MX2008002914A - Use of an aureolysin inhibitor for the treatment of inflammatory skin conditions characterised by colonisation with staphylococcus aureus - Google Patents

Abstract

There is provided, inter alia, a method for the treatment or prevention of an inflammatory skin condition which is characterised by colonisation with Staphylococcus aureus, comprising the topical administration of an aureolysin inhibitor.

Description

USE OF AN AUREOLISIN INHIBITOR FOR THE TREATMENT OF INFLAMMATORY CONDITIONS IN THE SKIN CHARACTERIZED BY COLONIZATION WITH STAPHYLOCOCCUS AUREUS Introduction The present invention relates to the treatment of inflammatory conditions in the skin that are characterized by colonization with Staphylococcus a ureus. Atopic dermatitis (AD), sometimes referred to as eczema, is a recurrent, chronic condition that is characterized by puritus, erythema, dry skin and inflammation. The pathogenesis of AD is not yet fully understood, although excessive T cell activation in response to antigen stimulation and hyperstimulation of T cells by Langerhans to topical cells will be important factors. Levels of IgE production correlate well with the severity of the disease, and although allergen-specific IgE can be observed in many patients, it is not clear that this discovery indicates sensitization to a specific allergen. The prevalence of the disorder varies widely, but it has been estimated that it is as high as 20% among children in some Western countries. AD is frequently seen in families with a history of topical diseases (asthma, allergic rhinitis and AD). The topical application of the antibiotic mupirocin has provided significant improvements in patients with poorly controlled AD, suggesting that the bacteria may be involved in the perpetuation of the disorder (Lever, R et al, Br. J. Derma Tol. 119: 189-198, 1988). Staphylococcus au ceus has been found to colonize the skin lesions of more than 90% of AD patients (Leyden, JE, Marples, RR and Kligman AM Br. J. Derma tol. 90: 525-530, 1974), while which is present in only 5% of normal subjects. Bacteria have been shown to be important in the exacerbation and chronicity of AD through the release of toxins (for example enterotoxins A, B, C and D, toxic shock syndrome toxin), many of which are highly antigenic in nature, thus exacerbating inflammatory responses on the skin (Leung, DYM et al., J. Clin. Inves, No. 92 1374-80, 1993). A particular study in children found that 81% of patients had colonization of Staphylococcus a ureus (compared to 4% of the control group) showing that the severity of the disease would be correlated with colonization by toxigenic strains (Bunikowski, R et al. J. Allergy Clin Immunol. 105 (): 814-819, 2000). A unifying link between the body of evidence suggests a function of environmental factors, including food allergens or aeroallergens, and the literature suggests an involvement of colonization of Staphylococcus aureus was found when mice exposed to enterotoxin B and home dust mite antigen Der p produced an additive inflammatory response (Herz, U J. Tnvest, Dermat 110 (): 224-231, 1999). In addition, Staphylococcus a? It has been shown that it preferentially binds to sites on the skin that involve Th-2 type inflammation (Cho, S-ll et al, J. Lnvest, Dermat, 16 (5): 658-663). , 2001). Current treatments typically involve a number of procedures (i) hydration of the skin - which includes lot formation and use of moisturizer (11) the use of medications to reduce or modulate the immune response, such as spheroids (g Lucocort i co i des) and immunosuppressants (eg ciclospopna A, tacrol imus and pi mecrol i mus) (n) elimination of contributing factors - irritants, allergens, emotional stress factors and infectious agents. Although current treatment can effectively treat acute phases of disorder, there are questions about its long-term use due to the potentially severe side effects associated with prolonged use of spheroids and immunosuppressants. Antibiotics are frequently used to treat fec-ons, although the general use of antibiotics, especially topical antibiotics, is generally discouraged due to the risk of the development of antibiotic-resistant bacterial strains.

Aureolysin (EC 3.4.24.29) is a metalloprotease which is secreted by Staphylococcus aureus (Dubin, G. Biol. Chem. 383: 1075-1086, 2002). Aureolysin is a member of the thermolysin protein family, which is dependent on zinc and calcium for its activity and has low substrate specificity. The aureolysin crystal structure was published in 1998, showing that the protein consists of an individual chain of 301 amino acids (Banbula, A et al., Structure 6 (9): 1185-1193, 1998). Aureolysin is encoded by the ur gene, genetic analyzes of which indicate that the protein is highly conserved and therefore can play an important role in the life cycle of the bacteria (Sabat, A et al., Jnfecfc. Immun 68 (2): 973-976, 2000). Researchers have discovered that aureolysin inhibitors should decrease the pathogenicity and potential colonization of Staphylococcus to ureas in atopic dermatitis. This limits the virulence of the organism through disturbing host-aureolysin interactions. The exact function of aureolysin is not clear, although it has been implicated in the processing of protease V8 (a secreted serine protease) and has been shown to inactivate the human proteinase inhibitors ax-anti-chymotrypsin and inhibitor ai-proteinase in vi t ro. Strains of Staphylococcus a? Reus that produce significant amounts of aureolysin or less susceptible to catheter ic L-37, a human bacterial peptide with potent activity against SLaphylococci (Sieprawska-Lupa, M et al.,? nL ími crob Agents ChemoLher. 8 (1?): 46 / 3-679 , 2004). However, proteolysis of anti- i-i-crobid peptides has yet to be tested as a mechanism for in vivo bacterial resistance (Brogden, KA Nature 3: 238-250, 2005). Secreted toxins are generally known to be significant virulence factors, however, aureoloma is not considered to be a defined virulence factor (Supuran, CT, Scozzafava, A and Clare, BW Med. Res. Rev. 22 ( 4): 329-372, 2002; Dubin, G Biol. Chem. 383: 10 / 5-1086, 2002). The Investigation of the Protected Life! In a series of SLaphylococcus aureus strains of patients with AD found that of those strains showing moderate to high proteolytic activity, aureolysin contributed between 25-100% of the proteolytic activity (Mi ed / obrodzki, J et al., Eur J Clin Microb io L Infect Dis 21: 269-276, 2002). Cl WO 02/089/30 discloses compounds and methods for the modulation of CD154 activity, such methods which include lowering the CD154 ratio by administering metal oppressor inhibitors to block the movement of CD154 from cell surface by endogenous human metalloproteases. Although Mainly discuss such procedure as an antithrombotic therapy, the treatment of AD is suggested, however there is no supporting evidence for this claim. In addition, no aureolysin, nor the function of aureolysin in AD, is discussed. Also, WO 02/089730 discloses the inhibition of metalloproteases but none are defined by the description or examples. The defined inhibitors are known as matrix metalloprotease, MMP inhibitors (Clan MA (M), MIO family) and 5 examples are given that they are inhibitors of broad spectrum MMPs or MMP2 / 9 gelatinase inhibitors. Therefore, they have indirectly shown that MMPs can segment CD154 but they have not defined which metalloproteases segment CD154. The description also mentions shedasas (adamalysin family of metalloproteases, M12 family) but does not provide evidence for their role in shedding CD154. Grobelny et al., (1992) Biochemistry 31, 7152-7154 discusses the inhibition of collagenase in human skin, thermolysin and elastase of Pseudomonas aeruginosum by hydroxamic peptide acids. A considerable number of MMPs are known in the art due to the fact that these have long been of interest as drug targets in cancer and inflammatory diseases. MMPs (MIO) and the family Adamalysine (M12) of mel to 1 -presetases are methyzcines (characterized by the fold of hnetzincin 'near the portion linking / i nc (Bode et al., L ~ CBS Lett, 331, 134-40, 1993)) and are other than JMs (characterized by the presence of a Glu residue