estimate of the number of viable bacteria or fungal cells in a sample. Viable is defined as the ability to multiply via binary fission under the controlled conditions.

(16) 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.

(17) The term C.sub.m-C.sub.n refers to a group with m to n carbon atoms.

(18) The term C.sub.1-C.sub.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. C.sub.1-C.sub.4 alkyl similarly refers to such groups containing up to 4 carbon atoms.

(19) The term optionally substituted refers to either groups, structures, or molecules that are substituted and those that are not substituted.

(20) Where optional substituents are chosen from one or more groups it is to be understood that this definition includes all substituents being chosen from one of the specified groups or the substituents being chosen from two or more of the specified groups.

(21) Where a moiety is substituted, it may be substituted at any point on the moiety where chemically possible and consistent with atomic valency requirements. The moiety may be substituted by one or more substituents, e.g. 1, 2, 3 or 4 substituents; optionally there are 1 or 2 substituents on a group. Where there are two or more substituents, the substituents may be the same or different.

(22) Substituents are only present at positions where they are chemically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without undue effort which substitutions are chemically possible and which are not.

(23) 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.

(24) 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.

(25) 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.

(26) Suitable or preferred features of any compounds of the present invention may also be suitable features of any other aspect.

(27) The invention contemplates pharmaceutically acceptable salts of the halogenated salicylanilide compounds of the invention. These may include the acid addition and base salts of the compounds. These may be acid addition and base salts of the compounds. Suitable acid addition salts are formed from acids which form non-toxic salts. Suitable base salts are formed from bases which form non-toxic salts.

(28) Pharmaceutically acceptable salts of the halogenated salicylanilide compounds may be prepared by for example, one or more of the following methods:

(29) (i) by reacting the compound of the invention with the desired acid or base; or

(30) (ii) by converting one salt of the compound of the invention to another by reaction with an appropriate acid or base or by means of a suitable ion exchange column.

(31) These methods are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised.

(32) Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed isomers. Isomers that differ in the arrangement of their atoms in space are termed stereoisomers. Stereoisomers that are not mirror images of one another are termed diastereomers and those that are non-superimposable mirror images of each other are termed enantiomers. When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or ()-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a racemic mixture. Where a compound of the invention has two or more stereocentres any combination of (R) and (S) stereoisomers is contemplated. The combination of (R) and (S) stereoisomers may result in a diastereomeric mixture or a single diastereoisomer. The compounds of the invention may be present as a single stereoisomer or may be mixtures of stereoisomers, for example racemic mixtures and other enantiomeric mixtures, and diastereomeric mixtures. Where the mixture is a mixture of enantiomers the enantiomeric excess may be any of those disclosed above. Where the compound is a single stereoisomer the compounds may still contain other diastereoisomers or enantiomers as impurities. Hence a single stereoisomer does not necessarily have an enantiomeric excess (e.e.) or diastereomeric excess (d.e.) of 100% but could have an e.e. or d.e. of about at least 85%

(33) The compounds of this invention may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)-stereoisomers or as mixtures thereof. Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see discussion in Chapter 4 of Advanced Organic Chemistry, 4th edition J. March, John Wiley and Sons, New York, 2001), for example by synthesis from optically active starting materials or by resolution of a racemic form. Some of the compounds of the invention may have geometric isomeric centres (E- and Z-isomers). It is to be understood that the present invention encompasses all optical, diastereoisomers and geometric isomers and mixtures thereof that possess activity against Clostridium bacteria, for example C. difficile.

(34) It is also to be understood that certain compounds of the invention, or salts or esters thereof, may exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms that possess activity against Clostridium bacteria, for example C. difficile.

(35) It is also to be understood that the halogenated salicylanilides of the invention may exhibit polymorphism, and that the invention encompasses all such forms that possess activity against Clostridium bacteria, for example C. difficile.

(36) It is further to be understood that the halogenated salicylanilide may be used in the form of suitable pharmaceutically-acceptable pro-drug of the compound and that such prodrugs are intended to be encompassed by the invention. Accordingly, halogenated salicylanilide may be administered in the form of a pro-drug, that is a compound that is broken down in the human or animal body to release a compound of the invention. A pro-drug may be used to alter the physical properties and/or the pharmacokinetic properties of a compound of the invention. A pro-drug can be formed when the compound of the invention contains a suitable group or substituent to which a property-modifying group can be attached. Examples of pro-drugs include in vivo cleavable ester derivatives that may be formed at a hydroxy group in a compound.

(37) Accordingly, the present invention includes the halogenated salicylanilides as defined hereinbefore when made available by organic synthesis and when made available within the human or animal body by way of cleavage of a pro-drug thereof. Accordingly, the present invention includes those halogenated salicylanilide compounds that are produced by organic synthetic means and also such compounds that are produced in the human or animal body by way of metabolism of a precursor compound, that is the halogenated salicylanilide may be a synthetically-produced compound or a metabolically-produced compound.

(38) A suitable pharmaceutically-acceptable pro-drug of a halogenated salicylanilide compound is one that is based on reasonable medical judgement as being suitable for administration to the human or animal body without undesirable pharmacological activities and without undue toxicity.

(39) Various forms of pro-drug have been described, for example in the following documents:

(40) a) Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985);

(41) b) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985);

(42) c) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 Design and Application of Pro-drugs, by H. Bundgaard p. 113-191 (1991);

(43) d) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992);

(44) e) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988);

(45) f) N. Kakeya, et al., Chem. Pharm. Bull., 22, 692 (1984);

(46) g) T. Higuchi and V. Stella, Pro-Drugs as Novel Delivery Systems, A.C.S. Symposium Series, Volume 14; and

(47) h) E. Roche (editor), Bioreversible Carriers in Drug Design, Pergamon Press, 1987.

