F eXd. of tJ.¾ Invention

The invention relates to the use of a sulphur-containing compound in the rea men of a patient with a drug resistant microbial infection, in particular an antibiotic-resistant infection .

Backg onixl to this Xnvsnt oti

Drug-resistant infectious agents _r namely those that are not efficiently killed or substantially growth-inhibited by antimicrobial compounds, are an increasingly important public health concern. Tuberculosis, gonorrhea, malaria and childhood ear infections are examples of diseases which have become more difficult to treat due to the emergence of drug- resistant pathogens. Antimicrobial resistance is becoming a factor in virtually all hospital-acquired (nosocomial) infections. It has been estimated that the annual cost of treating antibiotic resistant infections in the United States alone may be as high as $30 billion.

Resistance has been recognized since the introduction of penicillin nearly 50 years ago, when penicillin-resistant infections caused by Staphylococcus aureus rapidly appeared, Strains of muitidrug-resistant tuberculosis (MDR-TB) have emerged over the last decade and pose a particular threat to people infected with HIV. Drug-resistant strains are as contagious as those that are susceptible to drugs. Diarrheal diseases cause almost 3 million deaths a year - mostly in developing countries, where resistant strains of highly pathogenic bacte ia such as Shigella dysenteriae, Campylobacter _f Vibrio cholerae, Escherichia coli and Salmonella are emerging. Colistin, the most common polymyxin , is a last-resort treatment for: infections with bacteria such as E. coll and Klebsiella that resist all other available antibiotics. Researchers at South China Agricultural University in Guangzhou recently discovered a gene for resistance to colistin in infected livestock,, meat and humans (Yi-Yun Liu et al. , The Lancet, vol. 16, no. 2, px61~168, Feb 2016), The flier-1 gene can pass easily between bacteria. This is particularly concerning in that gram-negative bacteria, which cause common gut, urinary and blood infections in humans, can now become "pan-resistant", with genes that defeat, ail antibiotics now available.

Given the escalating problems associated with poorly treatable infections caused by an increasing variety of resistant infectious agents, such as antibiotic-resistant bacteria, there is a great need for improved anti-microbial treatments . The two ma or avenues for research into such treatments are development of novel antimicrobial compounds and the alternative approach of developing agents which serve to reduce or reverse the resistance displayed by the pathogens. The present inventors have focused on the alternative approach, as disclosed by the present invention.

Statements of the Invention

The present inventors have previously demonstrated the utility of cysteamine as an antimicrobial agent in cystic fibrosis. The present inventors have now shown that cysteamine has broader potential in a wider range of bacterial infections. Surprisingly, the present inventors have now shown that cysteamine has an application in reversing drug resistance and thus improving therapeutic treatment of infections associated with resistant pathogens,

According to a first aspect of the present invention, there is provided a sulphur-containing compound for use in the prophylaxis or treatment of an infection associated wit at least one microbe or microbial strain displaying at least some degree of drug resistance. In one embodiment, the infection is a drug-resistant .microbial infection. In one embodiment, the infection is a bacterial infection. In a further embodiment in the bacterial infection is an antibiotic resistance bacterial infection.

In a further aspect, the invention provides a prophylactic or curative treatment of an infection associated with at least one microbe or microbial strain displaying at least some degree of antimicrobial resistance comprising administering to a subject a sulphur-containing compound. In one embodiment, the patient is infected with a bacteria which has at least some or some degree of resistance to an antibacterial agent such as an antibiotic. The infection may be an antibiotic-res stant infection . Typically the sulphur-contai ing compound restores a degree of sensitivity of the antibiotic-resista t infection (or causative bacterium of the infection) to the antibiotic.

According to a further aspect of the present invention there is provided a product comprising a sulphur-containing compound and a second agent being an antimicrobial agent . The sulphur-containing agent and antimicrobial agent , although both have antimicrobial properties, are not the same. According to one embodiment , the antimic obial agent of the product of the present invention does not comprise peptides. Suitably, the product of the present invention does not comprise peptides.

In a preferred aspect of the invention, the sulphur- containing compound and antimicrobial agent are not administered in a singular dosage unit but are each instead in separate dosage units. The dosage units are preferably administered simultaneously but may be administered in a non-simultaneous fashion due to differential absorption at of the sulphur-containing agent and antimicrobial agent.

In a preferred aspect of the invention, the product is for use in the prophylaxis or treatment of an infection associated with at least one microbe or microbial strain displaying at least some degree of drug resistance,

In a further aspect _f the invention provides a prophylactic or curative treatment of an infection associated with at least one microbe or microbial strain displaying at least some degree of antimicrobial resistance comprising administering to a subject a product comprising a sulphur- containing compound and a second agent being an antimicrobial agent. The invention allows for the use of lower amounts of antimicrobials such as antibiotics when combined with a sulphur-contai ing compound.

A further aspect of the invention rovides a kit comp ising a first dosage unit comprising a sulphur-containing compound and a further dosage unit comprising an antibacterial agent. In the kit, the antibacterial agent may be an antibiotic.

As used herein '''sulphur-containing compound/'' is intended to cover cysteamir.e, cystamine or a derivative thereof. The sulphur-co taining compound may be an aminothiol . Examples of aminothiols include cysteamine and derivatives thereof. The term "derivative thereof" may encompass 2-methylthio ethylamine (cinnamate) , 2-methyl thio ethylurea, N- (2- methylthio ethyl) p-acetamido benzamide, 2-aminoethanethiol, N- ( 2-methylthio ethyl } p-acetamido benzenesulfonamide, N- ( 2- propylthioethyl) -p-methoxy benzamide, N- (butylthio ethyl) nicotinamide, N- {2-dodecylthio ethyl) p-butoxybenzamide, N- (2-methylthio ethyl) p-toluenesulfonamide , K- (2- isopropylthio ethyl) propionamide, N- (2-octyithio ethyl) acetamide, N- (2-butylthio ethyl) methanesulfonamide, N- (2- isopentylt ioethyl ) butane , bis 1, 4- (2-acetamido ethylthio) , 2 , 3-butanediol , 2~hexadecylthio ethylamine hydrochloride, 2- allylthio ethylamine alate, 9-octadecene 2-ylthio ethylamine hydrochloride, 2-dodecylthio ethylamine hydrochloride, 2- isopentylthio ethylamine mandelate, 2-octadecylthio ethylamine salicylate, 2- . beta . -hydroxyethyl thio ethylurea, 2- . beta . -hydroxyethylthio ethylamine hydrochloride, 2- (2, 3- dihydroxy propylthio) ethylamine p-toluenesulfonate, 2- (2- hydroxypropylthio ) ethylamineoxalate, N- {2-methylthio ethyl ) phenylacetamide, 2- ( 2 , 2-dimethoxy ethylthio) ethylamine hydrochloride, 2- (2, 2-dimethoxy ethylthio) ethylamineandecylenate , 2- (2, 2-diethoxy ethylthio) ethylamine undecylenate, 2- (2, 2-diethoxy ethylthio} ethylamine acetate, 2-undecenylthio ethylamine, 2-. beta. -ureidoethylthio ethylamine hydrochloride, 2- . beta , -acetamidoethylthio ethylamine tropate, 2,2' -thio diethylamine fumarate, 2,2'- t io diethy1urea, 3-.beta. -ami oethy11hio propylamine hydrochloride, S- . beta . -ureidoethyl thiocarbamate,