in the domain that in a / a zinc) of what aureolysin (M4) is a member. Thus, aureole is not categorized as an MMP. It has also been known for a long time that MMPs and particularly adamaJ sinas can segment membrane proteins that release active molecules (for example TNFalfa, Tas, CD30, CD40 etc). In summary, while the technique has recognized a role for Staphylococcus sau reus in the exacerbation of AD, proposed treatments have been directed at controlling either the inflammatory response that results in a combination of implicit disease and colonization. of Staphyl ococc? sa ureus or to directly control Staphylococcus a ureus. inhibition of a secreted protein, such as aureohsin is not contemplated in the art. Also, while the technique has recognized a role for the inhibition of some metalloproteases in the treatment of ΔD, this has been in the context of inhibition of meta endogenous matrix Loproteases and adama lysines and non-exogenous metalloproteases of colonizing bacteria. There is a clear need for new methods to treat inflammatory conditions in the skin that are characterized by colonization with S taphylococcus aureus, such as AD. Furthermore, in view of the well-known points considered the development of resistance to conventional antibacterial treatments, it is desirable that the new methods for the treatment of inflammatory conditions in the skin that are characterized by colonization with Staphylococcus a ureus involve a novel mechanism of action . BRIEF DESCRIPTION OF THE INVENTION In accordance with the present invention there is provided a method for the treatment or prevention of an inflammatory condition in the skin in a mammal that is characterized by colonization with Staphylococcus aureus, which comprises topical administration of an inhibitor of aureolysin In a second aspect of the present invention there is provided the use of an aureolysin inhibitor in the manufacture of a topical medicament for the treatment or prevention of an inflammatory condition in the skin in a mammal which is characterized by colonization with Staphylococcus aureus. . Also provided is a topical pharmaceutical composition comprising a pharmaceutically acceptable aureolein inhibitor and carrier or excipient, for use in the treatment of an inflammatory condition in the Skin that is characterized by colonization with Staphylococcus a? reus. The methods, uses and compositions are expected to be useful in veterinary applications (ie where the mammal is a domestic mamife.ro or cattle eg cat, dog, horse, pig etc). However, the main use or expected method is in pharmaceutical applications (ie where the mammal is a human). An advantage of the methods, uses and compositions of the invention is that until now the treatment is directed to aureolysin, since the treatment is directed to a secreted protein that is apparently not critical for bacterial survival, it is much less likely to originate from the selective pressure that can result in the production of resistant mutants of the bacterium in relation to procedures that involve the use of conventional antibiotics. In addition, since it is directed to an exogenous protein (i.e., a protein not present in mammals) then it can be expected to result in a mechanism related to side effects (i.e. the side effects that result from the inhibition mechanism before the agent inhibitory itself). BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows gels obtained for washed samples of the skin taken from acute AD sites. (zi ographic analysis with and without Compound 11) Figure 2 shows inhibition of proteolytic activity by Compound 3 in the milk agar plate assay. DETAILED DESCRIPTION OF THE INVENTION By the term 'an inflammatory condition characterized by co-occurrence with Staphylococcus aureus', a condition such as atopic dermatitis is proposed where the skin is colonized by Staphylococcus au reus in most cases. cases and where an increase in colon ation or cutaneous infection results in aggravation of the implicit condition and an increase in the inflammatory response. An additional inflammatory condition is Netherton's syndrome, a severe autosomal regressive skin disorder characterized by epmetroderma i Ctios L forme, atopy (atopic dermatitis and very high levels of igE) and tpchorrhexis invaginata. Most patients experience recurrent or persistent bacterial infections. By the term "atopic dermatitis" or "AD" a chronic recurrent inflammatory skin disease characterized by intense pruritus and cutaneous hyperresponsiveness associated with elevated serum levels of IgE and eosinophils is proposed In a preferred embodiment, the method or use of the II present invention is for the treatment or prevention of atopic dermatitis. The inhibitor of aureus I i s i na can be any inhibitor of meta Loprotease that is capable of inhibiting the proteolytic activity of aureol ßsin. The preferential inhibitor nh bi to the type A I i i i co and more preferably inhibit both allelic forms, aureol i sma type 1 and type II, the TL type that is previ ous in skin diseases.

(Sabbath A, Influence [mmun 68, 9 / 3-6, 2000). This inhibitor may or may not also directly inhibit endogenous metzincine metalloproteases; for example, the MMPs (MIO) (for example MMPs 1, 2, 8, 9) and / or adama l isines (M12). The types I and TT of aureol i s i a 1 i i ca are hereinafter referred to as "aureole if 1" and "aureolysin II" respectively. The ability of a given substance to inhibit aureus Lisin can be determined using the "Aureol enzyme inhibition assay" given in the Examples below. By "inhibition of aureol i si na" or "aureolisma mh i bidor" the researchers propose that it gives an IC50 value of less than 50 mi chromium! a r in the aureol enzyme enzyme assay (for example the aureolysin II enzyme assay), preferably less than 5 mi chromium lar specifically less than 0.5 micromolar.

By "inhibition of endogenous metzincine metalloproteinases" or "endogenous metzicin endoprotease metal inhibitors", the researchers proposed to give an IC50 value of less than 50 micromolar in the endogenous methoxymethrin-responsive methotrexate assay, preferably less than 5 micromolar especially less than 0.5 micromolar. As L in one embodiment of the invention the aureolysin inhibitor does not significantly inhibit endogenous meth / incine metalloproteases for example MMP-9. By "inhibitor not only" it is proposed that the inhibition resistance (for example as measured by IC50) of the inhibitor against mepha 1 or endogenous methanol (for example MMP-9) is at least 5 times more weak preferably by at least 10 times for example at least 50 times weaker than the inhibition resistance of the inhibitor against aureol isine (for example aureloisin II). In another embodiment of the invention, the aureolemic inhibitor significantly inhibits endogenous mefzincma oprofeases (for example, MMP-9). By "significantly inhibit" it is proposed that the inhibiting resistance (for example as measured by LC50) of the inhibitor against endogenous methzincine metalloproteases (for example MMP-9) is at least 0. O times for example at least 1 ve / the resistance of inhibition of the inhibitor against aureolysin (for example aureLoisine 11). I.a resistance inhibition (for example as measured by IC50) of the inhibitor against endogenous methzincine metalloproteases (for example MMP-9) can be for example at least 10 times, perhaps 100 times or even 1000 times the inhibition resistance of the inhibitor against aureolysin (for example aureloisin II). Known examples of endogenous MMPs are discussed in Mande et al., (2004) Clinical Cancer Research 10, 909-915. These include MMP-2 (gelatinase A), MMP-9 (gelatinase B) and MMP-14 (MT-MMP-1, a membrane-binding enzyme) MMP-1 (collagenase-1), MMP-3 (it is t romelisin-1), MMP-7 (matrilysin), MMP-11 (is t-romelysin-3) and MMP-13 (collagenase-3). Another example is MM -8 (collagenase-2).

