WO2008142397A1

WO2008142397A1 - Serine protease inhibitors for use in the treatment of bacterial infections

Info

Publication number: WO2008142397A1
Application number: PCT/GB2008/001717
Authority: WO
Inventors: Guy Timothy Layton; John Martin Clements; Stephen Rupert Chandler
Original assignee: Serentis Limited
Priority date: 2007-05-17
Filing date: 2008-05-19
Publication date: 2008-11-27
Also published as: GB0808690D0

Abstract

Use of a serine protease inhibitor selected from camostat, the metabolite of camostat, gabexate, nafamostat or sepimostat, or a pharmaceutically acceptable salt thereof, as an inhibitor of pathogenic bacterial growth.

Description

SERINE PROTEASE INHIBITORS FOR USE IN THE TREATMENT OF BACTERIAL INFECTIONS

Field of the Invention

The present invention relates to the use of serine protease inhibitors as inhibitors of bacterial growth. Background of the invention

Bacterial infections are a leading cause of death in children and the elderly. The emergence of antibiotic-resistant bacteria poses a significant threat to general welfare and health, and requires new forms of treatment.

Atopic dermatitis (AD), sometimes referred to as atopic eczema, is a chronic, relapsing condition which is characterised by pruritus, erythema, dry skin and inflammation. Staphylococcus aureus is found to colonise the skin lesions of more than 90% of AD patients, while being present in only 5% of normal subjects. The density of bacteria has been shown to correlate with the severity of lesion. Acne {Acne Vulgaris) is an inflammatory disease of the skin, caused by changes in the pilosebaceous units (skin structures consisting of a hair follicle and its associated sebaceous gland).

Rosacea is another skin condition believed to be caused or exacerbated by bacteria. For instance, rosacea is commonly treated with tetracycline antibiotics such as doxycycline and tetracycline.

Perioral dermatitis is a common facial skin problem in adult women. The exact cause of perioral dermatitis is not understood, but research suggests it may be related to proliferating bacteria in the hair follicles. Perioral dermatitis is commonly treated with antibiotics. An ulcer starts as reddened skin but gets progressively worse, forming a blister, then an open sore, and finally a crater. One category of ulcers is pressure ulcers. Signs of infection include foul odour, redness or tenderness around the ulcer and the skin close to the ulcer being warm and swollen. A number of bacteria are known to be associated with infected ulcers, including Peptostreptococcus spp, Prevotella spp and sometimes S. aureus. Infected ulcers are commonly treated with antibiotics.

Bacterial skin infections may be treated with antibiotics. However, this is unsatisfactory as widespread use of antibiotics may lead to antibiotic resistance. Camostat is chemically 4-[[4-[(aminoiminomethyl)amino]benzoyl] oxy]benzeneacetic acid 2-(dimethylamino)-2-oxoethyl ester. Camostat mesilate (FOY-305) is known as an oral inhibitor of multiple serine proteases and has been used for many years in the clinical therapy of pancreatitis and post- operative reflux eosophagitis.

The metabolite of camostat is chemically 4-[[4- [(aminoiminomethyl)amino]benzoyl]oxy]benzeneacetic acid. The metabolite of camostat (FOY-251) is a known serine protease inhibitor which is believed to have therapeutic effect in the treatment of pancreatitis and post-operative reflux eosophagitis by virtue of being the active metabolite of camostat.

Camostat, camostat metabolite and methods of preparing them are described, e.g. in US4021472 and US4224342.

Gabexate is chemically 4-[[6-[(aminoiminomethyl)amino]-1- oxohexyl]oxyl]benzoic acid ethyl ester. Gabexate mesilate (FOY) is known as an intravenous inhibitor of multiple serine proteases and has been used for many years in the clinical therapy of pancreatitis and disseminated intravenous coagulation. Gabexate and methods of preparing it are described, e.g. in US3751447.