(48) The halogenated salicylanilide may be used in the form of a prodrug of the compound for example, an in vivo cleavable ester thereof. An in vivo cleavable ester of a compound may be, for example, a pharmaceutically-acceptable ester which is cleaved in the human or animal body to produce the parent compound.

(49) A suitable pharmaceutically-acceptable pro-drug of a halogenated salicylanilide that possesses a hydroxy group is, for example, an in vivo cleavable ester or ether thereof. An in vivo cleavable ester or ether of a compound containing a hydroxy group is, for example, a pharmaceutically-acceptable ester or ether which is cleaved in the human or animal body to produce the parent hydroxy compound. Suitable pharmaceutically-acceptable ester forming groups for a hydroxy group include inorganic esters such as phosphate esters (including phosphoramidic cyclic esters). Further suitable pharmaceutically-acceptable ester forming groups for a hydroxy group include C.sub.1-10alkanoyl groups such as acetyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl groups, C.sub.1-10alkoxycarbonyl groups such as ethoxycarbonyl, N,N(C.sub.1-6).sub.2carbamoyl, 2-dialkylaminoacetyl and 2-carboxyacetyl groups. Examples of ring substituents on the phenylacetyl and benzoyl groups include aminomethyl, N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl, piperazin-1-ylmethyl and 4-(C.sub.1-4alkyl)piperazin-1-ylmethyl. Suitable pharmaceutically-acceptable ether forming groups for a hydroxy group include -acyloxyalkyl groups such as acetoxymethyl and pivaloyloxymethyl groups. Accordingly reference to a pharmaceutically acceptable ester of a compound encompasses the esters described above.

(50) Halogenated Salicylanilides

(51) The halogenated salicylanilide used in the present invention may be any of the halogenated salicylanilides described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable sat thereof, or a pro-drug of any thereof.

(52) Particular halogenated salicylanilides include the compounds of formulae (I) and (II) or a pharmaceutically acceptable salt thereof as described herein.

(53) More particularly the halogenated salicylanilide is selected from the group consisting of tetrachlorosalicylanilide, closantel, rafoxanide, oxyclozanide, resorantel, clioxanide, dibromosalan, tribromosalan and niclosamide.

(54) Niclosamide

(55) In a one embodiment of the invention the halogenated salicylanilide is niclosamide or a pharmaceutically acceptable salt thereof. Niclosamide (2,5-dichloro-4-nitrosalicylanilide) exhibits the following acute toxicity:

(56) LD.sub.50, mice, p.o., >5000 mg/kg

(57) LD.sub.50, rats, p.o., 5000 mg/kg

(58) LD.sub.50, rats, dermal, 2000 mg/kg

(59) LD.sub.50, rabbits, p.o., 5000 mg/kg

(60) LD.sub.50, cats, p.o., >1000 mg/kg

(61) Niclosamide thus exhibits low toxicity. The compound is poorly soluble in water and shows low intestinal absorption. Once in the bloodstream niclosamide is quickly cleared via the urinary tract or by enzymatic metabolism in the liver.

(62) Niclosamide Derivatives

(63) It is believed that a number of niclosamide analogs will act in a manner similar to niclosamide in the treatment of the Clostridium infections described herein. Illustrative niclosamide analogs include, but are not limited to closantel (CAS #: 57808-65-8), oxyclozanide (CAS #: 2277-92-1), rafoxanide (CAS #: 22662-39-1), clioxanide (CAS #: 14437-41-3). Other suitable niclosamide analogs include brotianide (CAS #: 23233-88-7), 4-chloro-3-nitrosalicylanilide, 4-chloro-5-nitrosalicylanilide, 2-chloro-5-methoxy-3-nitrosalicylanilide, 2-methoxy-3,4-dinitrosalicylanilide, 2,4-dimethyl-3-nitrosalicylanilide, 2.sup.l-chloro-3,4-dinitrosalicylanilide, 2-ethyl-3-nitrosalicylanilide and 2-bromo-3-nitrosalicylanilide; or a pharmaceutically acceptable salt thereof. Further niclosamide derivatives include those described in WO 2008/021088, particularly those described in Table 1 therein, which are incorporated herein by reference.

(64) Particular niclosamide analogues include closantel, rafoxanide and oxyclozanide. These compounds are expected to have a suitable toxicity profile for the use described herein.

(65) Acute Toxicity of Closantel:

(66) LD.sub.50, rats, p.o., 262-342 mg/kg (depending on the study), median 302 mg/kg

(67) LD.sub.50, rats, s.c., 67 mg/kg

(68) LD.sub.50, mice, p.o., 331 mg/kg

(69) LD.sub.50, mice, i.m., 57 mg/kg

(70) Acute Toxicity of Rafoxanide:

(71) LD.sub.50, rats, p.o., 980->2000 mg/kg (depending on the study), median >1490 mg/kg

(72) LD.sub.50, mice, p.o., 232-300 mg/kg (depending on the study), median 266 mg/kg

(73) LD.sub.50, rabbits, p.o., 3200 mg/kg

(74) Acute Toxicity of Oxyclozanide.