Examples of adamalysins include ADAM10, ADAM17 and ADAM33. As pointed out above, a number of these enzymes have been previously linked to cancer and inflammation and ADAM33 is genetically linked to asthma. The aureolysin inhibitor can also indirectly inhibit other proteases that damage the tissue in the skin. Proteases are often expressed as inactive zymogens that require proteolytic cleavage to become active. In fact, aureolysin itself is believed to be responsible for initiating the activation of extracellular proteases secreted by Staphylococcus a ureus (Sha et al., Microbiology, 150, 217-28, 2004). By M thus the aureole is also similar to activate endogenous host cell proteases present in the skin and therefore to exacerbate diseases such as AD. It is known, for example, that other 3 members of the M4 family (Elastase Pseudomonas aeruginosa, Viral Proteinase and Thermolysin) can activate human MMPs (Okamoto et al., J. BioL. Chem.? /? 6059-66, 199 /). An enzyme closely related to aureol isine, bací Loli sina, is known to activate pro-u-kinasin which converts plasminogen to plasmin (Narasaki et al., J Biol. Chem. 280, 14278-8 /, 2005). This reference also discloses that bacilol sine converts plasminogen to a molecule similar to my ni-plasminogen that is more susceptible to conversion to plasm na. The dalos show that aureolisma activates pro-urokinase and the inhibition of aurol Lsina can prevent this activation. Activation of pro-uroquinase leads to the activation of the plasminogen pathway that results in the production of pl asm ina pro-i n f 1 amatop a. Researchers have also shown that aureol is active pro-MMP- and the inhibition of aurol i s i na can prevent this activation. The activation of MMP-1 leads to increased degradation of collagen in the skin, which decreases the normal skin barrier. Aureol isine inhibitor can be, for example, selected from inhibitors of Lcrmol isine for example known thermo] isine inhibitors; for example acyclic succinyl hydroxamates as disclosed in Marcotte et al., J. Enzyme Lnh b 11 i on 14, 425-435, 1999 (see especially Table 1) which is incorporated herein by reference in its entirety. The ability of a given aureoiisin inhibitor to inhibit other enzymes for example MMPs can also be determined by standard assays employing the purified enzyme. The inhibitors of aureol isin include, or are expected to include, the following compounds: l ornas tat (compound 1, see US 5,183,900), marimastat (compound 3, see WO 94/02447), compound 5 (see WO 95 / 19957), soli astat (compound 7, see EP 1030842), compound 9 (see WO 95/19956), compound 11 (Ro 31-9790, see EP0664284 and Whittaker et al., Chemical Revie s, 99, 2735-2776, 1999) and its various forms (compound 2, compound 4, compound 6, compound 8, compound 10 and compound 12), compound 13 (CaLbiochem) and stereoisomers thereof, compound 14 (Calbiochem) and phosphoramidon ( compound 15) (see 'L'abJ a 1 straight away). Additional examples include compounds 16 and 17. Table 1 Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 Compound 8 Compound 9 Compound 10 Compound 1 1 Compound 12 Compound 13 Compound 14 Compound 15 Compound 16 Compound 17 Compounds 11, 12, 16 and 17 are novel and are claimed per se, together with pharmaceutically acceptable salts and solvates thereof, as one aspect of the invention. The compounds are claimed as solids in either amorphous or crystalline forms, which include all polymorphic forms. The crystalline forms can be prepared by recirculating the appropriate solvent compounds. The amorphous forms can be prepared for example by means of a spray drying solution of the compounds These compounds can be, for example, prepared as described in the Examples. As can be seen from the data contained within the Examples, compound 12 is particularly interesting since it has a very balanced inhibition against aureolysin as well as against MMPs (especially MMPs 1, 2, 8 and 9). That is proposed when administered at a level that should inhibit aureolysin, its effect on these other MMPs would be expected to be similar. This would be expected to provide an advantage in terms of reduced tendonitis, a systemic toxicological effect common to many compounds that have significant MIB inhibitory activity (e.g., compounds 3, 7 &11). As can also be seen from the data contained within the Examples, compound 16 is particularly interesting since it has remarkably potent aureolysin inhibitory activity. In fact it was much more potent as an aureolysin inhibitor than any of the other compounds tested. As these novel compounds are claimed per se, the researchers also claim processes for their preparation, their use as pharmaceuticals, pharmaceutical compositions containing them together with a pharmaceutically acceptable diluent or carrier (particularly, a topically acceptable diluent or carrier) as well as methods of treatment for inflammatory diseases in the skin that employ them and their use in the manufacture of a medicament for the treatment of inflammatory diseases in the skin. It is preferred that the inhibitor be formulated for topical administration and can be administered to a patient in an amount such that from 0.00001 to 10 g, preferably from 0.0001 to 1 g of active ingredient is delivered per m2 of the area being treated. Typically, the total amount of the inhibitor is 0. 001 to 12% by weight for example from 0.0018 to 11.6% by weight, suitably from 0.0088 to 1.4% by weight, for example 0.01-1.0% by weight, more suitably from 0.05 to 0.2% by weight, for example approximately 0.1% by weight weight, based on the total weight of the formulation. The topical formulation can, for example, take the form of a gel, ointment, cream or lotion. Other example presentations include impregnated dressings, pastes, dusting powders, rubbers, oils, t ansdermal devices, etc. The topical formulation will preferably maximize surface exposure and minimize systemic exposure to the active network (s). When the formulation is a gel it typically comprises a hydrophilic polymer such as polyethylene glycol. reticulated, starch or poliv nil p i r ro 1.1 reticuiada dona. An ointment, cream or lotion typically contains an aqueous phase and an oil phase in admixture. They can be generally characterized as oil-in-water emulsions or water-in-oil emulsions. 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 C 8 -C 22 alkoxy, 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% by weight to about 30% by weight and more preferably about 5% by weight to about 10% by weight based on the total weight of the formulation. The spicy emulsion is typically an active agent on the nonionic surface, for example, polysorbate 60 (available from 1C1? Merics), sorbitan tea rings, polyglyceryl o-4 oleate and polyoxyethyl ether at (4) 1 auric . Generally, the total amount of emulsion if sufficient is about 2% by weight to about 14% by weight and more preferably about 2% by weight to about 6% by weight per weight based on the total weight of the formulation. The pharmaceutically acceptable thickeners, such as Veegum.TM.K (available from R. T. Vanderbilt Company, Inc.) and long chain alcohols (ie C8-C22 alcohols such as cetyl alcohol, stearyl alcohol and cetearyl alcohol) can be used. The total amount of the thickener present is preferably about 3% by weight to about 12% by weight based on the total weight of the formulation. Preservatives such as methylparaben, propylparaben and benzyl alcohol may be present in the formulation. Other example preservatives are phenoxyethanol and chlorocresol. The appropriate amount of conservative tale (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% by weight to about 12% by weight based on the total weight of the formulation. Optionally, the formulation may 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 an individual ingredient can perform more than one function in a cream, ie, cevic alcohol can serve both as an emollient and as a thickener. Preferably, the formulation or the medicament is a cream. The cream typically consists of an oil phase and a water phase mixed together to form an emulsion. Preferably, the cream comprises an emulsion of oil in water. Preferably, the amount of water present in a cream of the invention is about 45% by weight to about 85% by weight based on the total weight of the cream. Where the formulation or medicament is an ointment, it typically comprises a pharmaceutically acceptable ointment base such as petrolatum or polyethylene glycol 400 (available from Union Carbide) in combination with polyethylene