Nafamostat is chemically 6-amidino-2-naphthyl 4-guanidino-benzoate. Nafamostat mesilate (FUT-175) is known as an intravenous inhibitor of multiple serine proteases and has been used for several years, in the clinical therapy of pancreatitis, disseminated intravenous coagulation and prevention of blood coagulation during extracorporal circulation. Nafamostat and methods of preparing it are described, e.g. in US4454338. Sepimostat is chemically 6-amidino-2-naphthyl 4-[(4,5-dihydro-1 H- imidazol-2-yl)amino]benzoate. Sepimostat mesilate (FUT-187) is known as an oral inhibitor of multiple serine proteases and has been evaluated as a possible drug for pancreatitis and other gastrointestinal conditions. Sepimostat and methods of preparing it are described, e.g. in US4777182 and US4820730. Summary of the invention

According to the present invention, a serine protease inhibitor selected from camostat, the metabolite of camostat, gabexate, nafamostat, sepimostat and pharmaceutically acceptable salts thereof is used as an inhibitor of pathogenic bacterial growth. For example, such a compound is used in the manufacture of a medicament for the topical treatment or prevention of a pathogenic bacterial skin infection in a mammal.

The methods, uses and compositions of the invention are expected to be useful in veterinary applications (i.e. wherein the mammal is a domestic or livestock mammal e.g. cat, dog, horse, pig etc). However, the principal expected use or method is in pharmaceutical applications (i.e. wherein the mammal is a human). Detailed Description of the Invention

The expression "inhibitor of bacterial growth" embraces inhibition of bacterial growth by any mechanism including (but not limited to) a bacteriocidal action. Effect of a compound as an inhibitor of bacterial growth can be evaluated by determining MIC values or by counting colonies as described in the Examples.

The term "pharmaceutically acceptable salt" refers to a salt, for example an acid addition salt or, in certain cases salts of an organic and inorganic base such as carboxylate, sulphonate and phosphate salt. All such salts are within the scope of this invention, and references to serine protease inhibitors described herein include the salt forms of the compounds. Examples of pharmaceutically acceptable salts are provided in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. ScL, Vol. 66, pp. 1-19.

The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are used. Examples of acid addition salts include salts formed with an acid selected from acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulphonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)-(1 S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1 ,2-disulphonic, ethanesulphonic, 2-hydroxyethanesuiphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L- glutamic), α-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (-)-L- malic, malonic, (±)-DL-mandelic, methanesulphonic (mesilate), naphthalenesulphonic (e.g. naphthalene-2-sulphonic), naphthalene-1 ,5- disulphonic, 1 -hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, (+)-L-tartaric, thiocyanic, toluenesulphonic (e.g. p-toluenesulphonic), undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

The serine protease inhibitors described herein may exist as mono- or di- salts.

If the serine protease inhibitor described herein is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO^"), then a salt may be formed with a suitable cation. Non-limiting examples of suitable inorganic cations include alkali metal ions such as Na⁺ and K⁺, alkaline earth metal cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺. Non- limiting examples of suitable organic cations include ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R⁺, NH₂FV, NHR₃ ⁺, NR₄ ⁺).

Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

Where the serine protease inhibitors described herein contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the invention.

Specific salts of camostat, gabexate, nafamostat or sepimostat that may be mentioned include the hydrogen succinate, succinate, phosphate, acetate, hydrogen tartrate, glycolate, hippurate, 1 -hydroxy-2-naphthoate, adipate and glutarate salts. The respective mesilates are preferred. It will be appreciated that the serine protease inhibitors described herein may exist in a number of different geometric isomeric, and tautomeric forms and references to compounds of the invention include all such forms.

It will also be appreciated that where the serine protease inhibitors described herein contain one or more chiral centres, they may exist in the form of two or more optical isomers, references to serine protease inhibitors described herein include all optical isomeric forms thereof (e.g. enantiomers, epimers and diastereoisomers), either as individual optical isomers, or mixtures (e.g. racemic mixtures) of two or more optical isomers. The optical isomers may be characterised and identified by their optical activity (i.e. as + and - isomers, or c/and /isomers) or they may be characterised in terms of their absolute stereochemistry using the "R and S" nomenclature developed by Cahn, lngold and Prelog, see Advanced Organic Chemistry by Jerry March, 4^th Edition, John Wiley & Sons, New York, 1992, pages 109-114, and see also Cahn, lngold & Prelog, Angew. Chem. Int. Ed. Engl., 1966, 5, 385- 415.