(75) LD.sub.50, rats, p.o., 980-3519 mg/kg (depending on the study), median 2250 mg/kg

(76) LD.sub.50, mice, p.o., 300 mg/kg

(77) LD.sub.50, rabbits, p.o., 3200 mg/kg

(78) Brominated Halogenated Salicylanilides

(79) In another embodiment the halogenated salicylanilide is a brominated halogenated salicylanilide, for example 4,5-dibromosalicylanilide (also known as dibromsalan); 3,5-dibromosalicylanilide (also known as metabromsalan; and 3,4,5-tribromosalicylanilide (also known as tribromsalan).

(80) Synthesis

(81) The halogenated salicylanilides described herein are known or can be synthesised using known methods. For example using methods analogous to those described in WO2004/006906. The compounds of the Formula (I) herein may be prepared by coupling an amine of the formula (III) with an acid of formula (IV):

(82) ##STR00006##

(83) Necessary starting materials are known or can be prepared using standard procedures of organic chemistry.

Pharmaceutical Compositions

(84) The halogenated salicylanilide may be administered to the subject in the form of a pharmaceutical composition comprising the halogenated salicylanilide, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable excipient.

(85) Conventional procedures for the selection and preparation of suitable pharmaceutical formulations are described in, for example, PharmaceuticalsThe Science of Dosage Form Designs, M. E. Aulton, Churchill Livingstone, 1988.

(86) The composition may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insuffiation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intraperitoneal dosing or as a suppository for rectal dosing). Suitably the composition is in a form suitable for oral administration.

(87) The compositions may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.

(88) Dosage

(89) 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 treated and the particular route of administration. For example, a formulation in a unit dose form such as a tablet or capsule intended for oral administration to humans will generally contain, for example, from 0.1 mg to 5 mg, for example from 0.5 mg to 5 g, from 0.5 to 1000 mg or from 10 to 500 mg of the halogenated salicylanilide compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition.

(90) The size of the dose of the halogenated salicylanilide for the treatment of the Clostridium infections described herein will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well-known principles of medicine.

(91) The halogenated salicylanilide will generally be administered in a dose of about 0.001 to about 75 mg/kg, for example from about 0.013 to about 66.7 mg/kg, about 0.5 to about 30 mg/kg or from about 2.5 to about 30 mg/kg. The halogenated salicylanilide may be administered within these dosage ranges to the subject from 1 to 4 times per day. The dosage may be administered by any suitable route, for example parenterally, orally or rectally. A particular route of administration which is generally applicable to all of the uses of the halogenated salicylanilides described herein is the oral administration of the halogenated salicylanilide to the subject.

(92) The particular dosage regimen used to treat a subject will depend on a number of factors that may readily be determined, such as the severity of the condition and its responsiveness to initial treatment, but will normally involve one or more administrations per day on an ongoing basis. The effective dosage of the pharmaceutical composition of the present invention varies from the formulation, administration pathway, age, weight and gender of a human or animal or with a disease caused by Clostridium species, particularly Clostridium difficile colonizing or infecting the intestinal tract of a human or animal having a Clostridium difficile infection.

(93) Therapeutic Use

(94) As described hereinbefore the halogenated salicylanilide is used for the treatment of an infection caused by Clostridium bacteria, particularly C. difficile. The halogenated salicylanilide may act to kill or eradicate the infection from the subject, thus providing a bactericidal effect. Alternatively the halogenated salicylanilide may inhibit growth or replication of the bacteria thus producing a bacteriostatic effect. In the context of the present invention, treatment of a condition encompasses both therapeutic and prophylactic treatment, of either an infectious or a non-infectious condition, in a subject for example a mammal such as a human or animal, but in particular a human. It may involve complete or partial eradication of the condition, removal or amelioration of associated symptoms, arresting subsequent development of the condition, and/or prevention of, or reduction of risk of, subsequent occurrence of the condition.

(95) Generally the halogenated salicylanilide will be administered to a subject experiencing symptoms of a Clostridium infection (for example a C. difficile infection). Accordingly, the halogenated salicylanilide may be for use in the treatment of a C. difficile associated disease, for example the halogenated salicylanilide may be for use in the treatment of a C. difficile associated disease selected from diarrhoea and colitis (including pseudomembranous colitis.

(96) In an alternative embodiment the halogenated salicylanilide is for use in the treatment of a C. difficile in a subject, wherein the subject is asymptomatic. Such uses may be useful to eradicate or inhibit a C. difficile infection in a subject that is at risk of developing C. difficile associated disease. Such subjects could include, for example subjects which require surgical procedures in which prophylactic antibiotics may be administered (for example certain orthopaedic surgery). By eradicating the C. difficile infection prior to administration of further antibiotics, the risk of antibiotic induced diarrhoea may be reduced.

(97) Subjects who have previously suffered from an antibiotic induced C. difficile infection may be at a particular risk of developing a C. difficile infection if they are administered antibiotics in the future. Accordingly, in another embodiment the halogenated salicylanilide is for use in the treatment of a subject prior to the administration of an antibiotic other than the halogenated salicylanilide, wherein the patient is asymptomatic of a C. difficile infection prior to administration of the halogenated salicylanilide and where the subject has previously suffered from an antibiotic induced a C. difficile infection.