2-ethoxycarbonylthio ethylamine hydrochloride, 2™ dimethylamino carbonylthio ethylamine sulfate, 2- butoxycarbonyl methylthio ethylurea, 2- ethyioxycarbonylmethylthio ethylamine hydrochloride, 6- . beta . -aminoethylthio hexanoate of methyl hydrochloride, 5- . beta . -aminoethy11hio pentanoic acid,

2-phenylthio ethylamine dihydrogen phosphate, 2-p-t- butylphenylthio ethylamine trichloracetate, 2-p-methoxyphenylthio ethylamine ditartrate, 2-tolylthio ethylamine hydrobromide, 2- (1- biphenyl thio} ethylamine hydrochloride,

2-N-pentachlorophenylthio ethyl acetamide, 2 ^~ benz.ylth.io ethylamine malate, 2-benzylthio ethylamine nicotinate, 2-benzylthio 2-methyl propylamine hydrochloride, 2-benzylthio propylamine lactate, N- (2-benzylthio ethyl } nicotinamide hydrochloride, N- (2- benzylthio ethyl) 10-undecene amide, N- {2-benzylthio ethyl) hexadecanamide, S- . beta . -aminoethyl mercaptobutyric acid, - (2-benzyithio ethyl) formami.de, N- (2-benzylthio ethyl) phenylacetamide, N- [2- (2, 6--dimethyl phenyl) ethyl] hexanamide, 2-o-aminophenylthio ethylamine succinate, N~ (2- benzylthio ethyl) glutamine, S- . beta . -aminoethyl mercapto acetic acid ( 3-S- . beta . -aminoethyl5 ercapto propionic acid, ( 3-S- . gamma . -amino propyl) mercapto acetic acid, S {2-p- methoxybenzamido ethyl) mercapto 2- {2-naphtyl methylthio) ethylamine hydrochloride, 2- { -naphtyl methylthio) ethylamine disuccinate, (2-thenyl) 2-thio ethylamine hydrobromide, 2-N-acetyl (2-thenylthio~ ethylamine, 2-o- chlorobenzylthio ethylamine hydrochloride, 2-p- ch1orofoenzylthio ethy1amine g1ycolate, 2-o-fluorobenzylthio ethylamine hydrochloride, 2-furfurylthio ethylamine hydrochloride, 2-tetrahydrofurfurylthio ethylamine p-amino- benzoate, 2- . beta . -phenylethylthio ethylamine glutamate, 2- diphenylmeth 1thio ethylamine hydrochloride, 2-triphenyl methylthio ethylamine hydrochloride hemihydrate, 2- (2- pyridyl ethylthio) ethylamine hydrochloride, 2- (2-p-toluene sulfonamido ethylthio) pyridine N-oxide, 2-. eta. - aminoethylthiomethyl pyridine N-oxide dihydrochloride, 2 - .beta . -aminoethylthio pyridine N-oxide hydrochloride, 2,4- dichloro 2-benzylthio ethylamine aspartate, N- [2- (3, 4- dichloro benzylthio) ethyl] butyrarrd.de, N- [ 2 - (2, 6-dichloro benzylthio} ethyl] dodecanami.de, N- [2- (3, 5-dichloro benzylthio) ethyl] trifluoroacetamide hydrochloride, 2-p- ethoxybenzylthio ethylamine hydrochloride, N- [2-m- f1uorobenzylthio ethyl] chloroacetamide, 2-p-bromobenzylthio ethylamine succinate, 2- {3, 4-d.imethoxy benzylthio) ethylamine malate, 2- (3, -methylenedioxy benzylthio) ethylamine hydrochloride, 2- (2, 4-dichloro cetylthio) ethylamine, 2 (3, 4, 5-trimethoxy benzylthio) ethylamine hydrocinnamate, 2-p- methoxy benzylthio ethylamine salicylate, 2-o- methylbenzylthio ethylamine phenyl-acetate, - [2-p- dimethylaminobenzylthio ethyl] methane-sulfonamide, 2-p- phenoxybenzylthio ethylamine hydrochloride, 2-.beta. - aminoethylthio pyridine hydrochloride ,

2-benzylthio ethylamine citrate, N~ [2-benzylthio ethyl] 2,4- dihydroxy 3, 3-dimethyl butyramide, N- (2-benzylthio ethyl) 6, 8-d.ihydroxy 7, 7-dimethyl 5-oxo 4-aza octanami.de , N- [2- (2- pyridyl thio) ethyl] propionamide, 2-(2-pyridyl meth lthio) ethy1amine dihydrochloride, 2-benzy11.hio ethylamine pantothenate ,

S- ( . beta . -acetamidoethyi ) mercaptoacetate of beta . - morpholinoethyl, S- ( . beta . - phenylacetamidoethyl ) mercaptoacetate N ' -methyl 2-piperazino ethyl , S- { . beta . -ureidoethyl ) mercaptoacetate of beta , - pyrrolidino-ethy, S- ( .beta, -trifluoroacetamidoethyl ) -

. eta .mercapto-propionate of . beta . -dimethylaminoethyl , 2-p-nitrobenzy.lthio ethylamine crotona e, 2-. beta. - morpholinocarbonyl ethylthio ethylamine hydrochloride, N,N- di ( hydroxyethyl } S- ( . beta . -benzarrtido-ethyl ) mercapto- acetamido, [2- ^! -methyl piperazino carbonylthio ethyl] acetamide, 2- (1-naphthyl thio) ethylamine hydrochloride, N- ( 3- . beta . -ureidoethylthio propyl) succinamic acid, 3~allylthio propylamine, 3- (2 , 2 ' -dimethoxy ethylthio) ropylamine , 3- (2, 2 ' -dimethoxy ethylthio) propylamine sulfate, S- .beta . -aminoethylmercapto acetic acid, the hydrochloride of S-- , beta . -aminoethyl mercapto acetic acid,

N- ( 2-benzylthioethyl ) acetamide, N- (2- benzylthioethyl ) propionamide, N~ (2- benzylthioethyl) butyramide, N- (2- benzylthioethyl ) .methanesulfonamide, N~ ( 2- benzylthioethyl) ethanesulfonamide, N~ (2-benzylthioethyl- propanesulfonamide, N- (2~benzylthioethyl) butanesulfonamide, S- (2-p-acetamidobenzenesulfonamido ethyl) mercapto acetic acid, S- (2-p-acetamidobenzamido ethyl) mercapto acetic acid, N- ( 2-thenylthioethyl ) acetamide, 2- benzylthio propylamine, 2-benzylthio 2-methyl propylamine, 2- ( 2-p-toluenesulfonamido ethylthio) pyridine N-oxide, S- (2-p-butoxybenzamidoethyl)mercapto acetic acid, 2-t- butylthio ethylamine hydrochloride, 2-raethoxycarbonyl methylthio ethy1amine hydrochloride, 2-ethoxycarbony1methylthio ethylamine hydrochloride, 2-propoxycarbony1met.hy1 thio ethylamine hydrochloride , 2-butoxycarbonylmethylthio ethylamine hydrochloride, 2,2' -thio diethylamine dihydrochloride, 3- (2-aminoethylthio) alanine hydrochloride, 2-benzylthio ethylammonium diacid phosphate, 2-methyithio ethylamine, N- (methylthioethyl) p-acetamidobenzarnide ,

N- (2-methylthioethyl) nicotinamide, N- (2- methylthioethyi ) benzamide, N- (2-methylthioethyl) p- butoxybenzamide , N- (2-methylthioethyl) butyramide,

N- (2-methylthioethyl) propionamide, N- (2~methylt.hioet.hyi) acetamide, N- (2-methylthioethyl ) butanesulfonaird.de, N- (2- octylthioethyl ) rn.ethanesulfonam.ide , 2-cetylthio ethylamine nydroch1oride, 2- (2-hydroxyethy1thio) ethylaraine hydrochloride, 2-methylthio ethylamine phenylacetatesnd 2- methylthio ethylamine undecylenat .