It will be appreciated that optical isomers may be separated by a number of techniques which are well known to the person skilled in the art, for example, chiral chromatography (chromatography on a chiral support). References to serine protease inhibitors described herein include references to these compounds as solids in either amorphous or crystalline form, including all polymorphic forms. Crystalline forms may be prepared by recrystallisation of the compounds from appropriate solvents. Amorphous forms may be prepared e.g. by spray drying a solution of the compounds. Examples of polymorphic forms include solvates (e.g. hydrates), complexes (e.g. inclusion complexes or clathrates with compounds such as cyclodextrins, or complexes with metals of the compounds), and pro-drugs of the compounds.

In a preferred embodiment, the invention relates to inhibition of bacterial growth associated with a pathogenic bacterial skin infection. In another preferred embodiment, the invention relates to the treatment or prevention of a pathogenic bacterial skin infection.

Bacterial skin infections can cause a range of diseases including cellulitis, erythrasma, folliculitis, skin abscesses, and carbuncles, hidradenitis suppurativa, impetigo, necrotizing skin infections, Staphylococcal scalded skin syndrome, acne (e.g. acne vulgaris), rosacea, wound infections, surgical site infections, and infected ulcers (e.g. diabetic leg ulcers) or blistering conditions. Skin colonisation with Staphylococcus aureus is found in most cases of atopic eczema or dermatitis and is associated with the severity of the skin lesions. Many types of bacteria can infect the skin. The most common pathogens found in skin infections are Staphylococcus species and Streptococcus species, in particular Staphylococcus aureus and Steptococcus pyogenes.

Other bacterial skin pathogens include Corynebacterium spp. (e.g.

Corynebacterium minutissimum), Enterococcus spp (e.g. Enterococcus faecalis), Propionibacterium acnes Haemophilus spp, Pseudomonas aeruginosa,

Escherichia coli, Bacillus anthracis, Clostridium spp, Bartonella spp,

Mycobacterium spp and Bacteroides spp (e.g. Bacteroides fragilis). Another example is Propionibacterium granulosum.

Amongst the bacterial pathogens, resistant strains have evolved/arisen which makes them particularly difficult to eradicate. Examples of such strains are methicillin resistant Staphylococcus aureus (MRSA), methicillin resistant coagulase negative Staphylococci (MRCNS), penicillin resistant Streptococcus pneumoniae and multiply resistant Enterococcus faecium.

In one embodiment, the pathogenic bacterial infection is caused by Escherichia coli. We have found that nafamostat is particularly suitable for inhibition of growth of Escherichia coli and therefore is indicated for the treatment of bacterial infections caused by Escherichia coli.

In one embodiment, the pathogenic bacterial skin infection is caused by

Staphylococcus aureus, which is responsible for a number of skin disorders including cellulitis, impetigo, folliculitis, and furuncles and carbuncles. Another skin disorder in which infection or colonisation by Staphylococcus aureus is an exacerbatory factor is atopic dermatitis.

Data demonstrating the effect of serine protease inhibitor, in inhibiting growth of Staphylococcus aureus, are shown in the examples. It has been shown that gabexate, nafamostat and camostat are also capable of inhibiting the activation of keratinocytes by Staphylococcus aureus. Activated keratinocytes produce IL-8 and other inflammatory cytokines which exacerbate underlying inflammatory skin conditions. In a further embodiment the pathogenic bacterial skin infection is caused by Propionibacterium acnes, which can contribute to acne. Propionibacterium granulosυm may also be a contributor to acne.

For the treatment of AD, acne, rosacea, perioral dermatitis and ulcers, especially infected ulcers, it is preferred to use, as the serine protease inhibitor, camostat or nafamostat.