(98) When the Clostridium infection is an antibiotic induced Clostridium infection (for example a C. difficile infection) further administration of the antibiotic causing the induced infection is suitably halted. Alternatively, the dosage of the antibiotic may be reduced or gradually tapered so as to reduce the risk of exacerbating the antibiotic induced Clostridium infection. Accordingly the halogenated salicylanilide may be administered to the subject concurrently with another antibiotic being used to treat a primary infection in the subject. For example it may be that the halogenated salicylanilide is administered to the subject concurrently with an antibiotic being used to treat a primary infection other than a C. difficile infection. The antibiotic used to treat the primary infection may, for example, be one or more antibiotics selected from a penicillin, a cephalosporin, a carbapenem, a monobactam (for example a -lactam antibiotic), a fusidane, a fluoroquinolone, a tetracycline, a glycylcycline, phenicol (for example chloramphenicol), a macrolide, a macrocyclic (for example fidaxomicin), a rifamycin, a ketolide, a lincosamide, an oxazolidinone, an aminocyclitol, a polymyxin, a glycopeptide, an aminoglycoside, a lipopeptide, an antimycobacterial, a nitromidazole, bacitracin, mupiricin, a pleuromutilin, a rifamycin, a sulphonamide and trimethoprim, or a combination of two or more thereof. However, preferably the halogenated salicylanilide is used alone or together with a reduced or tapered dose of the antibiotic(s) responsible for the induced Clostridium infection. More preferably the halogenated salicylanilide is administered to the subject in the absence of any other antibiotic.

(99) In one embodiment the halogenated salicylanilide is administered to the subject concurrently with another therapeutic agent in any of the treatments of the Clostridium infections (particularly the C. difficile) infections described herein. The other therapeutic agent may be, for example, an antibiotic active against C. difficile, a microbiome therapeutic or faecal transplant, or a vaccine or an antibody therapy for e.g. C. difficile.

(100) Accordingly in may be that the halogenated salicylanilide is administered to the subject concurrently with another antibiotic active against a Clostridium infection, particularly a C. difficile infection, other than the halogenated salicylanilide itself. Examples of such antibiotics include a nitroimidazole, for example metronidazole; a benzimidazole, for example ridinilazole (SMT19969); a glycopeptide, for example vancomycin; a macrocyclic antibiotic, for example fidaxomicin; an oxazolidinone, for example cadazolid; a lipopeptide, for example surothromycin or daptomycin; a glycylcycline, for example tigecycline; a DNA Minor Groove Binder (for example MGB-BP-3) a glycolipodepsipeptide (for example ramoplanin); CRS3123 (a Methionyl-tRNA synthetase (MetRS) inhibitor, Crestone Inc); and a rifamycin such as rifaximin, or a combination of two or more thereof. It may be that the halogenated salicylanilide is administered concurrently with metronidazole (for example concurrently with intravenous metronidazole).

(101) It may be that the halogenated salicylanilide is administered to the subject concurrently with a vaccine, for example concurrently with a vaccine which induces an immune response to C. difficile toxins, for example toxins A and B (e.g. ACAM-CDIFF, Sanofi, or VLA84 (a fusion protein containing cell binding domains of Toxins A and B, Valneva), or a vaccine which prevents a C. difficile infection, for example PF06425090 (Pfizer).

(102) It may be that the halogenated salicylanilide is administered to the subject concurrently with an antibody therapeutic, for example actoxumab and beziotoxumab or a combination thereof.

(103) It may be that the halogenated salicylanilide is administered to the subject concurrently with a faecal transplantation or microbiome therapeutics, for example concurrently with a faecal transplant, spores from a non-toxigenic C. difficile strain (e.g. VP-20621); spores from microbiome organisms (e.g. SER-109), a microbiota suspension (e.g. RBX2660), a probiotic (e.g. lactobacillus reuterei) or a lactamase, for example SYN-004.

(104) Reference to administration concurrently herein includes the separate, simultaneous or separate administration of the halogenated salicylanilide with the other therapy. The halogenated salicylanilide may be administered to the subject administered by the same or different routes of administration, for example oral, intravenously, subcutaneously, or rectally). The halogenated salicylanilide and the other therapy may be administered as a combined preparation; however, generally they will be administered as separate dosage forms to enable the dose and dosing regimen of each to be tailored accordingly.

(105) The presence of a Clostridium infection (for example a C. difficile infection) in a subject may be diagnosed using convention methods, for example infection may be suspected from subject exhibiting symptoms of a C. difficile associated disease. Infection may also be diagnosed using known methods for example A complete blood count to test for the presence of leukocytosis Measurement of albumin levels to check for hypoalbuminemia, which may accompany severe disease. Testing for elevated serum lactate levels (5 mmol/L), which can be a sign of severe disease. Stool examination. Stools may be positive for blood in severe colitis. Faecal leukocytes are present in about half of cases.

(106) Stool assays for C. difficile, may also be used including the following: Stool culture: This is a sensitive test. However, the results are slow and may lead to a delay in the diagnosis. Glutamate dehydrogenase enzyme immunoassay (EIA): This is a very sensitive test and detects the presence of glutamate dehydrogenase produced by C. difficile. Real-time polymerase chain reaction (PCR) assay: This test is an alternative gold standard to stool culture. The assay may be used to detect the C. difficile gene toxins. EIA for detecting toxins A and B produced by C. difficile.

(107) Imaging studies and procedures may also be used to detect infection in a subject. Suitable methods include abdominal computed tomography (CT) scanning. This method is particularly suitable when pseudomembranous colitis or other complications of CDI are suspected. In subjects with sepsis due to suspected megacolon, abdominal radiography may be performed instead of CT scanning to establish the presence of megacolon.

(108) The use of halogenated salicylanilides for the treatment of a Clostridium infection (for example a C. difficile infection) as described herein is expected to provide a wider therapeutic window than conventional treatments such as vancomycin, metronidazole or fidaxomicin. Accordingly, the use of halogenated salicylanilides may result in reduced side effects compared to known C. difficile treatments.