glycol 3350 (available from Union Carbide). The amount of ointment base present in an ointment of the invention is preferably about 60% by weight to about 95% by weight based on the total weight of the ointment. An exemplary formulation is a cream comprising an emulsification ointment (for example about 30% by weight) comprising white soft paraffin, emulsifying wax and for fine liquid made of 100% purified water and containing preservative (for example phenoxyethanol). This formulation can also be a buffer solution at the required pH (for example with citric acid and sodium phosphate). The concentration of active can typically be between 0.01 and 1.0% by weight. In a preferred embodiment, the formulation is a cream comprising an oil-in-water cream base comprising isostearic acid, cetyl alcohol, stearyl alcohol, white petrolatum, polysorbate 60, sorbitan monostearate, glycerin, xanthan gum, purified water, benzyl alcohol, methylparaben and propyl-paraben. Such cream may be in the form of Aldara imiquimod cream containing 5% imiquimod. Compound 12 has been found to be particularly soluble in water, particularly when the solid is in amorphous form. Formulated in oil-in-water or water-in-oil emulsions, since they can be taken in the water phase before emulsification with the oil phase (for example paraffin). The aureolysin inhibitor can be administered in conjunction with additional drugs, such as conventional therapies for the treatment or prevention of inflammatory conditions in the skin, for example antibiotics, steroids (such as hydrocortisone, clobetasone butyrate, betamethasone valerate, hydrocortisone butyrate, clobetasol propionate, fluticasone propionate, mo etasona furoate and dexamethasone), anti-inflammatory drugs spheroidal, macrolide immunosuppressants (such as cyclosporin A, tacrolimus and pimecrolimus), leukotriene antagonists and phosphodiesterase inhibitors. These additional treatments can be administered by any convenient route. Topical and oral routes are preferred. The active agents can be, where appropriate, administered in the form of pharmaceutically acceptable salts or solvates, for example hydrates. Accordingly, a method is also provided for the treatment or prevention of an inflammatory condition in the skin that is characterized by colonization with Staphylococcus a? reus, which comprises the topical administration of an aureolysin inhibitor in combination with the administration of an additional medicament. In a further aspect of the present invention there is provided the use of an aureolysin inhibitor in the manufacture of a topical medicament for the treatment or prevention of an inflammatory condition in the skin that is characterized by colonization with SLaphy lococcus a? reus in combination with an additional medication. The combination treatments can be administered simultaneously, separately or separately, by the same or different routes. In an exemplary embodiment, the additional medicament can be administered orally. For example, the additional medicament can be administered topically, for example, in a combined preparation with the aureol inhibitor i si na. For example, the additional drug may be an antibiotic substance that is bactericidal for Staphy lococcus aureus and that is administered either immediately or topically. The appreciation of the aureolide function in AD allows a novel method of identification for the identification of novel substances that are inhibitors of aureolysin for the treatment of? D. Thus in another aspect of the present invention there is provided an ip vil ro method of classification for an agent for use in the treatment or prevention of an inflammatory condition in the skin that is characterized by colonization with SLaphylococc? Sau rcus, comprising: ( ) contact the agent with aurcolysin; (L) Determining the aureole and if it is inhibited. The inhibition of aureol i s i na can be determined by a standard test, for example by means of the aureolysin inhibition assay or also by means of the milk agar plate assay described in the Examples. Also provided is a method of classifying an agent for use in the treatment or prevention of an inflammatory condition in the skin that is characterized by colonization with Staphylococcus a ureus, which comprises: (i) obtaining skin washes from patients (ii) ) contacting the agent with skin washes (iii) determining (eg by zymography or fluorogenic enzyme assay) if the proteolytic activity is inhibited. This activity may be due to aureolysin and optionally endogenous etzincin metalloproteases. By "agent" any chemical substance is proposed, either a "small molecule" (for example a molecule having a molecular weight of less than 1000 Da, especially less than 600 Da), peptide, protein or antibody. Small molecules are preferred (for example those having a molecular weight of less than 600 Da). Small peptides are preferred (eg containing less than 16 amino acid residues). These peptides can be linear or cyclized. In a suitable method for carrying out the methods and uses according to the invention, the presence of activity of metalloprotease in skin lesions associated with AD or another inflammatory condition is verified before treatment. Thus according to this aspect of the invention there is provided a method for the treatment of a skin lesion associated with an inflammatory condition in the skin in a mammal characterized by colonization with Staphylococcus aureus comprising (i) determining the presence of metalloprotease activity in skin washes from the site of the skin lesion and if the presence of metalloprotease activity is then confirmed (ii) topically administering an aureolysin inhibitor, to the skin lesion. Also provided is the use of an aureolysin inhibitor in the manufacture of a topical medicament for the treatment of a skin lesion associated with an inflammatory condition in the skin in a mammal that is characterized by colonization with Staphylococcus a ureus, wherein the skin lesion has been pre-determined to contain the metalloprotease activity. In certain embodiments, the aureolysin inhibitor is also an inhibitor of endogenous metzincine metalloproteinase. In another method suitable for carrying out the methods and uses according to the invention, the presence of S aureus in the skin lesions, associated with AD or another condition inflammation is verified before treatment. Thus according to this aspect of the invention a method is provided for the treatment of a skin lesion associated with an inflammatory condition in the skin in a mammal characterized by colonization with Staphylococcus a ureus comprising (i) determining the presence of Staphylococcus au reus at the site of the skin lesion and if the presence of Staphylococcus au reus is then confirmed (ii) topically administering an aureol inhibitor Lsina to the skin lesion. Also provided is the use of an aureolysin inhibitor in the manufacture of a topical medicament for the treatment of a skin lesion associated with an inflammatory condition in the p, in a mammal that is characterized by colonization with Staphyl ococcus au reus , where the skin lesion has been pre-determined by containing Staphy lococcus a ureus. In certain embodiments, the aureolysin inhibitor is also an inhibitor of endogenous metzincine endoprotein metaJ. In fact, the method and use according to a first aspect of the invention proceeds by a method step that involves confirming the presence of S taphylococcus a ureus at the site of the skin lesion. By "the site of the skin lesion" is proposed in and within the skin lesion or in the surrounding area. The presence of Staphylococcus a? Reus can be determined directly by showing the skin of patients and determining the presence of Staphylococcus aureus through microbiological or genetic methods. In the simplest form of assay, the affected skin is cleaned and the cleanliness is inoculated on the blood agar plates and the colonies of Staphylococcus a? Reus identified through standard microbiological procedures. A quantitative methodology can also be applied to determine the level of colonization. Genetic methods such as quantitative PCR can also be used to demonstrate the presence of Staphy lococcus