Alternatively, the sulphur-containing compound may be an organic disulphide, such as cystamine.

The sulphur-containing compound of the invention may be administered in the form of pharmaceutically acceptable salts. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanoi, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed. , Mack Publishing Company, Easton, Pa., US, 1985, p. 1418, the disclosure of which is hereby incorporated by reference; see also Stahl et. al, Eds, "Handbook of Pharmaceutical Salts Properties Selection and Use", Verlag Helvetica Chiraica Acta and Wiley-VCH, 2002. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions,- and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or, as the case may be, an animal without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The invention thus includes pharmaceutically-acceptable salts of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof for example the conventional non-toxic salts or the quaternary ammonium salts which a e formed, e.g., from inorganic or organic acids or bases . Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, blsulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate , dodecylsulfate, et anesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesu1fonate , 2-naphtha.lenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylprop.ionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups may be quaternized with such aqents as lower alkyl halides, such as methyl, ethyl , propyl, and butyl chloride , bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain ha1ides such as decy.i , lauryl, myristyl and stearyi chlorides, bromides and iodides, aralkyl ha I ides like benzyl and phenethyl bromides and others.

The terms "antimicrobial resistance" and "resistance" are used interchangeably to describe a situation where a pathogenic microbe has undergone some sort of change that reduces or eliminates the effectiveness of drugs, chemicals, or other agents to cure or prevent infections.

The terms "microbes" is used in its common meaning, i.e. to co er pathogen!c organi sms so sma11 that a microscope is required to see them. Microbes are also called microorganisms, and include bacteria, viruses, fungi, and parasites, out of which the former two, especially bacteria are the most relevant, for the purposes of the present invention .

As used herein, the term "antimicrobial agent" is intended to cover drugs, chemicals, or other substances that either kill or slow the growth of microbes. Among the antimicrobial agents in use today are antibacterial drugs, antiviral agents, antifungal agents, and antiparasitic drugs.

Preferably the antimicrobial agent is an antibacterial agent, for example an antibiotic agent. Antibiotic agents ma be bactericidal and/or bacteriostatic.

The antibiotic agent may contain a β-lactaxn ring. The β- lactam ring is part of the core structure, of several antibiotic families, the principal ones being the penicillins, cephalosporins, carbapenems, and monobactam . These antibiotic agent are called β-lactam. antibiotics.

Generally the antibiotic agent is of the group consisting of aminoglycosides, ansamycins, carbacepheirt, β-lactams carbapenems, cephalosporins, (including first, second, third, fourth and fifth generation cephalosporins) , penicillin, monobactams) , glycylcyclines, lincosamides, iipopeptides, macrolides, nitrofurans, oxazolidinones, quinolones, sulfonamides, polypeptides and tetracyclins .

The antibiotic agent may be of the group consisting of aminoglycosides, ansamycins, carbacepbem, carbapenems, cephalosporins (including first, second, third, fourth and fifth generation cephalosporins), lincosamides, macrolides, monobactams, nitrofurans, quinoiones, penicillin, sulfonamides, polypeptides and tetracyclins. Alternatively or additionally the antibiotic agent may be effective against .mycobacteri .

The antibiotic agent may be an aminoglycoside such as Amikacin, Gentamicin, Kar.amyc.in, Neomycin, Netilmicin, Tobramycin or Paromomycin.

The antibiotic agent may be an such as Geldanamycin and Herbimycin

Alternatively the antibiotic agent may be a carbacepheirt such as Loracarbef.

The antibiotic agent is a carbapenem such as Ertapenem, Doripenem, Imipenem/Cilastatin or Meropenem.

Alternatively the antibiotic agent may be a cephalosporins (first generation) such as Cefadroxil, Cefazolin, Cefalexin, Cefaiotin or Cefalcthin, or alternatively a Cephalosporins (second generation) such as Cefaclor, Cefamandole, Cefoxitin, Cefprozii or Cefuroxime . Alternatively the antibiotic agent may be a Cephalosporins (third generation) such as Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftibuten, Ceftizoxime and Ceftriaxone or a Cephalosporins (fourth generation) such as Cefepirtie and Ceftobiprole .

The antibiotic agent may be a lincosamides such as Clindamycin and Azithromycin, or a macrolide such as Azithromycin, Clarithromycin, Dirithromycin, Erythromycin , Roxithromycin, Troleandoraycin, Telithromycin and

Spectinomyci . Alternatively the antibiotic agent may be a monobactams such as Aztreonam, or a nitrofuran such as Furazolidone or Nitrofurantoi .

The antibiotic agent may be a penicillin such as Amoxicillin, Ampicillin, Azlociliin, Carbenicillin, Cloxacillin, Dicioxacillin, Flucloxacil Lin, Mezlocillin, Nafcillin , Oxacillin, Penicillin G or V, Piperacillin, Temocillin and Ticarcillin. The antibiotic agent may be an oxazolidinone such as iinezolid or tedizolid.

The antibiotic agent may be a sulfonamide such as Mafenide, Sulfonamidochrysoidine, Sulfacetamide, Sulfadiazi e, Si1 er sulfadiazine, Sulfamethizole, Sulfamethoxazole,

Sulfanilamide, Sulfasalazine, Sulfisoxazole, Trimethoprim, and Trimethoprim-Sulfamethoxazole (Co-trimoxazole) ( MP- SMX) . The antibiotic agent may be a quinolone such as Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trcvafloxacin, Grepafloxacin, Sparfloxacin and Teniafloxacin ,

The antibiotic agent may be a polypeptide, Examples of such polypeptides include Bacitracin, Colistin and Polymyxin B. In one embodiment, the antibiotic agent is not a polypeptide .

The antibiotic agent may be a lipopeptide . Examples of such lipopeptides include Daptomycin and Surfactin.

Alternatively,: the antibiotic agent, may be a tetracycline such as Demeclocycline, Doxycycline, Minocycline and Oxytetracycline

Alternatively the antibiotic agent may be a glycylcycline . Examples of such giycyicyclines include ticjecycline .

Alternatively or additionally the antibiotic agent may be effective against mycobacteria. In particular the antibiotic agent may be Clofazimine, Lamprene, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid,

Pyrazinamide, Rifampicin, Rifabutin, Rifapentine or St.reptomycin .

In one embodiment, the antibiotic agent is a macrolide and/or an aminoglycoside and/or suiphonamid.es.

In one embodiment, the antibiotic agent is a macrolide and/or an aminoglycoside.

In one embodiment, the antibiotic agent is a macrolide and/or sulphonamide . In one embodiment, the antibiotic agent is an aminoglycoside and./or sulphonamide .

In one embodiment, the antibiotic is selected from tobramycin, azithromycin,, telithromycin, ciprofiaxin, ceftazidime ,

In one embodiment, the antibiotic agent is not ciproflaxin. In another embodiment the antibiotic is not tobramycin.

The antibiotic agent may be active in the treatment or prophylaxis of infections caused by Enterobacteriaceae {e.g. E.coli or Klebsiella spp. , such as K, pneumoniae) or non- Enterobacteriaceae bacteria such as Burkhoideria spp.