The ability of a given serine protease inhibitor to inhibit bacterial growth can be determined using the methods given in the Examples below.

As can be seen from the Examples below, the claimed compounds are effective in inhibiting bacterial growth in both Gram -ve and Gram +ve bacteria.

Suitably the serine protease inhibitor is formulated for topical administration and it may be administered to a patient in an amount such that from 0.01 to 10 g, preferably from 0.01 mg to 1 g, active ingredient is delivered per m² of the area being treated. Typically, the total amount of inhibitor is from 0.0001 to 10 wt%, suitably from 0.001 mg to 1.0 wt% based on the total weight of the formulation,

The topical formulation may, for example, take the form of a gel, ointment, cream or lotion. Other examples of presentations include impregnated dressings, pastes, dusting powders, sprays, oils, transdermal devices etc. When said formulation is a gel, it typically comprises a hydrophilic polymer such as cross-linked polyethylene glycol, cross-linked starch or polyvinyl pyrrolidone.

An ointment, cream or lotion typically contains an aqueous phase and an oleaginous phase in admixture. They may generally be characterised as oil-in- water emulsions or water-in-oil emulsions. Alternatively, the formulation may be entirely oleaginous, consisting of an oleaginous base or oil such as fractionated coconut oil, sesame oil, avocado oil, corn oil or primrose oil, and may be thickened with white soft paraffin or other suitable thickener.

The formulation may additionally contain one or more emollients, emulsifiers, thickeners and/or preservatives, particularly when it is a cream or ointment.

Emollients suitable for inclusion in creams or ointments are typically long chain alcohols, for example a C8-C22 alcohol such as cetyl alcohol, stearyl alcohol and cetearyl alcohol, hydrocarbons such as petrolatum and light mineral oil, or acetylated lanolin. The total amount of emollient in the formulation is preferably about 5 wt% to about 30 wt%, and more preferably about 5 wt% to about 10 wt% based on the total weight of the formulation.

The emulsifier is typically a nonionic surface active agent, e.g., polysorbate 60 (available from ICI Americas), sorbitan monostearate, polyglyceryl-4 oleate and polyoxyethylene(4)lauryl ether. Generally the total amount of emulsifier is about 2 wt% to about 14 wt%, and more preferably about

2 wt% to about 6 wt% by weight based on the total weight of the formulation.

Pharmaceutically acceptable thickeners, such as Veegum K (available from R. T. Vanderbilt Company, Inc.), and long chain alcohols (i.e. C8-C22 alcohols such as cetyl alcohol, stearyl alcohol and cetearyl alcohol) can be used.

The total amount of thickener present is preferably about 3 wt% to about 12 wt% based on the total weight of the formulation.

Preservatives such as methylparaben, propylparaben and benzyl alcohol can be present in the formulation. Other example preservatives are phenoxyethanol and chlorocresol. The appropriate amount of such preservative(s) is known to those skilled in the art.

Optionally, an additional solubilizing agent such as benzyl alcohol, lactic acid, acetic acid, stearic acid or hydrochloric acid can be included in the formulation. If an additional solubilizing agent is used, the amount present is preferably about 1 wt% to about 12 wt% based on the total weight of the formulation.

Optionally, the formulation can contain a humectant such as glycerin and a skin penetration enhancer such as butyl stearate, urea and DMSO. It is known to those skilled in the art that a single ingredient can perform more than one function in a cream, i.e., cetyl alcohol can serve both as an emollient and as a thickener.

Where said formulation or medicament is a cream, it typically consists of an oil phase and a water phase mixed together to form an emulsion. Preferably, the cream comprises an oil-in-water emulsion. Preferably, the amount of water present in a cream of the invention is about 45 wt% to about 85 wt% based on the total weight of the cream.