a ureus. The presence of Staphylococcus a ureus can also be determined indirectly by determining the presence of metalloprotease activity for example in skin washes of patients. The presence of metalloproteases and metalloprotease activity can be detected in skin washes of patients by gelatin zymography or enzyme assay. Examples Synthetic Examples 1. Synthesis of Compounds 11 and 12 Two interchangeable synthesis routes were used to generate compounds 11 and 12. The first was exemplified by The synthesis of compound 1 of (R) -2 - (2-methox-2-oxoet?) -4-meph1-pentanoic acid. The second was exemplified by the synthesis of compound 12 of L-leuc na. The first route can be used to synthesize compound 12 by using (S) -2- (2-methox? -2-oxoet? L) -4-methylpentanoic acid and the second can be used to synthesize compound 11 to the uti 1 i / ar D-lucine instead of L-leucine. A. Synthesis of (R) -NI- ((S) -3,3-d? Met? Ll- (methylamino) -1-oxobutan-2-? L) -N4-h? Drox? -2-? Sobut ? lsucc? nam? da (Composite 11) i) A mixture of (R) -2- (2-methoxy-2-oxoet-l) -4-methyl-pentanoic acid (0.5 g, 2.4 mmol), DCC (1.2 eq, 0.61 g) and HOBT (1.02 eq, 0.34 g) in d-chloroform (5 ml) was stirred at room temperature for 10 rain. Was added (S) -2-ammo-N, 3, 3-tr? Met? The volume was stirred (1.1 eq, 0.39 g) and the mixture was stirred at room temperature overnight. The resulting white precipitate was filtered and the filtrate was concentrated to dryness under reduced pressure to remove an oil. The crude oil was dissolved in ethyl acetate (50 ml) and extracted sequentially with 2N hydrochloric acid (2x50 ml), saturated sodium bicarbonate (2x50 ml) and brine (50 ml), dried with MgSO4 and concentrated to dryness. This residue was recrystallized from diethyl ether to dr 3- ((S) -3,3-d? Met? Ll- (methylamide) -1-oxobutan-2-? 1 carbamo i 1) -5-me ti lLexanoa to (R) -methyl as a white solid (0.60 g, 74%). A second reaction of 1.05 g of acid (R) -2- (2-met ox? -2-oxoet? L) -4-methylpentanoic dLo 1.06 g (65%) of L product. XH NMR (400MHz, CDC13): 0.83 (d, 311), 0.87 (d, 311), 0.96 (s, 9H), 1.22 (m, 111), 1.51 (m, 211), 2.40 (d, III, J = 14.5 Hz), 2.65 (m, 211), 2. 1 1 (s, 311), 3.63 (s, 311), 4.22 (d, 1H, j = 9.15), 6.15 (d, 111, j = 4.39), 6.40 (d, 111, j = 9.52). ii) 3 - ((S) -3,3-d? met? ll- (met 1 am i no) -1-oxobutan-2-ylcarbamoyl) -5-met? J hexanoate of (R) -met? lo it was converted to compound 12 using the method of Levy et al., (J. Med Chem 41: 199-223, 1998). Hydroxylamine hydrochloride (7.2 eq, 0.91 g) was dissolved in methanol (8.8 ml) and cooled to 0 ° C. Potassium hydroxide (11.4 eq, 1.17 g) in methanol (b.8 ml) was added and the mixture was stirred at 0 ° C for 1 h. The mixture was filtered and the filtrate was added to a solution of 3- ((S) -3,3-d? Met 11-1- (methy1 rm no) -1-oxobutan-2-Icarbamoi I) -5- The mixture was stirred at room temperature for 30 ml ng of methanol (0.6 g, 3.6 mJ) and methanol (R) -methanol in methanol (0.6 g, 3.6 mJ). The reaction mixture was concentrated to dryness under reduced pressure and was dissolved in water (7 ml), acidified to pH 5 with 6 N hydrochloric acid and then neutralized with saturated sodium bicarbonate to pH 7. The resulting precipitate was collected by filtration and purified by flash chromatography eluting with methane 1 / dic. The 5% rornetin at % methanol / dichloromethane. The product containing fractions were combined and concentrated to dryness under reduced pressure. The solid was triturated in isopropyl alcohol: ethyl acetate (1: 1) and filtered to give (R) -N1- ((S) -3,3-dimethyl-1- (methylamino) -1-oxobu tan- 2-yl) -N 4 -hydroxy-2-isobutylsuccinamide as a white solid (68 mg, 7%). A second reaction using 1.06 g of 3- ((S) -3,3-dimethyl-1- (methylamino) -l-oxobutan-2-ylcarbamoyl) -5-methylhexanoate of (i?) -methyl in which the mixture of concentrated reaction was purified directly by flash chromatography gave 0.25 g (24%) of the product. ^ -I NMR (400MHz, DMSO): 0.76 (d, 311), 0.80 (d, 3H), 0.86 (s, 9H), 1.05 (m, 1H), 1.40 (m, 2H), 2.09 (m, 2H ), 2.54 (s, 3H), 2.82 (m, 1H), 4.13 (m, 1H, = 9.5Hz), 7.64 (d, 1H, j = 9.89), 7.84 (d, 1H, = 4.76), 8.68 ( br s, 1H), 10.35 (br s, 1H). B. Synthesis of (S) -Ni- ((S) -3,3-dimethyl-l- (methylamino) -1-oxobutan-2-yl) -N 4 -hydroxy-2-isobutylsuccinamide (Compound 12) i) added sodium nitrite (84.2 g, 1.22 mol) stepwise at 0 ° C to an ice-cooled solution of L-leucine (100 g, 0.762 mol) and 48% aqueous HBr (836 g) and water (360 ml) ). When the addition was complete the reaction was left at 0 ° C for 1 h and then allowed to warm to room temperature overnight. The reaction mixture was then re-cooled to 0 ° C and cooled by the addition of stage to stage of sodium carbonate (-135 g) until the reaction mixture reached pH 4.5. The mixture was then extracted into dichloromethane (2 x 500 ml), dried with MgSO 4 and the solvent removed in vacuo to yield (S) -2-bromo-4-methylpentanoic acid as an orange oil (115.8 g, yield). 80%) that loses its color at rest. H NMR (CDC! 3): d 0.92 (311, d, J = 6.5Hz), 0.96 (3H, d, J = 6.5IIz), 1.80 (III, m), 1.91 (211, m), 4.28 (III , L, J = 7.85Hz), 11.76 (1H, br s). aD -39.6 °, c = 2.118 in MeOII. LI) tp dieterate boron fluoride was added (7.9 g, 0.0559 mol) to a solution of (S) -2-bromo-4-methylpentanoic acid (108 g, 0.559 mol) in tert-butyl acetate (450 ml) and the reaction mixture was stirred overnight under nitrogen at room temperature. The mixture was drained in saturated sodium bicarbonate and the basic pll was maintained by the addition of more sodium bicarbonate. The organic phase was separated, washed with brine (2 x 200 mJ), dried over MgSO 4 and evaporated to dryness to give 135 g of crude oil. The oil was then purified by vacuum distillation (55 ° C to 4 mbar to give a theoretical boiling point of 190 ° C). 2-Bromo-4-met i 1 pentanoate of (S) -tert-but was collected as a colorless oil (91.7 g, 66% yield). XH NMR (CDC13): d 0.89 (3H, d, J = 6.5llz), 0.93 (3H, d, = 6.5Hz), 1.46 (911, s), 1.72 (JH, m), 1.84 (211, m) 4.15 (1H, t, J = 1.65Hz). aD -29.91 °, c = 1906 in MeOH. iii) Potassium tert-butoxide (38.8 g, 0.346 mol) was added step by step at 0 ° C under nitrogen to a solution of dibenzyl malonate (98.3 g, 0.346 mol) in dry dimethylformamide (169 ml) until dissolved . The solution was kept at 0 ° C and a solution of 2-bromo-4-methylpentanoa to (S) -tert-butyl (86.8 g, 0.346 moles) in dry dimethylformamide (160 ml) was added dropwise over a period of time. 1 h and then stirred at 0 ° C for 4 days. The reaction was warmed to room temperature, diluted with ethyl acetate (600 mL) and saturated, aqueous ammonium chloride (400 mL) was added. The organic layer was separated and the aqueous layer was re-extracted with ethyl acetate (400 ml). The organic layers were combined and washed with 1.0% sodium chloride solution (500 ml) and dried over MgSO4. The solvent was removed to give 195 g of pale yellow oil. The oil was purified by flash column chromatography (heptane: ethyl acetate 10: 1) to give 63 g (40% yield) of 4-methylpentan-1, 1,2-tricarboxylate of (S) - 1, 1-dibenzyl-2-er-butyl as a clear oil that solidified as a white solid at rest. XH NMR (CDC13): d 0.82 (6H, m), 1.37 (9H, s), 1.50 (3H, m), 3.03 (1H, dt, ^ = 4.281- ^., J2 = 10.32Hz), 3.73 (III , d, = 10.20Hz), . 11 (4H, m), /.29 (1011, m). aD -15.93 °, c = 1.695 in MeOII. iv) TFA (100 ml) was added to a solution of 4-methylpentan-1,2, tri-carboxy and all (S) -1, 1-di-benzyl-2-tert-butyl (60 g, 0.132). mol) in di-1-oromethane (500 ml) and the reaction was stirred at room temperature overnight. The solvent was removed under vacuum and the resulting oil re-dissolved in dichloromethane (300 ml). The material was then washed with water (300 ml), dried with MgSO ^ and the solvent was removed to give a pale orange oil (51.1 g, aD -7.44 °, c = 2.554 in MeOIl). The oil was dissolved in 180 ml of diethyl ether, 520 ml of n-hexane was added and the solution was cooled in an ice bath. The resulting precipitate was filtered and the filtrate was concentrated in vacuo to yield (S) -2- (J, 3-b? S (benzyl-1-oxy) -1,3-d? Oxopropan-2-? L) acid) -4-methylpentanoic as a pale oil (31.06 g, 60% yield). 