Generally the antibiotic agent is active in the treatment or prophylaxis of infections caused by gram-negative or gram- positive bacteria, such as Pseudomonas spp., for example Pseudomonas aeruginosa, 3urk.hold.eria spp., Escherichia coli, Klebsiella spp., for example K. pneumoniae, Staphylococcus spp., for example S. aureus.

In one embodiment of the invention, the antibiotic is not a β-iactam antibi ot ic..

In one embodiment of the invention, the antibiotic is not a penicilli .

In one embodiment of the invention, the antibiotic is not. a cephalosporin .

In one embodiment of the invention, the antibiotic is not a carbapene .

In one embodiment of the invention, the antibiotic is not a monobactam. In one aspect of the invention the normal dosing regime of the antibiotic can be reduced by up to 10%; such as by up to 20%; such, as by up to 30%; such as by up to 40%; such as by up to 50%; such as by up to 55%. Thus the dosing of an antibiotic in the present invention can be reduced, according to the dose provided by the MIC values exemplified in present invention.

In another aspect of the invention the risk of developing antibiotic resistance can be dramatically reduced. By combining the active principle of antibiotics with a sulphur-containing compound such as cysteamine, the effect of the antibiotic may be increased up to 10 times giving two alternative advantages; the micro-organisms are up to 10 times as susceptible increasing the efficiency of the therapy; alternatively the therapeutic dose can be reduced concurrently with 90% while maintaining the therapeutic effect .

In a preferred aspect of the invention,, the sulphur- containing compound and the additional antimicrobial agent may be administered simultaneously, sequentially or separately. The sulphur-containing compound and the additional antimicrobial agent may be provided as a combination package. The combination package may further instructions for simultaneo s, separate or sequential administration of each of the sulphur-containing compound and additional antimicrobial agent. For sequential administration,, the sulphur-containing compound and the additional antimicrobial agent may be administered in any order. In one embodiment , the sulphur-containing compound is administered before the additional antimicrobial agent. Pharmaceutical Product

The present invention provides a product comprising a first active agent being sulphur-containing compound and a second agent being an antimicrobial agent.

The above mentioned active agents may be administered as free or fixed combinations. Free combinations may be provided as combination packages containing all the active agents in free combinations. Fixed combinations are often tablets or capsules.

The active agents may be administered simultaneously, sequentially or separately. The active agents may be provided as a combination package. The combination package may contain the product of the invention together with instructions for simultaneous, separate or sequential administration of each of the active agents. For sequential administration, the active agents can be administered in any order .

The active agents of the product of the invention may be provided as pharmaceutical compositions additionally containing one or more pharmaceutically acceptable, diluents, excipients and/or carriers, This applies to both fixed and free combinations.

The active agents of the present invention may be administered by any suitable route known to those skilled in the art, preferably in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. The active compounds and composition may, for example, be administered parenterally, orally, intranasal, intrabronchial, enterally, transdermally, sublinquaily, rectally, vaginally, ocularly, or topically. Both local and systemic administration is contem 1ated , For the purposes of parenteral administration {"parenteral" as used herein, refers to modes of administra ion which include intravenous, intramuscular, enteral, intraperi oneal, intrasternal, subcutaneous and intraarticular injection and infusion of which intravenous (including continuous intravenous administration) is most preferred) solutions in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble salts. Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic, with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.

The sulphur-containing compound may be administered parenteraliy before parenteral administration of the additional antimicrobial agent. Alternatively, the sulphur- containing compound may be administered parenter liy simultaneously with parenteral before administration of the additional antimicrobial agent.

The products of the invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray, atomiser, nebuiiser, with or without the use of a suitable propel!ant ,

Alternatively the products of the invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or powder. The products of the invention may be dermally or transdermal ly administered, for example, by use of a skin patch, depot or subcutaneous injection. They may also be administered by pulmonary or rectal routes.

For oral administration, the pharmaceutical composition may be. in the form of; for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredient. Examples of such dosage units are capsules, tablets, powders, granules or a suspension, with conventional additives such as lactose; marmitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose; and with lubricants such as talc or magnesium stearate. The active ingredient may also be administered by injection as a composition wherein, for example, saline, dextrose or water may be used as a suitable carrier.

The products of the invention may also find application as /in an oral formulation wherein the product is formulated in a carrier, for example selected from, films, tapes, gels, microspheres, lozenges, chewing gum, dentrifices and mouthwash .

The amount of therapeutically active compound that is administered and the dosage regimen for treating a disease condition with the compounds and/or compositions of this invention depends on a variety of factors, including the age, weight, sex and medical condition of the subject, the severity of the disease, the route and frequency of administration, and the particular compound employed, as well as the pharmacokinetic properties of the individual treated, and thus may vary widely. The dosage will generally be lower if the compounds are administered locally rather than systemically, and for prevention rather than for treatment. Such treatments may be administered as often as necessary and for the period of time judged necessary by the treating physician. One of skill in the art will appreciate that the. dosage regime or therapeutically effective amount of the inhibitor to be administrated may need to be optimized for each individual. The pharmaceutical compositions may contain active ingredient in the range of about 0.1 to 2000 mg, preferably in the range of about 0.5 to 500 mg and most preferably between about 1 and 200 mg. A daily dose of about 0.01 to 100 mg/kg body weight, preferably between about 0.1 and about 50 mg/kg body weight and most preferably from about 1 to 20 mg/kg body weight, may be appropriate. The daily dose can be administered in one to four doses per day.

The products of the invention may be administered to the respiratory tract. Thus, the present invention also provides aerosol pharm.aceut.ical formulations comprising a product of the invention. Also provided is a nebuliser or inhaler containing a product of the invention.

Additionally, the products of the invention may be suited to formulation as sustained release dosage forms and the like. The formulations can be so constituted that they release the active agents, for example, in a particular part of the intestinal or respiratory tract, possibly over a period of time. Coatings, envelopes, and protective matrices may be made, for example, from polymeric, substances, such as polylactide-glycolates , liposomes , microemulsions , microparticles, nanoparticles, or waxes. These coatings, envelopes, and, protective matrices are useful to coat indwelling devices, e.g. stents, catheters, peritoneal dialysis tubing, draining devices and the like. Methods and Use

The invention provides the use of a sulphur-containing compound in the prophylaxis or treatment of an infection associated with at least one microbe or microbial strai displaying at least some degree of drug resistance. In one embodiment, the infection is a drug-resistant microbial infection. In one embodiment, the infection is a bacterial infection. In a further embodiment in the bacterial infection is an antibiotic resistance bacterial infection.

The invention also provides a prophylactic, or curative treatment of an infection associated with at least one microbe or microbial strain displaying at least some degree of antimicrobial resistance comprising administering to a subject a sulphur-containing compound.

The bacterial infection may include an infection caused by mere than one microorganism, for example bacteria and any one of fungi, yeast, viruses and protozoa.