(109) Halogenated salicylanilides (e.g. niclosamide) are poorly absorbed following oral administration. Accordingly, the concentration of the halogenated salicylanilide in the faeces is expected to be high compared to for example oral administration of similar doses of vancomycin or metronidazole. A high local concentration of the halogenated salicylanilide in the GI tract, especially the colon, would be expected to enhance the potency and or efficiency of the antibacterial effect locally in the intestine and colon.

(110) As illustrated in the examples, a number of the halogenated salicylanilide tested had similar or higher potencies than vancomycin, metronidazole or fidaxomicin and may therefore be expected to provide an antibacterial effect on Clostridium infections at a similar or lower dose than conventional treatments such as vancomycin.

(111) Clostridium bacteria (for example C. difficile) are expected to exhibit a low frequency of spontaneous mutation in the presence of the halogenated salicylanilides described herein. Therefore, it is expected that the risk of resistance to the halogenated salicylanilide emerging will be low.

EXAMPLES

(112) In the examples below, the effects of various halogenated salicylanilides are compared to vancomycin (a currently approved compound for the treatment Clostridium difficile infections).

Example 1 MIC Determinations

(113) C. difficile MICs are determined according to CLSI guideline using microbroth dilution as described in Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved StandardEighth Edition CLSI, ISBN: 1-56238-789-8, except isovitalex was used in the place of lysed horse blood.

(114) The media used for the C. difficile MIC tests was Brucella broth supplemented with hemin (5 g/ml), vitamin K and Isovitalex (according to the manufacturer's instructions). Inoculated plates were incubated for 44 to 48 hours at 36 C. under anaerobic conditions (anaerobic chamber or anaerobic jar with gaspack).

(115) The C. difficile strain ATCC 700057 was used as a reference control during the C. difficile MIC determinations. This strain has an expected MIC towards vancomycin of 1 ug/mL.

(116) The results are shown in Tables 2 and 3.

(117) TABLE-US-00002 TABLE 2 MIC of clinical isolates of C. difficile against niclosamide and vancomycin C. difficile isolate Niclosamide (ug/mL) Vancomycin(ug/mL) 7-6011209 0.06 0.5 7-7150288 0.125 1 7-7152701 0.25 2 7-7154992 0.25 1 7-5779928 0.25 2 7-6778909 0.25 0.5 7-6870430 0.25 0.5 7-7154712 0.25 2 7-7104022 0.125 1 7-7153872 0.5 2 7-5085357 0.125 0.5 7-7150997 0.125 0.5 7-6008526 0.25 2 7-7124449 0.125 0.5 7-6854508 0.125 0.5 7-7363761 0.125 0.5 7-7200552 0.25 0.5 7-7150318 0.125 0.5 7-7150628 0.125 1 7-7149204 0.125 1 7-7154712 0.25 2 7-7116411 0.125 2 7-7151551 0.25 2 7-7156197 0.25 2 12055 0.125 0.5 12060 0.125 0.5 12061 0.06 0.5 12062 0.06 0.5 12063 0.06 0.5 12064 0.06 1

(118) Table 2 illustrates that niclosamide has a lower MIC than vancomycin against the C. difficile isolates tested.

(119) TABLE-US-00003 TABLE 3 C. difficile C. difficile Strain 7-6011209 Strain 12055 Compounds MIC (g/ml) MIC (g/ml) Clioxanide 0.031 <0.08 Closantel 0.125 0.125 Oxyclozanide 0.25 0.25 Rafoxanide <0.08 <0.08 Tribromsalan 0.125 0.25 Vancomycin 0.125 0.25

Example 2

(120) Table 4 compares the properties of niclosamide with those of vancomycin, metronidazole, fidaxomicin. The data shown in Table 4 was obtained from published data together with data from the examples herein.

(121) TABLE-US-00004 TABLE 4 Vancomycin Metronidazole Fidaxomicin Niclosamide Class Glycopeptide Nitroimidazole Macrolide Halogenated salicylanilide Dosing TID, oral TID, oral or IV BID, oral 1-4 times daily, oral MIC90 (g/mL) 2.sup.a 1.sup.a 0.5.sup.a 0.06-0.25.sup.b Highest MIC observed 16.sup.a >32.sup.a 1 0.5 (g/mL) Spectrum of activity Gram+ Gram+/ Gram+ Gram+ Side effects.sup.c Bladder pain, Abdominal or Nausea, Nausea, retching, bloating, bloody stomach cramps, vomiting, abdominal pain urine, painful dizziness, abdominal pain, urination, fever, heartburn. gastrointestinal dry mouth, irregular Spinning sensation. hemorrhage, heartbeat, loss of Trouble sleeping, anemia, appetite, mood congestion, dry neutropenia changes, muscle mouth pain or cramps, numbness, rapid weight gain, shortness of breath, tiredness Footnotes: .sup.aANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2002, p. 1647-1650 & CID 2012: 55 (Suppl 2) S143 .sup.bData from Tables 3 and 4 herein. .sup.cData from product labels.

Example 3: Additional MIC Determinations

(122) The MIC of the halogenated salicylanilides clioxanide, closantel, oxyclozanide, rafoxanide and tribromsalan was determined against 24 clinical isolate strains of C. difficile. Fidaxomycin, metronidazole and vancomycin were used as comparator compounds in the study. The comparator compounds represent the antibiotics most often used in current treatments of C. difficile infections.