1H NMR (CDC13): b 0.81 (311, d, J = 3.47IIz), 0.83 (3H, d, J = 3.47Hz), 1.16 (1H, m), 1.59 (211, m), 3.18 (111, m ), 3.79 (1H, d, J = 9.79Hz), 5.14 (411, m), /.il (1011, m). aD -40.10 °, c = 2.319 in MeOIl v) I10BT (11.2 g, 0.083 mol) was added to a solution of (S) -2- (1, 3-b? s (benc i 1 ox i) -1) , 3-d? Oxopropan-2-? L) -4-methylpentanoic acid (30 g, 0.0 / 5 mol) in ethyl acetate (200 ml) and dimethyl formamide (lOml). The reaction cooled to 0 ° C and a solution of DCC (19.2 g, 0.093 mol) was added in ethyl acetate (40 ml, 2 vol) for 15 min. The reaction was allowed to warm to room temperature and was stirred for 1 h. The DCU was removed by filtration, the reaction was again cooled to 0 ° C, a solution of (S) -2-amino-N, 3, 3-trimethylbutanamide (10.86 g, 0.075 mol) in ethyl acetate (20%) was added. ml, 2 vol) and the reaction was stirred at room temperature for 2 days. The reaction was then washed with 2 M sodium carbonate (200 ml), water (200 ml), 2 M sodium carbonate (200 ml) again, brine (200 ml) and water (200 ml). The organic layer was dried with MgSO 4, filtered and concentrated in vacuo to give 35 g of pale yellow waxy solid. The solid was suspended in diethyl ether and 2- ((S) -1- ((S) -3,3-dimethyl-1- (methyarnino) -1-oxobu tan-2-ylamino) -4-methyl-l- oxopentan-2-yl) dibenzyl malonate was filtered as a white solid (24.64 g, 62% yield). XH NMR (CDC13): d 0.81 (3H, d, J = 3.06Hz), 0.82 (3H, d, J = 3.06Hz), 0.98 (9H, m), 0.99 (1H, m), 1.57 (11-1) , m), 1.69 (1H, m), 2.67 (31-1, d, J = 4.90Hz), 2.94 (1H, dt, Jl = 3.67Hz, J2 = 10.20Hz), 3.78 (1H, d, J = 10.20Hz), 4.13 (1H, d, J = 8.77Hz) 5.08 (4H, m), 6.22 (11-1, d, J = 4.49Hz), 6.33 (1H, d, J = 8.77Hz) 7.29 (10H , m). aD -36.00 °, c = 1750 in MeOH. P.F 98-99 ° C. vi) 2- ((S) -1- ((S) -3,3-dimethyl-l- was dissolved Dibenzyl (methylamino) -1-oxobu tan-2-ylamino) -4-methyl-1-oxopentan-2-yl) malonate (24.5 g, 0.047 mol) in ethanol (300 ml) and the solution was purged with nitrogen. The flask was evacuated, purged with nitrogen again and 10% palladium was added over the carbon catalyst (2.45 g, 10% by weight). The flask was re-evacuated, purged with nitrogen once and then evacuated and purged three times with hydrogen. The reaction was left under a hydrogen atmosphere and was stirred at room temperature during the week. The catalyst was filtered and the solvent was removed under vacuum to give 15 g of 2- ((S) -1- ((S) -3,3-dimethyl-l- (methylamino) -l-oxobutan-2-ylamino acid. ) -4-methyl-l-oxopentan-2-yl) alonic as a white solid (100% yield). XH NMR (DMSO): d 0.82 (3M, d, J = 6.53Hz), 0.90 (12H, m), 1.05 (1H, m), 1.51 (2H, m), 2.57 (3H, d, J = 4.49Hz ), 3.09 (1H, dt, Jx = 3.88Hz, J "2 = 8.32Hz), 3.33 (1H, d, J = 10.20Hz), 4.07 (1H, d, J = 9.18Hz), 7.55 (1H, m ) 8.12 (1H, d, J = 9.18Hz) aD -80.50 °, c = 1.913 in MeOH, P.F 101 ° C, vii) 2- ((S) -1- ((S) -) acid was dissolved 3, 3-dimethyl-l- (methylamino) -l-oxobutan-2-ylamino) -4-methyl-l-oxopentan-2-yl) malonic acid (14.5 g) in ethanol, carbon (1.45 g) was added and the The reaction was stirred overnight at 80 ° C. The carbon was filtered and the solvent was removed in vacuo to yield 11.51 g of (S) -3- ((S) -3,3-dimethyl-l- ( methylamino) -l-oxobutan-2- ilcarbamoi I) -5-met? 1 hexanoi co as a pale gray solid. 1 H NMR (DMSO): d 0.81 (311, d, J = 6.32IIz), 0.87 (1211, m), 1.09 (1H, m), 1.45 (211, m), 2.19 (III, dd, ^ = 6.5311 / , J2 = 16.12Hz), 2.36 (1H, dd, J1 = 7.75Hz, 2 = 16.12ll), 2.54 (311, d, J = 4.49Hz), 2.91 (1H,), 4.12 (III, d, /.78 (111, m) 7.95 (1H, d, J = 9.18Hz). aD -42.21 °, c =]. 919 in McOH. P.F. 204-205 ° C. vi 11) 0-Benzylhydroxy hydrochloride (9.30 g, 0.058 mol), NMM (5.93 g, 0.059 mol), 1IOB1 (6.42 g, 0.048 mol) and ID? C (9.11 g, 0.048 mol) were added to a solution agitated acid (S) -3- ((S) -3, 3-d? rnef? 1 -1 - (I am not) -1 -oxobul an-2-? read rbamo i 1) - -met i Ihexanoic (11.51 g, 0.038 mol) on dimethyl formamide (161 ml) and dichloromethane (205 ml) at 0 ° C. The reaction mixture was allowed to warm to room temperature and was stirred overnight. It was then diluted with oromethane (500 ml) and washed sequentially with water (500 ml), 0.6 N 1IC1 (500 ml), saturated sodium carbonate (500 ml) and water (4 x 500 ml). The organic layer was dried and the solvent was removed in vacuo to give (S) -N4- (benzyloxy) -N1- ((S) -3, 3-d? Methyl I-1 - (rnef and 1 amino) - 1 -oxobutan-2-? L) -2-? Sobut i 1 succi nami gives like a white gone (7.35 g, 43% yield). XH NMR (DMSO): d 0.81 (311, d, J = 6.3? Ilz), 0.87 (3H, d, J = 6.53IIz), 0.90 (911, m), 1.00 (III, m), 1.42 (211, m), 1.97 (1H, dd, J1 = 7.34Hz, ^ = 1 .4Hz), 2.11 (1H, dd, J1 = 7.14Hz, J2 = 15.44Hz), 2.52 (311, d, J = 4.49llz), 2.96 (1H, m), 4.12 (1H, d, J = 9.38Hz), 4.73 (211, q, Jx = 11.02117, J2 = 9.7llz), 7.38 (5H, m), 7.85 (III, m), 7.97 (III, d , = 9.18hz). aD -19.37 °, c = 1497 in MeOH. P.F. 128-129 ° C. x) It was dissolved (S) -N4- (benz i 1 x i) -N1- ((S) -3, 3-dimet 1-1- (met Lla? no) -l-oxobutan -? -? l) The mixture was succinamide (7.35 g) in ethanol (100 ml) and the solution was purged with nitrogen. The flask was evacuated, purged with nitrogen again and 10% palladium on carbon catalyst (735 mg, 10% by weight) was added. The flask was re-evacuated, purged with nitrogen once and then evacuated and purged three times with hydrogen. The reaction mixture was then left under a hydrogen atmosphere and stirred at room temperature during the week. The catalyst was filtered and the solvent was removed under vacuum to give 5.12 g of (S) -N1- ((S) -3,3-dimethyl-J- (methylamino) -! -oxobuLan-2-yl) -N4 -h? drox -2-? sobu-tilsucc nami da (compound 12) as a white solid (98% yield). XH NMR (D4-MeOH): d 0.89 (3H, d, J = 6.53llz), 0.94 (3H, d, J = 6.32Hz), 1.01 (9H, s), 1.17 (111, m), 1.5 / ( 2H, m), 2.14 J2 = 14.68Hz), 2.71 (311, s), 2.93 (III, rn), 4.10 (III, s). aD -33.1 °, c = 1.60 in MeOII. 2. Synthesis of Compounds 16 and 17 (S) -2-amino-? / - methyl-4-phenylbutanamide was prepared as follows: i) To a stirred solution of (S) -2-amino-4-phenylbutanoic acid (5.0 g) , 27.9 mmol) in methanol (25 ml) was added thionium chloride (2.26 ml, 30.69 mmol) at 0 ° C. The mixture was warmed to room temperature then heated at 65 ° C for 2 h. After concentration in vacuo the residue was triturated with diethyl ether (10 ml) and the solid was collected by suction filtration, washed with diethyl ether (5 ml) and air-dried to give 2-amino-4-. phenylbutanoa to (S) -methyl as its hydrochloride salt (6.1 1 g, 95%). LCMS (3 min) purity = 97%, tr = 1.08, m / z. 194 [M -? - H] +. ii) To a solution of 8 M methylamine in ethanol (8.7 ml, 69.6 mmol) was added 2-amino-4-phenylbutanoa hydrochloride of (S) -methyl (4.0 g, 17.4 mmol) at room temperature. The agitation was continued overnight. The reaction mixture was concentrated in vacuo, diethyl ether (5 ml x 3) was added and the evaporation was repeated. The solid was suspended in dichloromethane (30 ml), washed with saturated aqueous sodium bicarbonate (10 ml) and water (10 ml), dried (Na 2 SO 4), filtered and concentrated in vacuo to give (S) -2-amino-? -methyl-4-phenylbutanamide as a white solid (2.77 g, 83%).