The bacterium may be a Gram-positive or a Gram-negative bacterium. A bacterial pathogen may be derived from a bacterial species selected from the group consisting of:

Staphylococcus spp., e.g. Staphylococcus aureus such as

Methiciliin resistant S. aureus, Staphylococcus epidermidis;

Enterococcus spp., e.g. Enterococcus faecalis; Streptococcus pyogenes; Listeria spp.; Pseudo onas spp,; Mycobacterium spp., e.g. Mycobacterium tuberculosis; Enterobacter spp.;

Campylobacter spp.; Salmonella spp.; Streptococcus spp., e.g. Streptococcus Group A or B, Streptococcus pneumoniae;

Helicobacter spp. , e.g. Helicobacter pylori; Neisseria spp., e.g. Neisseria gonorrhea, Neisseria meningitidis; Borrelia burgdorferi; Shigella spp., e.g. Shigella flexneri;

Escherichia coli; Haemophilus spp., e.g. Haemophilus influenzae; Chlamydia spp.. e.a. Chlamydia trachoma i . Chlamydia pneumoniae _? Chlamydia psittaci ; Francisella fularensis; Bacillus spp. , e.g. Bacillus anthracis; Clostridia spp,, e.g. Clostridium botulinum; Yersinia spp. , e.g. Yersinia pestis; Treponema spp.; Burkholderia spp.; e.g. Burkholderia mallei and B pseudomallei ,

In particular the bacterium may include Pseudomonas spp., for example Pseudomonas aeruginosa; Staphylococcus spp., for example Staphylococcus aureus and Staphylococcus epidermidis Haemophilus spp., for example Haemophilus influenza; Burkholderia spp., for example Burkholderia cepacia; Streptococcus spp., Propionibacterium spp,, for example Propionibacteriurn acnes. Preferably the bacterium is selected from Pseudomonas spp., for example Pseudomonas aeruginosa and Staphylococcus spp,, for example Staphylococcus aureus and Staphylococcus epidermidis .

In one embodiment of the invention, the bacterial infection is caused by Enterobacteriaceae (e.g. E.coli or Klebsiella spp., such as K. pneumoniae) or non-Enterohacteriaceae bacteria such as Burkholderia spp., for example B. cepacia or B .muitivorans .

In a further embodiment of the invention, the bacterial infection is caused by gram-negative or gram-positive bacteria, such as Pseudomonas spp., for example Pseudomonas aeruginosa, Burkholderia spp., Escherichia coli, Klebsiella spp., for example K. pneumoniae, staphylococcus spp., for example S. aureus, in particular ethicillin resistant S . aureus .

In one embodiment of the invention, the bacterial infection is caused by a bacterium no including Burkholderia spp. In another embodiment of the invention, the bacterial infection is caused by a bacterium not including Pseudomonas spp. for example Pseudomonas aeruginosa. The method of the invention may be used to minimise and prevent the formation of bacterial colonies, in particular bacterial biofilras in a variety of environments including, but not limited to, household, workplace, laboratory, industrial environment, aquatic environment (e. g. ipeline systems), medical devices including indwelling devices such as defined herein, dental devices or dental implants, animal body for example human body.

The method of the invention may be used to prevent or restrict, the formation of a bacterial colony. The method of the present invention may be used to prevent or treat bacterial infections including topical infections, oral infections and systemic infections. Topical infections may include wounds, ulcers and lesions for example, cutaneous wounds such cuts or burns, and conditions associated therewith .

Oral infections may include gingivitis, periodontitis and mucositis .

Systemic infections may include cystic fibrosis, COPD and other conditions associated with mucosal infections, for example, gastrointestinal, urogenital or other respiratory infectio s .

The product of the invention may be useful in the prevention of, delay of progression of, or treatment of a disease or condition selected from the group consisting of skin and wound infections, middle-ear infections, gastrointestinal tract infections, peritoneal membrane infections, urogenital tract infections, oral soft tissue infections, formation of dental plaque, eye infections (including contact iense contamination) , endocarditis, infections in cystic fibrosis, and infections of indwelling medical devices such as described herein.

Throughout the description and claiins 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.

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 th rewith .

The invention will now be described by way of Examples only with reference to the following Figures in which:

Figure 1 is a graph showing that cysteamine chemopotentiates the activity of ciprofloxacin in the neutropenic mouse thigh model of infection with P. aeruginosa LES431. Legend::!, Vehicle control, 2. Colistin [5 mg./kg] (positive control), 3. Cysteamine only [1.25 mg/kg] , 4. Ciprofloxacin only [15 mg/kg] , and 5, Ciprofloxacin + Cysteamine. One way Anova with Tukey' s post hoc test analysis . *** = p < 0.001, ** ~ p < 0.01, ns = not significant.

EXMPLSS :

Met od

MICIoo determination

The MICioo { concentration at which 100% of bacteria were killed) of all Burkholderia cepacia complex (Bcc) isolates was determined versus cysteamine and the antibiotics tobramycin, ciprofloxacin, ceftazidime and trimethoprim/sulfamethox zole using the CLSI broth microdilution procedure (LSI. 2012a. Methods for dilution ant iraicrobial susceptibility tests for bacteria that grow aerobically; Approved Standard - Ninth Edition; 07-A9. Wayne, PA: Clinical and Laboratory Standards Institute) .

MICioo values for Burkholderia strains are described as the concentration of antibiotic {in micrograms/ml) required, to inhibit the growth of all of the bacteria tested,, cultured over 48 hours at 37 °C in cation-adjusted Muller-Hinton broth. The initial inoculum is prepared from a single colony recovered from frozen stocks (after confirmation of culture purity) which is transferred via sterile inoculation loop to 10 ml cation-adjusted Muller-Hinton broth in a 30 ml universal container. This is incubated aerobically and statically for 48 h at 37 °C prior to the experiment.

The inoculum. is standardised by comparison with 0,5 McFarland standard absorption at 625 nm. This is done by serially diluting 100 μΐ of the liquid culture 2-fold in phosphate buffered saline on a 96-well microtitre plate and comparing with the mean value of triplicate 100 μΐ volumes of 0.5 McFarland standards on the same plate. The closest dilution is then diluted a further 1:150 in sterile, twice- concentrated, cation-adjusted Muller-Hinton broth.

In the test plate, antibiotics of choice (tobramycin, ciprofloxacin, ceftazidime and trimethoprim/sulfamethoxazole) are serially diluted 2-fold in sterile distilled water to achieve 2x the relevant concentrations used in each experiment, by diluting 50 μΐ of each antibiotic into 50 μΐ volumes of distilled water. Negative controls contain 50 μΐ of sterile distilled water only. To these plates 50 μΐ of 1:150 diluted inoculum is added to appropriate wells, Negative culture controls are also prepared by the addition of 50 μΐ of sterile cation-adj usted Miiller-Hinton broth. This brings the final concentration of Miiller-Hinton broth to 1 x in 100 μΐ volumes in each well each containing the required concentration of test antibiotic. Typically, each 96-well plate will contain 3 experimental replicates for each Burkholderia strain at each concentration of antibiotic.

The plates are then read at 625nm using the Biotek plate reader to obtain a time 0 h baseline absorbance reading. The plates are then incubated for 48 h at 37 °C. At 48 h the plates are read at 625 ran using the Biotek plate reader to determine growth of bacteria over time in relevant concentrations of test antibiotic. Mean absorbance values are calculated using Microsoft Excel and base line optical density values taken at time 0 h are subtracted. The concentration of antibiotic required for complete inhibition of bacterial growth (MICioo) is determined as the concentration with absorbance the same as or below those for the uninoculated controls.

Checkerboard Assays

Checkerboard assays of cysteamine and antibiotics were conducted according to the method of Burkhart, et al 2006. Antibiotic susceptibility profiling of Bcc (resistant, intermediate or sensitive to antibiotics) was performed using CLSI PerfoITin3.nce Standards for Antimicrobial Susceptibility Testing using other nGri-EnterGbacteriaceae interpretive standards, Wayne PA, 2012b.