(123) Stock solutions of the test compounds and comparators were made in DMSO at a concentration of 1 mg/mL.

(124) Microorganisms

(125) The C. difficile strains used are shown in Table 5:

(126) TABLE-US-00005 TABLE 5 Name MLST Name MLST 7-6011209 ST002 7-7363761 ST008 7-7150288 ST003 7-7200552 ST017 7-7154992 ST139 7-7150318 ST059 7-6778909 ST016 7-7150628 ST034 7-6870430 ST001 7-7149204 ST006 7-7104022 ST103 7-7116411 ST005 7-5085357 ST028 7-7150997 ST049 7-7124449 ST009 7-6854508 ST013 12055 12063 12060 12064 12061 12065 12062 12066
Culture Medium

(127) C. difficile strains were grown on Brucella blood agar+hemin+vitamin K [1], plates were incubated 44 to 48 hours at 36 C. under anaerobic conditions. Broth cultures were performed in Brucella bouillon supplemented with Isovitalex [2] according the instructions from the supplier (BBL). All cultures were performed under anaerobic conditions in an anaerobic chamber [3].

(128) Antibacterial Activity

(129) The antibacterial activity of the study compounds was determined using the following protocol. 1. Day 1: bacterial strain is isolated and incubated at 37 C. on Brucella blood agar+hemin+vitamin K. 2. Day 2: Inoculate 5 ml of Brucella bouillon supplemented with Isovitalex (BBI) with one isolated colony in 15 ml Falcon tube and incubated overnight at 36 C. in anaerobic conditions. 3. Day 3: Dilute the antibiotics in BBI to their highest concentration (8 g/ml in 2 ml). Make a series of two fold dilutions in deep well 96 well plates. Transfer 150 l of the antibiotics solution to 96-well plates. After 5-6 hours, the culture was stopped and OD.sub.600 was measured. The culture is diluted to 10 CFU/ml About 1 l of this diluted culture is added in all wells in order to have 10 cells per well. Plates are incubated at 44 to 48 hours at 36 C. under anaerobic conditions. 4. Day 5: OD.sub.600 is measured after incubation. Inhibition calculated as follows:

(130) $\mathrm{Inhibition}=1-\frac{\mathrm{OD}\mathrm{antibiotic}-\mathrm{OD}\mathrm{negative}\mathrm{control}}{\mathrm{OD}\mathrm{positive}\mathrm{control}-\mathrm{OD}\mathrm{negative}\mathrm{control}}$
Results

(131) The MIC values of the tested compounds against the 24 strains of C. difficile are shown in Table 6.

(132) TABLE-US-00006 TABLE 6 Strain Clioxanide Closantel Oxyclozanide Rafoxanide Tribromsalan Fidaxomycin Metronidazole Vancomycin 7-6011209 <0.008 0.016 <0.008 <0.008 <0.008 <0.008 0.031 0.031 7-7150288 <0.008 0.031 <0.008 <0.008 <0.008 <0.008 0.063 0.5 7-7154992 <0.008 0.063 <0.008 0.016 0.063 <0.008 1 0.5 7-6778909 <0.008 0.031 <0.008 <0.008 <0.008 <0.008 0.25 05 7-6870430 <0.008 0.031 <0.008 <0.008 0.016 0.016 1 0.5 7-7104022 <0.008 0.25 <0.008 <0.008 1 <0.008 >8 2 7-5085357 <0.008 0.016 0.016 0.016 0.016 <0.008 1 0.5 7-7150997 <0.008 0.016 <0.008 <0.008 0.16 <0.008 0.031 <0.008 7-7124449 <0.008 0.25 0.031 0.031 0.063 <0.008 2 0.5 7-6854508 <0.008 0.25 <0.008 <0.008 0.031 <0.008 1 0.5 7-7363761 <0.008 <0.008 7-7200552 <0.008 0.5 0.25 0.063 0.25 <0.008 2 1 7-7150318 <0.008 0.63 0.016 <0.008 <0.008 <0.008 1 1 7-7150628 <0.008 0.063 0.31 0.31 0.031 <0.008 0.5 1 7-7149204 <0.008 0.31 <0.008 <0.008 <0.008 <0.008 0.5 0.5 7-7116411 0.125 0.25 0.125 0.031 <0.008 2 >16 2 12055 <0.008 0.063 0.016 <0.008 0.016 <0.008 1 1 12060 <0.008 0.125 0.031 0.031 0.063 <0.008 1 0.5 12061 <0.008 0.063 0.016 0.016 0.016 <0.008 1 0.25 12062 <0.008 0.063 <0.008 0.016 <0.008 <0.008 0.125 0.125 12063 <0.008 0.031 0.016 <0.008 0.016 <0.008 0.25 0.25 12064 <0.008 0.031 <0.008 <0.008 <0.008 <0.008 0.031 0.5 12065 <0.008 0.063 0.031 0.016 0.016 <0.008 1 0.5 12066 0.063 0.5 0.125 0.063 0.031 <0.008 1 0.5

(133) Table 6 shows that the tested halogenated salicylanilides were active against the tested strains. The most active compounds were clioxanide, rafoxanide and oxyclozanide, which compared favourably with the activity of the comparator compound fidaxomycin. All of the tested halogenated salicylanilides generally exhibited lower MIC values than the comparator compounds, metronidazole and vancomycin.