LCMS (3 min) purity = 94%, tr J.05, m / z. 193 [M + H], 1R NMR (MeOD) d 1.65 (III, m), 1.85 (III, m), 2.55 (2 H, m), 2.60 (3 H, s, CONHMe), 3.15 (111, m ), 7.00 - /. 20 (5 H, m, Ar). This material was used in the synthesis of both compounds 16 and 17 as described below. A. Synthesis of (S) -N 4 -hydroxy-2-isobutyl-N 1 - ((S) -1- (methylamino) -l-oxo-4-phenylbutan-2-yl) succinamide (Compound 16) they designated two synthesis routes for compound 16 route A and route B were used. Route A for Compound 1 6 i) To a mixture of (S) -3- (methoxycarbonyl) -5-methylhexanoic acid (250 rng, 1.33 mmol), EDC (331 mg, 1726 mmol) and HOBT (233 mg, 1726 rnmoi) in TIII "(5ml.) Was added (S) -2-amino-N-methyl-4-fem. 1 butanamide (281 mg, 1.46 mmol) followed by triethylamine (0.46 ml, 3.30 mmol). The reaction was stirred at room temperature overnight After the removal of the volatiles, the residue was taken up in ethyl acetate (10 mL), washed with 10% citric acid (5 mL), followed by sodium bicarbonate. aqueous sodium, saturated (5 ml) and water (5 ml) The ethyl acetate layer was dried (Na 2 SO 4), filtered and concentrated in vacuo to give 4-rnet? -1. 2- (2- ( (S) -1- (met l ami no) -l-oxo-4-fem lbutan-2.? I amino) -2-oxoethyl) penta-noate from (S) -met? Jo (380 mg, 79 %) LCMS (3 min) purity = 93%, tr 1.97, m / z 363 [M -? - H] +, 725 [2M + H] +. XH NMR (MeOD) d 1.80 (6H, m, isobutyl), 1.20 (1H, m, isobutyl), 1.55 (2 H, ra, isobutyl), 1.80 (1H, m), 2.05 (1H, m), 2.30 ( 1H, dd, J = 14.91 and 5.62 Hz), 2.40 (1H, dd, J = 14.91 and 9.29 Hz), 2.45 (2 H, m), 2.60 (3 H, s, CONl-IMe), 2.85 (1H, m), 3.55 (3 H, s, C02Me), 4.15 (1H, dd, J = 9.54 and 4.89 Hz), 7.00-7.20 (5 H, m, Ar). ii) To a solution of 4-methyl-2- (2- ((S) -1- (methylamino) -1-oxo-4-phenylbu tan-2-ylamino) -2-oxoethyl) penta-noate (S) ) -methyl (250 mg, 0.69 mmol) in a mixture of THF (2.0 ml) and methanol (2.0 ml) was added 2M aqueous sodium hydroxide (1.0 ml) at room temperature. After stirring at room temperature for 1 h the reaction mixture was concentrated to about 0.5 ml and acidified thoroughly with 1 M aqueous hydrochloric acid. The aqueous layer was extracted with ethyl acetate (2 x 3 ml) and the organic layers Combine were dried (Na2SO4), filtered and concentrated in vacuo to give a mixture of (S) -5-methyl-3- ((S) -1- (methylamino) -l-oxo-phenylbutan-2-acid. ilcarbamo-il) hexanoic (isomer A) and (S) -4-methyl-2- (2- ((S) -1- (methylamino) -l-oxo-4-phenylbutan-2-ilarnino) -2- oxoethyl) -pentanoic (isomer B) (210 mg, 87%); this was used directly in stage (iii). LCMS (3 min) purity = 72% (isomer A, tr = 1.69) + 10% (isomer B, tr = 1.67), m / z. 349 LM-HI] +. iii) To the mixture of carboxylic acids obtained from step (ii) (210 mg, 0.603 mmol), EDC (150 mg, 0.784 mmol) and HOBT (105 mg, 0.784 mmol) in THF (5 ml) was added followed by 0-tetrahydro-2H-pyran-2-yl-hydroxylamine (92 mg, 0.784 mmol) and triethylamine (0.20 mL, 1.50 mmol). The reaction mixture was stirred at room temperature overnight. After removal of the volatile products the residue was taken up in ethyl acetate (10 ml), washed with 10% citric acid (5 ml), followed by saturated aqueous sodium bicarbonate (5 ml) and water (5 ml). my) . The ethyl acetate layer was dried (Na 2 SO 4), filtered and concentrated in vacuo to give (2S) -2-isobutyl-N 1 - ((S) -1- (methylamino) -l-oxo-4-phenylbutan- 2-yl) -N 4 - (tetrahydro-2 H -pyran-2-yloxy) succinamide (isomer A) and (2S) -2-isobutyl-N 4 - ((S) -1- (methylamino) -l-oxo-4 phenylbutan-2-yl) -N1- (tetrahydro-2H-? iran-2-yloxy) succinamide (isomer B) (240 mg, 89%). LCMS (3 min) purity = 61%, (isomer A, tr = 1.77) -I- 13% (isomer B, tr = 1.74), m / z. 448 [M -? - HJ +, 364 [M + H -THP] +. iv) To a solution of (2S) -2-isobu il-N1- ((S) -1- (methylamino) -l-oxo-4-phenylbutan-2-yl) -N4- (tetrahydro-2H-pyran- 2-yloxy) succinamide and (2S) -2-isobutyl-N4- ((S) -l- (methylamino) -l-oxo-4-phenylbutan-2-yl) -N1- (tetrahydro-2H-pyran-2) -loxy) succinamide (240 mg, 0.537 mmol) in methanol (7 ml) was added Amberlyst resin 1-1-15 (200 mg). The mixture was stirred at room temperature for 3 h, filtered and concentrated in vacuo. The desired compound was then isolated by preparative reverse phase HPLC. The crude product in sulfoxide of di met i 1 or: aceton itri I or 2.1 (1.6 ml) was injected into a C18 l'hermollypers i 1-Keystone Ilyperprep HS column (12 μm, 100 x 21.2 mm) and discharged for 9.5 rain (30 ml / min) with a 20-100% gradient of aceton itri I or / 'IT? 0.1% (solvent B) in water / 1'F? 0.1% (solvent?) using UV protection at 215 nm to produce compound 16 as a white solid (60 mg, 32%).

Compound 16 LCMS (7 min) purity = 100% (Lr = 3.48), m / z 364 [MIH] +. XH NMR (MeOD) d 0.80 (3 H, d, J = 6.36 11 /, 11-23), 1.90 (3 H, d, J = 6.36 Hz, 11-23), 1.15 (111, m, 11-21) ), 1 45 (? II, m, 11-21 and H-22), 1.75 (III, m, H-8), 2.10 (III, dd, J = 15.16 and 4.15 Hz, H-16), 2.15 ( 1H, m, II-8), 2.35 (III, dd, J = 10.5 and 15.16 Hz, H-16), 2.50 (III, m, 11-7), 2 65 (III, m, 11-7), 2.65 (3 II, s, H-20), 2. / 0 (III, m, 11-15), 4.15 (III, dd, J = 11, 3.66, H-9 Hz), 7.05-7.20 (5 H , m,? r). 13C NMR (MoOD) d 23.29, 24.74, 27.52, 28.04, 34.43, 35.19, 31.10, 43.20, 44.18, 55.53, 128.18, 130.50, 130.52, 143.19, 1 /2.13 (C-1 /), 1 / 5.93 (C- 13), 179.17 (C-ll). Route B for Compound 16 j) To a stirred mixture of (S) -3- (methoxy ca rbom 1) -5-met i hexanoic acid (100 mg, 0.532 mmol) and Boc20 (139 mg, 0.638 mmoJ) in L-BuOH (99%, 1.5 ml) was added DMAP (19.5 mg, 0.159 mmol) at room temperature. After stirring for 1.5 h the reaction mixture was concentrated in vacuo, diluted with ethyl acetate (5 ml), washed with saturated aqueous citric acid (2 x 2 ml), followed by aqueous sodium bicarbonate, saturated (2 x 2 ml) and water (2 ml). The ethyl acetate layer was dried (Na 2 SO), filtered and concentrated so as to give 2-? sobuti 1 (S) -4- tert -butyl 1-methoxyl succinate as yellow yellow oil (100 mg). This compound is used in the next step without further purification. H NMR (CDC13) d 0.75 (6 H, m, / -butyl), 1.15 (III, m, 1-butyl), 1.20 (9 H, s, 1-bufyl), 1.35 (2 1-1, m, (-butyl), 2.15 (III, dd, J = J6.28 and 5.21 H /), 2.35 (III, dd, J = 16.28, 9.33 Hz), 2.65 (1I-I, m), 3.40 (3 II, s, C02Me.) 11) To a solution of 2-? sobut? The (S) -4-tert-butyl-1-methyl ester (100 mg, 0.41 mmol) in methanol (0.8 ml) was added with potassium carbonate (68 mg, 0.492 mmol) and water (0.2 ml). The mixture was c Slow at 55 ° C for 18 h. The reaction mixture was then concentrated, either 1 to 1 v 1 or in ethyl acetate (5 ml) and acidified completely with acid 1 M aqueous hydrochloric acid. The aqueous layer was extracted with ethyl acetate (2 ml) and the combined ethyl acetate layers dried (Na S04), filtered and concentrated in vacuo to give acid (S) -2- (2- /: er-butoxy-2-oxoethyl) -4-methylpentanoic as a crude product (60 mg) which was used directly in step (iii). iii) To a mixture of crude (S) -2- (2-tert-butoxy-2-oxoethyl) -4-methylpentanoic acid (90 mg, 0.39 mmol), EDC (97 mg, 0.507 mmol) and HOBT (68 mg) , 0.507 mmol) in DMF (1 ml) was added (S) -2-amino-N-phenyl-4-phenylbutanamide (90 mg, 0.468 mmol) followed by triethylamine (0.135 ml, 0.97 mmol). The reaction mixture was stirred at room temperature overnight. After removal of the volatile products the residue was taken up in ethyl acetate (5 ml), washed with 10% citric acid (2 ml), followed by saturated aqueous sodium bicarbonate (2 ml) and water (2 ml). my) . The ethyl acetate layer was dried (Na 2 SO 4), filtered and concentrated in vacuo to give 5-methyl-3- ((S) -1- (methylamino) -1-oxo-4-phenylbutan-2-ylcarbamoyl ) (S) -ter-butyl hexanoate (65 mg, 79%); this was used directly in stage (iv). LCMS (3 min) purity = 47%, tr = 2.16, m / z 405 [M-l-HJ +. iv) To a solution of (S) -ter-butyl 5-methyl-3- ((S) -1- (methylamino) -l-oxo-4-phenylbutan-2-ylcarbamoyl) hexanoate (65 mg, 0.161 mmol ) in dichloromethane (0.6 ml) was added TFA (0.24 ml) at room temperature. After the resting for 45 min. the reaction mixture was evaporated to dryness. Dichloromethane (2 x 0.5 ml) was added and the evaporation was repeated to give (S) -5-methyl-3- ((S) -1- (methylamino) -l-oxo-4-phenylbutan-2-ylcarbamoyl) hexanoic as a yellow viscous oil (58 mg, quantitative). LCMS (3 min) purity = 49%, tr = 1.69, m / z. 349 [M + H] +. v) To a mixture of (S) -5-methyl-3- ((S) -1- (methylamino) -l-oxo-4-phenylbutan-2-ylcarbamoyl) hexanoic acid (58 mg, 0.167 mmol), EDC (66 mg, 0.344 mmol) and HOBT (46 mg, 0.344 mg) in THF (2 ml) were added 0-t, ehydro-2H-pyran-2-yl-hydroxylamine (40.3 mg, 0.784 mmol) and triethylamine (0.084 mg). ml, 0.60 mmol). After stirring at room temperature for 18 h, the reaction mixture was evaporated, redissolved in ethyl acetate (2 ml), washed with 10% citric acid (0.5 ml), followed by saturated aqueous sodium bicarbonate. (0.5 ml) and water (0.5 ml). The ethyl acetate layer was dried (Na 2 SO 4), filtered and concentrated in vacuo to give (2S) -2-isobutyl-N 1 - ((S) -1- (methylamino) -l-oxo-4- crude phenylbutan-2-yl) -N 4 - (tetrahydro-2 H -pyran-2-yloxy) succinamide (50 mg, 67%). LCMS (3 min) purity = 44%, tr = 1.77, m / z. 448 [M + H] +, 364 [M + H-THP] +. vi) (2S) -2-isobutyl-N 1 - ((S) -1- (methylamino) -l-oxo-4-phenylbutan-2-yl) -N 4 - (tetrahydro-2 H -pyran-2-yloxy) succinamide (50 mg, O.llmmol) was deprotected to give compound 16 using the procedure described under Route A.