This involves combining two antibiotic dilution series on one 96-well microtitre plate to assess the effect of co- therapy on the growth of microorganisms determined by optical density at 625 nm, and was adapted from the Burkhart et al . , method mentioned above, A typical plate plan is illustrated in Figure 1 below. Antibiotics are prepared in two separate, sterile, 96-well microtitre plates at 4 x the final concentration by two-fold serial dilutions in sterile distilled water across the plates in different directions . Water only is added to the negative inoculum and no antibiotic controls. Various volumes may be used to perform the serial dilutions depending upon the amount of antibiotic required for the challenge experiments if performing multiple experimental replicates, or preparing the same antibiotic to challenge a number of different strains of Bcc. A total of 25 ul of each dilution of antibiotic, and controls is required for each challenge plate, so (for example) a two-fold serial dilution of antibiotic made using 150 μΐ volumes would be (in theory) enough volume for six challenge plates (150 ÷ 25 = 6), although in practice, due to volume retention by pipette tips this should be adequate for 5 challenge plates, Antibiotic plates can be prepared in advance of the experiment, depending upon the. stability of the antibiotic used, by performing the dilutions and freezing the plates at -20°C prior to the day of the experiment..

On the day of the experiment, 25 μΐ from each well on the antibiotic plates is transferred to the challenge plate giving a total volume of 50 μΐ . Negat ive, uninoculated, controls are prepared by adding 50 μΐ of cation-adjusted Miiller Hinton broth. Incolum are prepared as described above, using the referenced method (LSI. 2012a. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved Standard - Ninth Edition; M07-A9. Wayne, PA; Clinical and Laboratory Standards Institute). Cultures (grown previously for 48 h in cation- adjusted Miiller Hinton broth at 37 °C) are standardised by comparing to a 0.5 McFarland standard optical density at 625 nm using a Biotek plate reader. The appropriate dilution is then further diluted 1:150 in twice-concentrated cation- adjusted Muiler Hinton broth. Fifty microliters of this dilution is used to inoculate the plate to give final volumes of 100 μΐ per well of 1 x cation-adjusted Muiler Hinton broth and the appropriate concentrations of each antibiotic ,

A time 0 h reading is then taken at 625 ran using the Biotek plate reader to determine background absorbance. The plates are then incubated, statically, for a further 48 h at 37°C prior to another reading at 625 nm to determine growth and the efficacy of antibiotic co-therapy.

From each, plate it is possible to determine the MICioo for each antibiotic when used alone (single antibiotic controls) , as well as the MICioo for antibiotics in combinatio . This method is also used to calculate the Fractional Inhibitory Concentration Index (FICl) using the formula shown below but for our purposes we wanted to determine if co-therapy improved the efficacy of the clinically defined antibiotic. Using interpretive criteria defined by CLSI (CLSI Performance Standards for Antimicrobial Susceptibility Testing using Other non- Enterobacteriaceae interpretive standards, Wayne PA, 2012b) , we could determine if a strain was defined as resistant, intermediate or sensitive to monotherapy with the antibiotics tested using MICioo testing methods described above, as well as from single antibiotic control dilutions in the checkerboard experiments. The checkerboard experiments could then be used to determine if co-therapy altered sensitivity or reversed resistance to the clinical antibiotic as defined by CLSI interpretive, criteria.

Cloning of mcr-1 gene for phosphoethanolamine-mediated colistin resistance into E. coli NEB ^® Express laboratory protein expression strain The open reading frame sequence for the gene mcr-1 , a probable phosphoethanolamine transferase (accession number A0A0R6L508) was synthesised using the GeneArt. gene synthesis service {Thermo Fisher Scientific) . This sequence was amplified using the polymerase chain reaction (PGR) with flanking primers and digested with appropriate restriction enzymes (Ndel and Xhol) and ligated, in-frame, into the multiple cloning site of plasmid pET29b. Plasmids with, and without, mcr-1 insert were transformed into E. call NEB ^® Express laboratory strain of E. coll. Internal, detection primers were used to confirm presence or absence of the mcr- 1 insert in the transformed cells and expression of the mcr- 1 gene was confirmed due to phenotypic change in the MIC of this strain to colistin (using the method as described above) .

Etest ^® assessment of antibiotic MIC of Neisseria gonorrhoeae GC agar plates were prepared containing Vitox supplement (Oxoid- ^K Thermo Fisher Scientific, MA, USA) with and without a range, of concentrations of filter-sterilised cysteamine. This is done by autoclaving the required volume of GC agar in solution at 121 ^e C for 15 min. Following this the agar is allowed to cool to 60°C prior to the addition of vitox supplement (for the cultivation of fastidious N. gonorrhoeae) and cysteamine solutions as required to final concentration ranges of 0, 128, 256, and 512 mg/L in 100 ml volumes. The agar is then poured, evenly, under aseptic conditions and the plates are allowed to set and dry in a safety cabinet or lamina flow hood.

N, gonorrhoeae is prepared by culturing overnight at 37°C in a 5% CO2 atmosphere on GC agar plus vitox plate. A suspension of N. gonorrhoeae cells is made by asepticaily transferring loops of overnight growth from the surface of the GC agar plus vitox plate into sterile PBS under aseptic conditions. This is then serially diluted two-fold in PBS to reach the required optical density by comparison to 0.5 McFarland standard. A sterile swab is then inoculated with the appropriate dilution of suspended cells in PBS and this is used to create a spread plate by streaking over the full surface of the appropriately labelled plate and allowed to absorb .

As appropriate, an Etest ^® strip, containing a standardised gradient, of antibiotic is placed (using sterile forceps) onto the surface of inoculated plate. Plates are then incubated upside down at 37°C in a 5% CO ₂ atmosphere for 24 hrs to allow growth of N. gonorrhoeae and the appearance of a zone of clearance on each plate (where this occurs) . The MIC can be determined by the point at which along this gradient strip no further growth occurs leading to a zone of clearance on the surface of the agar. Experiments were conducted in triplicate on 3 separate occasions.

Mouse thigh model of infection

Mouse thigh infection model experiments were conducted at Surofins Panlabs, (Taipei, Taiwan) . Mice were rendered neut opoenic with cyclophosphamide prior to infection. They were separated into groups of 5 animals per treatment. Inoculation (confirmed by culture) was with 1.52 x 10" cfu/ml of P. aeruginosa strain LES4.31 and conducted 1 hour prior to treatment which was i. v. for saline vehicle controls, ciprofloxacin, and cysteamine treated mice, and SC for colistin (used as a positive control) . Animals were sacrificed 25 h post infection (24 h post-treatment) and thigh weights and cfu/g of tissue were calculated and recorded.

Microbiology from Aberdeen clinical study Patients 18 years of age, weighing >50 kg with stable CF lung disease were commenced on oral cysteamine bitartrate (Cystagon®) 450 mg once daily, increased weekly to 450 mg four times daily. Serial plasma cysteamine concentrations were measured for 24 h after the first dose. Participants were reviewed every week, for 6 weeks, except at 4 weeks. Plasma cysteamine concentrations were measured 8 h after dosing when reviewed at 1, 2 and 3 weeks and 6 h after dosing when reviewed at 5 weeks. Sputum cysteamine concentration was also quantified at the 5-week assessment. Routine monitoring of clinical microbiology and reporting of speciation and resistance profile of major colonising microorganisms from patients continued as normal, prior to, during where necessary and after the trial by the hospital microbiology .labo atory.