One exemplary formulation is a cream which comprises an emulsifying ointment (e.g. around 30 wt%) comprising white soft paraffin, emulsifying wax and liquid paraffin made to 100% with purified water and containing preservative (e.g. phenoxyethanol). This formulation may also be buffered to the required pH (e.g. with citric acid and sodium phosphate). The concentration of active may typically be between 0.001 and 1.0 wt%. Alternatively, the cream may be more complex, for example an oil-in-water base comprising isostearic acid, cetyl alcohol, stearyl alcohol, white petrolatum, polysorbate 60, sorbiton monostearate, glycerin, xanthum gum, purified water, benzyl alcohol, methylparaban and propyl-paraban.

Where the formulation or medicament is an ointment, it typically comprises a pharmaceutically acceptable ointment base such as petrolatum, polyethylene glycol 400 in combination with polyethylene glycol 3350 (available from Union Carbide) or bases such as propylene glycol dicaprylocaprate, linoleoyl macrogolglycerides or caprylic/capric triglycerides (available from Gattefosse). The base may be an oil, such as fractionated coconut oil, sesame oil, avocado oil, corn oil or primrose oil. The amount of ointment base present in an ointment of the invention is preferably about 60 wt% to about 95 wt% based on the total weight of the ointment.

The serine protease inhibitor may be administered in conjunction with further medicaments, such as conventional therapies for the treatment or prevention of bacterial infections, for example antibiotics or anti-inflammatory treatments, for example steroids.

These further treatments may be administered by any convenient route. Topical and oral routes are preferred.

Combination treatments may be administered simultaneously, sequentially or separately, by the same or by different routes. In one embodiment, the further medicament may be administered orally. In a second embodiment, the further medicament may be administered parenterally, e.g. intravenously. In another embodiment, the further medicament may be administered topically, e.g. in a combined preparation with the serine protease inhibitor or separately at different sites.

For example, the further medicament may be an antibiotic which is administered orally or topically. It may be a steroid or other anti-inflammatory treatment which is administered orally, topically or parenterally. It may be a metalloprotease inhibitor which is administered, e.g. orally or topically. Another example is a retinoid which is administered, e.g. orally or topically.

For example, the further medicament may be a combination product which is administered, e.g. orally or topically, e.g. a combination of a steroid and antibiotic.

In one embodiment, the method and use described herein may be preceded by a method step involving confirming the presence of the bacteria.

For example, the presence of bacteria may be determined directly by sampling the skin of patients and determining the presence of bacteria through microbiological or genetic methods. In the simplest form of assay, the affected skin is swabbed and the swab is inoculated onto blood agar plates and colonies of the bacteria identified through standard microbiological procedures. A quantitative methodology may also be applied to assess the level of colonisation. Genetic methods such as quantitative PCR may also be used to demonstrate the presence of bacteria.

The following Examples illustrate the invention.

Example 1

Inhibition of Staphylococcus aureus 8325-4 growth

The inhibitory activities of camostat (Wako Chemicals GmbH, Neuss, Germany), gabexate (Tocris Bioscience, Bristol, UK; BIOMOL International LP,

Exeter, UK; Wako Chemicals GmbH) and nafamostat (BIOMOL International LP) were tested against Staphylococcus aureus 8325-4.

A.

Staphylococcus aureus 8325-4 was maintained on tryptone soya agar (Oxoid). Minimal inhibitory concentrations (MIC) were determined by a broth microdilution method.

Staphylococcus aureus 8325-4 was grown at 37°C in tryptone soya broth

(TSB) overnight. In a 96-well microtitre plate a dilution series of the serine protease inhibitors camostat, gabexate and nafamostat were added to a final volume of 100 μl of TSB with a starting inoculum of approximately 1 x 10⁵ colony-forming units/ml Staphylococcus aureus 8325-4. The plates were incubated with at 37°C for 20 hours. The minimal inhibitory concentration (MIC) was determined by the concentration of serine protease inhibitor at which there was no visible bacterial growth in the wells.

The data in Table 1 shows that Staphylococcus aureus 8325-4 was inhibited by both camostat and nafamostat.