REFERENCES

(134) [1] H.-P. Schau, J. F. MacFaddin, Media for IsolationCultivationIdentificationMaintenance of Medical Bacteria, Volume I. XI+929 S., 163 Abb., 94 Tab. Baltimore, London 1985. Williams and Wilkins. $90.00. ISBN: 0-683-05316-7, J. Basic Microbiol., vol. 26, no. 4, pp. 240-240, 1986. [2] L. Pospisil, [Isovitalexa chemically definable enricher of culture media for Neisseria gonorrhoeae], Ceskoslovensk Dermatol., vol. 46, no. 1, pp. 23-25, February 1971. [3] A. N. Edwards, J. M. Suarez, and S. M. McBride, Culturing and Maintaining Clostridium difficile in an Anaerobic Environment, J. Vis. Exp. JoVE, no. 79, p. e50787, September 2013.

Example 5: Sporulation Study

(135) Clostridium difficile is a spore forming bacteria that causes severe diarrhea in healthcare settings. The spore is the infective agent, and is implicated in disease transmission and recurrence. Prevention or inhibition of spore formation may therefore minimise the risk of transmission and recurrence of infection, particularly in a hospital environment. Currently the main treatments used for the treatment of C. difficile infections are vancomycin, metronidazole, rifaximin and fidaxomicin. It has been shown that fidaxomicin inhibits C. difficile sporulation [1].

(136) The halogenated salicylanilide, rafoxanide was tested assess its ability to inhibit spore formation. Fidaxomicin was used as a comparator in the study.

(137) Methods

(138) Bacterial Strain

(139) The C. difficile strain used in the study was 7-6011209, a clinical isolate from the MLST group ST002.

(140) Antimicrobial Agents

(141) Rafoxanide and fidaxomicin (ex. Sigma-Aldrich) were prepared as 10 mg/mL stock solutions in dimethyl sulfoxide (DMSO). The compounds were diluted further to appropriate concentration in growth media prior to testing for their effect on sporulation.

(142) Culture Media and Culture Conditions

(143) C. difficile strains were grown and cultured in Brucella bouillon supplemented with Isovitalex as described above in Example 3.

(144) Sporulation was carried out using Clospore medium [2], comprising Special Peptone Mix (Oxoid) 10 g/L, yeast extract 10 g/L, (NH.sub.4).sub.2 SO.sub.4 0.6 g/L, MgSO.sub.4 7H.sub.2O 0.12 g/L, CaCl.sub.2 2H.sub.2O 0.08 g/L, K.sub.2CO.sub.3 3.48 g/L, KH.sub.2PO.sub.4 2.6 g/L, pH 7.90.1.

(145) The germination medium was BHIS medium [3] containing 1 g/L of sodium taurocholate. BHIS medium comprises: Brain Heart Infusion 37 g/L, yeast extract 5 g/L, agar 15 g/L, L-cysteine 0.1% (w/v), glucose 0.5% (w/v) and FeSO.sub.4 0.09% (w/v).

(146) All cultures were performed at 37 C. under anaerobic conditions in an anaerobic chamber as described in Example 3.

(147) Sporulation Kinetics

(148) C. difficile was grown overnight on blood agar plates. One colony was transferred to 10 mL of Brucella bouillon enriched with Isovitalex and grown overnight. Clospore medium containing the appropriate concentration of the test compound was inoculated at 1% with the overnight culture. Samples were withdrawn every 24 hours for quantitation of heat-resistant spores (survivors after incubation at 65 C. for 20 minutes). Spores were serially diluted in 0.09% NaCl and plated on BHIS agar supplemented with 0.1% sodium taurocholate to grow the spores for quantitation.

(149) Concentrations of the test compounds were normalized to the MIC such that they were at least 8-fold above the MIC of the respective compound (8-fold for rafoxanide, and >8-fold for fidaxomicin).

(150) Results

(151) The impact of the test compounds on sporulation kinetics is shown in FIG. 1.

(152) The negative control sporulates rapidly reached a value of 210.sup.5 by 24 hours and approached its maximum count of approximately 10.sup.7 by 48 hours sporulation. Rafoxanide, at 8-fold MIC, suppressed formation of spores throughout the 96 hour study period. The comparator compound fidaxomycin, at >8-fold MIC, suppressed formation of spores for the first 48 hours of the study, however, increased spore formation compared to rafoxanide occurred at the 72 and 96 hour time points.

(153) The data illustrated by FIG. 1 shows that rafoxanide suppresses spore formation more effectively than fidaxomycin at a fixed effect level relative to MIC in this study. These results suggest that rafoxanide may inhibit the shedding of C. difficile spores and as such be effective in controlling the spread of infection in, for example, a hospital environment. The compound may also be useful in minimising the risk of recurrent infections in patients.

REFERENCES

(154) [1] F. Babakhani, L. Bouillaut, P. Sears, C. Sims, A. Gomez, and A. L. Sonenshein, Fidaxomicin inhibits toxin production in Clostridium difficile, J. Antimicrob. Chemother., vol. 68, no. 3, pp. 515-522, March 2013. [2] J. Perez, V. S. Springthorpe, and S. A. Sattar, Clospore: a liquid medium for producing high titers of semi-purified spores of Clostridium difficile, J. AOAC Int., vol. 94, no. 2, pp. 618-626, April 2011. [3] C. J. Smith, S. M. Markowitz, and F. L. Macrina, Transferable tetracycline resistance in Clostridium difficile, Antimicrob. Agents Chemother., vol. 19, no. 6, pp. 997-1003, June 1981.