Formula for calculating FICI

_' loo dru A in combination HC _10C , drug B in combination iC _!00 drug A alone M!C _j oo drug 8 alone

All media and chemical reagents were purchased from Sigma. Bcc isolates used in this study were either type strains purchased from the American Type Culture Collection (AT'CC) , the National Collection of Type Cultures (NCTC) , of Public Health England, or Deutsche Sammiung von Mikroorganismen und Zelikulturen (DSMZ) . Clinical strains were gifted from either Aberdeen Royal Infirmary, or the University of Glasgow Dental School .

References :

CLSI . 2012a. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved Standard - Ninth Edition; M07-A9. Wayne, PA: Clinical and Laboratory Standards Institute. Burkhart CG, Burkhart. CN, Isham N. 2006. Synergistic antimicrobial activity by combining an aliylamine with benzoyl peroxide with expanded coverage against yeast and bacterial species. Br J Dermatol 154 : 341-3Ί 4.

CLSI, 2012b. Performance Standards for Antimicrobial Susceptibility Testing; Twenty second Informational Supplement. Clinical and Laboratory Standards Institute, Wayne, PA.

Results

Table 1. Antimicrobial activity (MIC100; mg/L) of Burkholderia isolates versus Azithromycin (AZM) and Azithromycin in combination with cysteamine. All result represent the median MICs from triplicate samples from triplicate experiments. All data manipulations were carried out in Microsoft Excel.

MIC100 Alone MIC100 in Combination

Fold

Species Strain Origin S/l/R* S/l/R* Reduction

B. cepacia NCTC 10744 human, bronchial washings 500 16 125 8

* CLSI & EUCAST interpretative criteria do not exist for Burkholderia or non- Enterobacteriaceae against AZM. The criteria applied here are CLSI guidelines for Enterobacteriaceae: S - <16 mg/L, R - >32 mg L

Table 2. Antimicrobial activity (MIC100; mg/L) of P. aeruginosa isolates versus Azithromycin (AZM) and Azithromycin in combination with cysteamine. All resul represent the median MICs from triplicate samples from triplicate experiments. All data manipulation were carried out in Microsoft Excel.

MIC100 Alone MIC100 in Combination Fold

Species Strain Origin S/l/R* S/l/R* Reduction

Cysteamine AZM Cysteamine AZM of AZM

P. aeruginosa DSMZ1128 human, outer ear infection 250 12 31 4 3

P. aeruginosa DS Z1299 human, sputum 250 16 63 8 2

P. aeruginosa PA01 Not disclosed 500 23 94 8 3

P. aeruginosa NH57388A Cystic fibrosis patient sputum 500 16 94 8 2

P. aeruginosa NH57388B Hoffmann et al., 2005 250 63 94 23 3

P. aeruginosa NH57388C Hoffmann et al., 2005 250 125 31 63 2

P. aeruginosa NH57388D Hoffmann et al., 2005 375 94 125 31 3

P. aeruginosa DSMZ50071 Type Strain 250 63 94 16 4

P. aeruginosa PA14 human, burn 250 47 63 16 3

P. aeruginosa Pa058 Not disclosed 500 125 125 31 4

P. aeruginosa Pa492a Not disclosed 250 31 125 8 4

P. aeruginosa PA0579 Not disclosed 375 31 125 8 4

* CLSI & EUCAST interpretative criteria do not exist for Pseudomonas or non- Enterobacteriaceae against AZM. The criteria applied here are CLSI

guidelines for Eniembacteriaceae: S - <16 mg/L, R -≥32 mg/L

Table 3. Antimicrobial activity (MIC100; mg/L) of Enterobacterlaceae versus Azithromycin (AZM) and Azithromycin in combination with cysteamine. All resul represent the median MICs from triplicate samples from triplicate experiments. All data manipulations were carried out in Microsoft Excel.

MIC100 Alone MIC100 in Combination Fold

England

The criteria applied here are CLSI guidelines for Enterobacteriaceae: S -≤16 mg/L, R -≥32 mg/L

Table 4. Antimicrobial activity (MIC100; mg/L) of Colistln (COL) and COL in combination with cysteamine versus bacterial pathogens. All results represent t

The criteria applied here are EUCAST breakpoints for enterobacteraciae (£. coli and . pneumoniae), as there are no CLSI breakpoints for collstin and enterobacteraciae: S -≤2 mg/L, R - >2 mg/L. For B. cenocepacia DSMZ16553, the CLSI breakpoints for Pseudomonas spp. were used as there are no CLSI or EUCAST breakpoints for colistin versus Burkholderia spp.

Table 5. Antimicrobial activity (MIC100; mg/L) of different antibiotics (ABX) and ABX in combination with cysteamine versus Staphylococcus aureus DSMZ117 (Human blood). All results represent the median MICs from triplicate samples from triplicate experiments. All data manipulations were carried out in Micros

The criteria applied here are CLSI breakpoints for Staphylococcus spp., except mupirocin for which the EUCAST guidelines are used as there is no

Staphylococcus spp. CLSI breakpoint for mupirocin. * - Rifampicin is also known as rifampin.

Table 6. Antimicrobial activity (MIC100; mg/L) of different antibiotics (ABX) and ABX in combination with cysteamine versus Staphylococcus aureus (Cystic fibros

The criteria applied here are EUCAST breakpoints for Staphylococcus spp. MRSA - Methicillin resistant S. aureus; MSSA - Methicillin sensitive S. aureus.

Table 7. Antimicrobial activity (MIC100; mg/L) of different Ciprofloxacin (CIP) and CIP in combination with cysteamine versus Klebsiella pneumoniae H 1544207 (Clinical isolate, Public Health England). All results represent the median MICs from triplicate samples from triplicate experiments. All data manipulations we carried out in Microsoft Excel.

MIC100 Alone MIC100 in Combination Fold

Antibiotic ABX CI S/l/R S/l/R Reduction

Cysteamine CIP Cysteamine CIP ABX

Ciprofloxacin Fluoroquinolone 512 64 256 32

The criteria applied here are CLSI breakpoints for enterobacteraclae.

Table 8. Antimicrobial activity (median MIC100; mg/L) of Burkholderia isolates versus tobramycin and tobramycin in combination with cysteamine. All resul represent the MIC from triplicate samples from triplicate experiments. All data manipulations were carried out in Microsoft Excel. S = sensitive, I = intermedia and R■ resistant as defined by CLSI interpretive criteria. A change in case indicates an effect on MIC which did not cross a boundary between CLSI criteria b did have a positive impact i.e. - R r = Less resistant and s -» S = more sensitive.