Table 1

Compound MIC uM

Gabexate >500

Nafamostat 62.5

Camostat 500

B.

The effect of the serine protease inhibitors on S. aureus growth in conditions optimum for the growth of human keratinocytes in culture was tested. To each well of a 24-well cell culture plate are added 0.5 ml Epidermal Keratinocyte Basal Medium with Keratinocyte Growth Supplement (TCS CellWorks) ± vehicle or test compounds. The S. aureus inoculum is prepared by growth of S. aureus 8325-4 in keratinocyte growth medium at 37°C overnight. The absorbance of the culture is measured and adjusted to an optical density at 600nm (OD₆oo) of 0.1 in keratinocyte growth medium. 10 μl the S. aureus 8325- 4 inoculum is added to the appropriate wells.

The cultures are incubated for 18-20hrs at 37°C in an atmosphere of 5%

CO₂ and the absorbance measured at 620 nm. Growth inhibition was determined by reference to the vehicle control and the minimal inhibitory concentration determined as the concentration at which there was >90% inhibition of growth.

Results of experiments are shown in Table 2.

Table 2

Compound MIC (uM) gabexate 400 nafamostat 50 camostat 200 The experiment shows that gabexate, nafamostat and camostat inhibit the growth of S. aureus when grown in conditions optimised for the in vitro growth of human epidermal keratinocytes (nafamostat shows the strongest effect under these conditions). Example 2

Inhibition of Propionibacterium acnes ATCC 11827

The inhibitory activities of camostat (Wako Chemicals GmbH, Neuss, Germany), gabexate (Tocris Bioscience, Bristol, UK; BIOMOL International LP, Exeter, UK; Wako Chemicals GmbH) and nafamostat (BIOMOL International LP) were tested against Propionibacterium acnes ATCC 11827.

Propionibacterium acnes ATCC 11827 was obtained from LGC Promochem (Teddington, UK). Cultures were grown on tryptone soya agar (Oxoid) in anaerobic conditions using an anaerobic jar with an anaerobic gas generating kit from Oxoid. Using a 25-well cell culture plate, 1.5 ml of tryptone soya agar was mixed with varying concentrations of each serine protease inhibitor (camostat, gabexate or nafamostat) in each well. A suspension of Propionibacterium acnes was made in tryptone soya broth (TSB) and adjusted to an optical density at 600nm (OD600) of 0.1. 2μl of this suspension was placed on the surface of the agar, allowed to dry. The plates were then incubated for 4 days at 37°C in anaerobic conditions. The minimal inhibitory concentration (MIC) was determined by the concentration at which there was no visible growth of Propionibacterium acnes.

The data in Table 3A shows that Propionibacterium acnes ATCC 11827 was inhibited by both nafamostat and camostat.

Table 3A Compound MIC uM

Gabexate >800

Nafamostat 100

Camostat 400 Example 3

Inhibition of Escherichia coii

The inhibitory activity of nafamostat (BiOMOL International LP) was tested against Escherichia coli strain TAM 1. Escherichia coli strain TAM1 (mcrA Δ(mrr-hsdRMS-mcrBC) ΦδOlacZΔ

M15 ΔlacX74 recA1 araΔ139 (ara-leu)7697 galU galK rpsL endA1 nupG) was obtained from Active Motif Europe (Rixensart, Belgium) and maintained on tryptone soya agar (Oxoid). Minimal inhibitory concentrations (MIC) were determined by a broth microdilution method. In a 96-well microtitre plate a dilution series of the serine protease inhibitor nafamostat was added to a final volume of 200 μl of tryptone soya broth (Oxoid) with a starting inoculum of approximately 5 x 10⁵ colony forming units/ml Escherichia coli TAM1. Microtitre plates were incubated at 37°C for 18 hours. The minimal inhibitory concentration (MIC) was determined as the concentration at which there was no visible bacterial growth in wells.

The results in Table 4 show that the growth of Escherichia coli was inhibited by nafamostat.