(155) The invention is further illustrated by the following numbered clauses:

(156) 1. A halogenated salicylanilide, or a pharmaceutically acceptable salt or ester thereof for use in the treatment of an infection in a subject caused by Clostridium bacteria.

(157) 2. The halogenated salicylanilide for the use of Clause 1 wherein the infection is caused by Clostridium difficile.

(158) 3. The halogenated salicylanilide for the use of Clause 2, wherein the infection is a Clostridium difficile associated disease.

(159) 4. The halogenated salicylanilide for the use of Clause 3, wherein Clostridium difficile associated disease is diarrhoea, colitis (for example pseudomembranous colitis) or toxic megacolon.

(160) 5. The halogenated salicylanilide for the use of any of Clauses 1 to 4, wherein the Clostridium infection is an antibiotic induced Clostridium infection, wherein the antibiotic is other than a halogenated salicylanilide.

(161) 6. The halogenated salicylanilide for the use of Clause 5, wherein the antibiotic other than a halogenated salicylanilide is selected from clindamycin, a cephalosporin (for example cefotaxime and ceftaidime), ampicillin, amoxicillin and a quinolone (for example a fluoroquinolone, optionally ciprofloxaxin or levofloxacin).
7. The halogenated salicylanilide for the use of any of Clauses 1 to 6, wherein the Clostridium infection has not been treated with an antibiotic prior to administration of the halogenated salicylanilide to the subject.
8. The halogenated salicylanilide for the use of any of Clauses 1 to 6, wherein the subject has a Clostridium infection which has recurred following treatment with an antibiotic other than a halogenated salicylanilide.
9. The halogenated salicylanilide for the use of Clause 8, wherein Clostridium infection has recurred after being treated with an antibiotic selected from metronidazole, vancomycin and fidaxomicin.
10. The halogenated salicylanilide for the use of any of Clauses 1 to 6, wherein the Clostridium infection is refractory to a prior antibiotic treatment other than a halogenated salicylanilide.
11. The halogenated salicylanilide for the use of Clause 10, wherein the prior antibiotic treatment is selected from metronidazole, vancomycin and fidaxomycin.
12. The halogenated salicylanilide for the use of any preceding Clause wherein the infection is caused by the NAP1/027/BI C. difficile strain.
13. The halogenated salicylanilide for the use of any preceding Clause, wherein the halogenated salicylanilide is of the formula (I):

(162) ##STR00007##
wherein
X is O or S;
R.sup.1 and R.sup.2 are at each occurrence independently selected from halo;
R.sup.3 and R.sup.4 are at each occurrence independently selected from H, C.sub.1-6 alkyl, OR.sup.A1, NO.sub.2 and CN;
R.sup.5 is H or -L.sup.1-R.sup.7;
R.sup.6 is H or C(O)R.sup.A2;
L.sup.1 is selected from a bond, O, S, or (CR.sup.A3R.sup.B).sub.o, wherein o is 1 or 2;
R.sup.6 is phenyl, unsubstituted or substituted with 1, 2, or 3 groups selected from halo, C.sub.1-4 alkyl, OR.sup.A4, NO.sub.2 and CN;
R.sup.A1, R.sup.A2, R.sup.A3 and R.sup.A4 are at each occurrence independently selected from H and C.sub.1-4 alkyl;
R.sup.B is at each occurrence selected from H, C.sub.1-4 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 thereof
14. The halogenated salicylanilide for the use of Clause 13, wherein X is O.
15. The halogenated salicylanilide for the use of Clause 13 or Clause 14, wherein R.sup.6 is H.
16. The halogenated salicylanilide for the use of any of Clauses 13 to 15, wherein R.sup.3 and R.sup.4 are at each occurrence independently selected from H, C.sub.1-4 alkyl, OR.sup.A1 and NO.sub.2.
17. The halogenated salicylanilide for the use of any of Clauses 13 to 16, wherein L.sup.1 is selected from O, CH.sub.2 and CH(CN).
18. The halogenated salicylanilide for the use of any of Clauses 13 to 17, wherein R.sup.7 is phenyl unsubstituted or substituted with 1, 2 or 3 groups selected from halo.
19. The halogenated salicylanilide for the use of any of Clauses 1 to 12, wherein the halogenated salicylanilide is selected from

(163) ##STR00008##
or a pharmaceutically acceptable salt or ester thereof.
20. The halogenated salicylanilide for the use of any of Clauses 1 to 12, wherein the halogenated salicylanilide is selected from the group consisting of niclosamide, clioxanide, closantel, oxyclozanide, rafoxanide, tribromosalan, or a pharmaceutically acceptable salt or ester thereof.
21. Use of a halogenated salicylanilide, or a pharmaceutically acceptable salt or ester thereof for the manufacture of a medicament for the treatment of an infection in a subject caused by Clostridium bacteria.
22. A method of treating an infection caused by Clostridium bacteria in a subject, the method comprising administering to said subject an effective amount of a halogenated salicylanilide, or a pharmaceutically acceptable salt or ester thereof.
23. The halogenated salicylanilide for the use of any of Clauses 1 to 20, the use of Clause 21 or the method of Clause 22, wherein the halogenated salicylanilide is orally administered to the subject.
24. The halogenated salicylanilide for the use of any of Clauses 1 to 20, the use of Clause 21 or the method of Clause 22, wherein the subject is a human or warm blooded animal, optionally wherein the subject is a human.
25. The halogenated salicylanilide for the use of any of Clauses 1 to 20, the use of Clause 21 or the method of Clause 22, wherein the subject is a human aged 65 years or older