MIC100 (ug/ml) Sensitivity screening

Lynovex alone Tobramycin alone Combination data

Genus & Species Strain S/l/R S/l/R

B. multivorans 1140 256.00 >64 256/32

B. multivorans 1142 >256 32.00 128/2

B. multivorans 1247 >256 >64 256/8

B. cenocepacia CFSYN 936 128 32 64/2

B. cenocepacia CFSYN 1045 256 64 128/8

B. cenocepacia CFSYN 1112 256 32 128/4

B. cepacia CFSYN 946 128 16 64/0.125

B. multivorans CFSYN 1081 >256 64 256/2

B. multivorans CFSYN 954 >256 32 256/16

B. multivorans CFSYN 945 >256 >64 256/16

B. cepacia NCTC 10743 >256 16 64/8

B. cepacia NCTC 10744 256 64

64/16

B. multivorans

05.41360rgB 256 64 128/32 (gen.ll)

B. multivorans

05.38686OrgB 256 1 128/0.5 (gen.ll)

B. cenocepacia

05.66335OrgA 256 1 128/0.25 (gen.lllA)

B. cenocepacia

07.37324AOrgA >256 0.5 64/0.25 (gen.lllA)

 Table 9. Antimicrobial activity (MIC100; mg/L) of Burkholderia isolates versus ciprofloxacin and ciprofloxacin in combination with cysteamine. All results represe the MIC from triplicate samples from triplicate experiments. All data manipulations were carried out in Microsoft Excel. S = sensitive, I = intermediate and R resistant as defined by CLSI interpretive criteria. A change in case indicates an effect on MIC which did not cross a boundary between CLSI criteria but did have positive Impact i.e. - R -> r = Less resistant, and s -> S = more sensitive.

41 Table 10. Antimicrobial activity (MIC100; mg/L) of Burkholderia isolates versus Trimethoprim/Sulfamethoxazole and Trimethoprim/Sulfamethoxazole in combinati with cysteamine. Ail results represent the MIC from triplicate samples from triplicate experiments. All data manipulations were carried out in Microsoft Excel. S sensitive, I = intermediate and R - resistant as defined by CLSI interpretive criteria. A change in case indicates an effect on MIC which did not cross a bounda between CLSI criteria but did have a positive impact i.e. - R -> r = Less resistant, and s - S = more sensitive.

Genus & Species Strain Lynovex alone Trimeth/Sulfa S/i R Combination data

(median) alone (median) (median MIC)

Tab)© 11. Antimicfobial activi y (WtCieo ^* , nsg L} sf ceftazidime aSKi ceftazidime in combination with cysteamifte versus Bufkhoi^Bria i$s!ates. AM results represe the MIC from triplicate samples from- triplicate experiments, A1! data manipulations were carried out in Microsoft Excel, S - sensiti e, S == intermediate and S¾ resistant as def ecJ CliSi interpretive criteria. A c ange in case indicates an effect on NIIC which did not cross a boyrKJstry beswserc CLSi criteria but did haw® positive impact Le. - R ^ r = Less resistant, and s § ~ mors sensitive.

B. cepacia NCTC10743 256 4 §1 128/4

Table 12. Antimicrobial activity (MIC100; mg/L) of Colistin (COL) and COL in combination with cysteamine versus E. coli NEB ^® Express laboratory strain of E. c transformed with plasmid vector pET-29b with or without cloned insert of mcr-1 gene for phosphoethanolamine-mediated colistin resistance. All results represe the median MICs from triplicate samples from triplicate experiments. All data manipulations were carried out in Microsoft Excel.

Table 13. Antimicrobial activity (MIC, mg/L) of azithromycin alone and in combination with cysteammo versus antimicrobial resistant -attain tioisseria gonorrhoea H16 620523 as detected using Etest strips on GC agar (plus vito supplement) containing different concentrations of cysteamine. Aii resuits represent thfe fSfliC from triplicate samples from ttipfeate experiments. All data msnipiilations were carried out in Microsoft ExesL S - sensitive, J « i termediate and R ~ resistant as defined by CLSI interpretive criteria; A change in case indicates an effect n MIC which d id not cros a boundary between CLSi criteria but did have a positive impact i.e. - R -> r ^■ Less resistant, and s -> S = more sensitive.

Tabie 14. Antimicrobial activity {MIC in g/l) of representative aminoglycoside antibiotics (A8X) against a range of dirsicatiy relevant antibiotic resistant intermediate) strains of bacteria. S/l/R denotes sensitivity, intermediate or resistant as efm& bv CLSi for each relevant bacteria! species. Fa Mycobacte abscessus the interpretive criteria for Nocardia sp» was used as recommended. ^Reduction In E¾|G is shown as the fraction of the .iMIp value for the ciirtic antibiotic found when used in combination with cysteamine, compared to the antibiotic used alone.

Antibiotic Species Strain MIC S/l/R* MIC in S/l/R* Reduction in

Table 15. Antimicrobial activity (MIC in g/l) of representative fluoroquinolone antibiotics (ABX) against a range of clinically relevant antibiotic resistant ( intermediate) strains of bacteria. S/l/R denotes sensitivity, intermediate or resistant as defined by CLSI for each relevant bacterial species. ^Reduction in MIC shown as the fraction of the MIC value for the clinical antibiotic found when used in combination with cysteamine, compared to the antibiotic used alone.

Antibiotic Species Strain MIC S/l/R* MIC in S l/R* Reduction in

 Table 16. Antimicrobial activity (MIC in g/l) of representative macrolide antibiotics (ABX) against a range of clinically relevant antibiotic resistant strains bacteria. S l/ denotes sensitivity, intermediate or resistant as defined by CLSI for each relevant bacterial species. ^† Reduction in MIC is shown as the fraction the MIC value for the clinical antibiotic found when used in combination with cysteamine, compared to the antibiotic used alone.

Antibiotic Species Strain MIC S/l/R* MIC in S/l/R* Reduction in

50 Table 17. Antimicrobial activity (MIC in g/l) of the polymyxin, colistin (ABX) against a range of clinically relevant antibiotic resistant (or intermediate) strains bacteria. S/l/R denotes sensitivity, intermediate or resistant as defined by CLSI for each relevant bacterial species. Reduction in MIC is shown as the fraction the MIC value for the clinical antibiotic found when used in combination with cysteamine, compared to the antibiotic used alone.

Antibiotic Species Strain MIC S l/R* MIC in S/l/R* Reduction in

mcr-1

Table 18. Antimicrobial activity (MIC in g/l) of the sulfonamide, sulfamethoxazole with trimethoprim (ABX) against a range of clinically relevant antibiotic resistan (or intermediate) strains of bacteria. S/l/R denotes sensitivity, intermediate or resistant as defined by CLSI for each relevant bacterial species, deduction in MIC shown as the fraction of the MIC value for the clinical antibiotic found when used in combination with cysteamine, compared to the antibiotic used alone

Antibiotic Species Strain MIC S/l/R* MIC in S/l/R* Reduction

aeruginosa

Table 19: The predominant pathogens reported in the sputum from three different patients before and after the Aberdeen clinical trial show a change in resistanc profile (MRSA = Methicillin-resistant Staphylococcus aureus; MSSA = Methicillin-sensitive Staphylococcus aureus; PR = pan-resistant; S = sensitive, Cip" = ciprofloxacin-resistant; Cip ⁸ = ciprofloxacin sensitive.

Patient with Pre trial Post trial

altered clinical microbiology microbiology

microbiology

Patient 1 MRSA MSSA

Patient 2 PR P. aeruginosa S P. aeruginosa

Patient 3 Cip ^R P. aeruginosa Cip ^s P. aeruginosa

Table 20: Accompanies Figure 1 below - The reduction of microbial burden in the neutropoenic mouse thigh model of infection (illustrated graphically in Figure shown as a Iog10 reduction of cfu g compared with vehicle control. Colistin (at 5 mg kg) was used a s a positive control.

Treatment Log™ reduction

in cfu/g

Vehicle only 0

Colistin (positive control) _{5 Q2}

[5 mg/kg]

Cysteamine [1.25 mg/kg] 0.74

Ciprofloxacin [15 mg/kg] 2.02

Ciprofloxacin + cysteamine 4.6