Table 4

Compound MIC uM

Nafamostat 500

Example 4

Inhibition of activation of human keratinocytes by S. aureus

Activated keratinocytes produce IL-8, a proinflammatory chemokine. Many bacterial products cause the activation of keratinocytes. In this experiment monolayers of human keratinocytes in culture are infected with S. aureus and the effect of serine protease inhibitors on the activation of keratinocytes measured via IL-8 production.

The S. aureus are prepared by growth in keratinocyte growth medium at 37⁰C overnight. The absorbance of the culture is measured and adjusted to an optical density at 600nm (OD₆oo) of 0.1 in keratinocyte growth medium. Normal human skin epidermal keratinocytes (TCS Cellworks, Botolph

Claydon, Buckingham, UK) are maintained in Epidermal Keratinocyte Basal Medium with Keratinocyte Growth Supplement (keratinocyte growth medium) as per instructions. Proliferating cultures are trypsinised, harvested, treated with a trypsin inhibitor and resuspended in keratinocyte growth medium (TCS Cellworks) at approximately 50,000 cells/well, to provide monolayers in 24-well cell culture plates. Cells are incubated overnight at 37⁰C in 5% CO₂ to allow recovery. The spent medium is aspirated from the wells, and replaced with fresh keratinocyte growth medium ± vehicle or test compound, with 10 μl of the S. aureus 8325-4 inoculum added to the appropriate wells. The keratinocytes are incubated at 37⁰C in 5% CO₂ for a further 18hr. The culture medium is removed from each well and centrifuged at >10,000 x g for 2 minutes and the supernatant removed. The concentration of IL-8 is determined using a human IL-8 enzyme- linked immunosorbent assay (ELISA) development kit from R&D systems (Catalog Number: DY208) using the manufacturers instructions. Results of the experiments are shown in Table 5A.

Table 5A

IL-8 (pg/ml)

Concentration Gabexate Nafamostat

(uM) Un- Un+ S. aureus + S. aureus stimulated stimulated

Vehicle 34 1089 25 1050

30 42 1343 50 1096

100 40 718 56 72

200 40 69 83 61

In a subsequent experiment the following results were obtained:

Table 5B

Concentration Camostat

(uM) Un- + S. aureus stimulated

Vehicle 10 790

25 8 734

50 2 975

100 4 916

200 3 582

400 7 135

The results show that gabexate, nafamostat and camostat inhibit the activation of keratinocytes by S. aureus. Example 5 Inhibition of human skin kallikreins by camostat, gabexate and nafamostat

The inhibitory activities of camostat (Wako Chemicals GmbH, Neuss, Germany), gabexate (Tocris Bioscience, Bristol, UK; BIOMOL International LP, Exeter, UK; Wako Chemicals GmbH) and nafamostat (BIOMOL International LP) were tested against a range of recombinant human kallikreins (R&D Systems Europe Ltd, Abingdon, UK). For use, kallikreins 7 and 14 were activated with thermolysin (R&D Systems) followed by the addition of EDTA as described by the manufacturer; control incubations confirmed that under the assay conditions described no residual thermolysin activity was detectable. Kallikrein 8 was activated with endoproteinase Lys-C (Sigma-Aldrich Co Ltd, Poole, UK) as described by the manufacturer; control incubations demonstrated that under the assay conditions described no residual Lys-C activity was detectable. Assay mixtures (0.1 ml) ± compound (dissolved in water) were as follows: kallikrein 5 (3.5 ng per assay), 9OmM sodium phosphate buffer pH 8.0, 0.045% Brij 35, 0.1 mM Boc-Val-Pro-Arg-AMC (Bachem GmbH, Weil am Rhein, Germany); kallikrein 7 (15 ng per assay): 45 mM Tris-HCI pH 8.5, 0.135 M sodium chloride, 0.045% Brij 35, 10 μM Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH₂ (R&D Systems); kallikrein 8 (10 ng per assay): 45 mM Tris-HCI pH 8.5, 0.135 M sodium chloride, 0.045% Brij 35, 0.1 m M Boc-Val-Pro-Arg-AMC; and kallikrein 14 (0.8 ng per assay); 45 mM Tris-HCI pH 8.0, 0.135 M sodium chloride, 0.045% Brij 35, 0.1 mM Boc-Val-Pro-Arg-AMC. Control incubations lacked either compound _CXl or enzyme. Assays were incubated at 37^QC/1h and stopped with 0.1ml 0.5M acetic acid. Because of background fluorescence a series of nafamostat controls was also done in which the enzyme was added after the acetic acid. The extent of substrate cleavage was assessed fluorimetrically using 39 Onm excitation/460 nm emission (kallikreins 5, 8 and 14) or 320 nm excitation/405 nm emission (kallikrein 7). Percentage inhibition values were calculated after correction for controls without enzyme and the compound concentration eliciting a 50% decrease in enzyme activity under assay conditions (the IC₅₀ value) was determined by curve fitting (XLfit, IDBS Ltd).

The data in Table 6 show that skin kallikreins 5, 7, 8 and 14 were all inhibited by the three compounds. Table 6

IC₅₀ value (uM)

Kallikrein

Camostat Gabexate Nafamostat

5 0.21 6.8 0.22

7 37 62 17

0.071 1.9 0.0033

14 1.0 1.3 0.014

Example 6

Time-kill analysis following exposure to nafamostat

S. aureus 8325-4 was maintained on tryptone soya agar (Oxoid). Cultures were grown at 37°C in tryptone soya broth (TSB) overnight then diluted with fresh broth to yield a starting inoculum of approximately 4 x 10⁶ CFU/ml. Nafamostat was added at a final concentration four- or eightfold above its minimal inhibitory concentration, and the cultures incubated with agitation at 37°C. A parallel culture containing no antibiotic served as a control. Colony counts were determined at intervals by serial dilution onto tryptone soya agar plates (see Table 7). Table 7

This analysis shows that nafamostat can rapidly kill S. aureus and has a bacteriocidal mode of action.

All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

Claims

1. A compound which is a serine protease inhibitor selected from camostat, the metabolite of camostat, gabexate, nafamostat, sepimostat and pharmaceutically acceptable salts thereof, for the treatment or prevention of a condition by acting as an inhibitor of pathogenic bacterial growth.

2. A compound according to claim 1 , for use as a topical medicament for the treatment or prevention of a skin disorder or a pathogenic bacterial skin infection or colonisation in a mammal.

3. A compound according to claim 2, wherein the infection is caused by Staphylococcus aureus.

4. A compound according to claim 2, wherein the infection is caused by Propionibacterium acnes.

5. A compound according to claim 2, wherein the infection is caused by Escherichia coli.

6. A compound according to claim 2, wherein the disorder is atopic dermatitis.

7. A compound according to claim 2, wherein the disorder is acne.

8. A compound according to claim 2, wherein the disorder is rosacea.

9. A compound according to claim 2, wherein the disorder is perioral dermatitis.

10. A compound according to claim 2, wherein the disorder is an infected ulcer.

11. A compound according to any preceding claim, which is camostat or a pharmaceutically acceptable salt thereof.

12. A compound according to claim 11 , which is camostat mesilate.

13. A compound according to any of claims 1 to 10, which is the metabolite of camostat or a pharmaceutically acceptable salt thereof.

14. A compound according to any of claims 1 to 10, which is gabexate or a pharmaceutically acceptable salt thereof.

15. A compound according to claim 14, which is gabexate mesilate.

16. A compound according to any of claims 1 to 10, which is nafamostat or a pharmaceutically acceptable salt thereof.

17. A compound according to claim 16, which is nafamostat mesilate.

18. A compound according to any of claims 1 to 10, which is sepimostat or a pharmaceutically acceptable salt thereof.

19. A compound according to claim 18, which is sepimostat mesilate.

20. A compound according to any preceding claim, administered in combination with an antibiotic.

21. A compound according to any preceding claim, administered in combination with a steroid or other anti-inflammatory treatment.