Yield = 1.8 mg, 4.5% (over 6 stages). LCMS (7 m n) purity = 100% (Lr = 3.48), m / z 364 | M UIJ. 1 \ i NMR was identical to that prepared by the Ru to AB Sintesxs of (R) -N4-hxdrox? -2-? Sobut? L-Nl- ((S) -1- (methylammo) -l- oxo-4-phen? lbutan-2-? l) succinamide (Compound 17) i) To a mixture of acid (K) -J- (meXox and carbonyl) -5-methylhexanoi co (250 mg, 1 3? mmol) , I DC (332 mg, 1.73 mmol) and IIOBT (234 mg, 1/3 mmol) in lili (5 ml) was added 0-tetrahydro-2H-p? an-2-? 1 -hi droxi 1 arm na (202 mg, 1.73 mmol) and Ct3N (0.46 ml, 3.30 mmol). The me / cl do reacc LO? it was stirred at room temperature overnight. After removal of the volatile products the residue was taken up in ethyl acetate (15 ml), washed with 10% citric acid (5 ml), followed by saturated aqueous sodium bicarbonate (5 ml) and water (5 ml). ml). The acetate layer was dried (Na 2 SO 4), filtered and concentrated in vacuo to give 4-mel i 1 -2- (2-oxo-2- (tetrahydro-2H-p? r n -2-? 1 ox i am i no) ot i 1) pen tanate to (2R) -methyl as a colorless viscous oil (0 33 g, 85%). LCMS (3 rain) purity = 67%, tr = 1 84, m // 288 [M t Hj +, 204 [M + H-THP] +. n) To a solution of 4-met? L-2- (2-oxo-2- (tetrahydro-211-p? Ran-2- i lox i ami no) ot i 1) (2R) -methyl pentanoate (320 mg, 1.11 mmol) in a mixture of TllI (3.0 ml) and methanol (1.5 ml) was added 2 M aqueous sodium hydroxide. (1.5 ml) at room temperature. After stirring at room temperature for 1 h the reaction mixture was concentrated to approximately 0.5 ml and acidified thoroughly with 1 M aqueous hydrochloric acid. The aqueous layer was extracted with ethyl acetate (2 x 5 ml) and the organic layers Combine were dried (Na2SO4), filtered and concentrated in vacuo to give a mixture of (2R) -4-methyl-2- (2-oxo-2- (tetra id ro-2H-piran-2-iJ .oxiamino) ethyl) pentanoic acid and (3R) -5-methyl-3- (tetrahydro-2H-pyran-2-yloxycarbamoyl) acid (260 mg, 86%) that was used directly in step (iii). LCMS (3 min) purity = 70%, tr = 1.59 (co-eluted isomers), m / z 274 [M + H] +, 190 [M-l-H-THP] +. iii) To a mixture of (2 R) -4-methyl-2- (2-oxo-2- (tetrahydro-2H-pyran-2-yloxyamino) ethyl) pentanoic acid and (3R) -5-methyl-3 acid - (Tetrahydro-2H-pyran-2-yloxycarbamoi) hexanoic acid (120 mg, 0.439 mmol), EDC (109 mg, 0.571 mmol) and HOBT (77 mg, 0.571 mmol) in DMF (3 mL) was added (S) - 2-amino-N-methyl-4-phenylbutanamide (102 mg, 0.53 mmol), followed by triethylamine (0.151 mL, 1.09 mmol). The reaction mixture was stirred at room temperature overnight then diluted with ethyl acetate (10 ml), washed with 10% citric acid (5 ml), followed by saturated aqueous sodium bicarbonate (5 ml) and water (5 ml). The ethyl acetate layer was dried (Na 2 SO 4), filtered and concentrated in vacuo to give a mixture of (2R) -2-Isobutyl-N 1 - ((S) -1- (methylamino) -l-oxo-4-phenylbutan-2-yl) -N 4 - (tetrahydro-2 H -pyran-2-yloxy) succinamide (isomer) A), (2R) -2-isobutyl-N4- ((S) -1- (methylamino) -l-oxo-4-phenylbutan-2-yl) -N1- (tetrahydro-2H-pyran-2-yloxy) succinamide (isomer B) and (3R) -3-isobutyl-1- (tetrahydro-2H-pi ran-2-yloxy) pyrrolidin-2,5-dione (80 mg). LCMS (3 min) showed 23% of A and B isomers (co-eluted at tr = 1.77), m / z. 448 [M + H] +, 346 [M + H-THP] + and 69% by-product (3.R) -3-isobutyl-1- (tet: rahydro-2H-pi ran-2-yloxy) pi rrolidin-2,5-dione (tr = 2.0), m / z 319 LM-l-Na -? - MeCN J +, 533 [2M + Na] +. Purification of preparative reverse phase HPLC of the crude product in dimethyl sulfoxide: acetonitrile 2: 1 (1.6 ml) was injected onto a C18 TLie column or Hyporsil-Keys tone Hyperprep HS (12 μm, 100 x 21.2 mm) isolated fractions containing a mixture of isomers A and B. The column was eluted for 9.5 min at 30 ml / min with a gradient of 20-100% acetonitrile / 0.1% TFA (solvent B) in water / 0.1% TFA (solvent A) using UV detection at 215 nm. iv) Fractions containing (2 R) -2-isobutyl-N 1 - ((S) -1- (methylamino) -l-oxo-4-phenylbu tan-2-yl) -N 4 - (tetrahydro-2 H -pyran- 2-yloxy) succinamide and (2 R) -2-isobuyl-N 4 - ((S) -1- (methylamino) -l-oxo-4-phenylbu tan-2-yl) -N 1 - (tetrahydro-2H -piran-2-yloxy) succinamide from step (iii) were allowed to establish at room temperature; overnight. Concentration in vacuo gave the deprotected products (10 mg), of which compound 17 (1.1 mg) was purified by preparative HPLC using the method described in step (ni) - LCMS (7 min) purity = 86%, tr = 3.21, m / z 364 [M + H] +, 386 [M + Na] +, XH NMR (MeOD) d 1.75 (3 II, d, J = 6.35 Hz, isobutyl), 1.85 (3 H, d, J = 6.35 Hz, isobutyl), 1.15 (III, m, isobutyl), 1.45 (2 H, m, isobutyl), 1.90 (1H, m), 2.10 (1H, dd, J = 14.45 and 5.85 Hz), 2.25 (1H, m), 2.40-2.60 (2 H, m), 2.60 (3 H, s, CONHMe), 2.75 (1H, m), 4.10 (1H , dd, J = 9.15 and 5.21 Hz), 7.00-7.15 (5 H, m,? r). Biological Examples 1. Aureolysin inhibition assays and MMP enzyme The inhibitory activity of the compound against purified aureolisma type 1 and type II (BioCentrum Ltd) was determined in mixtures (0.1 ml) containing buffer solution of 90 mM MOPS pH 6.8, chloride of calcium 4.5 mM, Bri 35 at 0.045%, Mca-Pro-Leu-Gl and-Leu-Dap (Dnp) -Ala-Arg-amide 10 μM (Bachem), aureol i si na and 2-dimethyl sulfoxide vehicle % with or without inhibitor. The activity of the compound against human MMPs 1, 2, 8 and 9 (Calbiochem) was determined in the same manner except that the regulatory solution used was 90 mM Tps-HCl pll 7.5 @ 25 ° C, 90 mM sodium chloride, 9 mM calcium and Bpj 35 0.045%. The reactions were incubated at 37 ° C for 1 hour, capped with 0.1 ml of 0.5 M acetic acid and fluorescence it was measured using excitation 320 nm and emission 405 nm. The concentration of the compound showed a 50% decrease in enzyme activity under test conditions (the IC50 value) was determined by curve fitting (XLfit, IDBS Ltd). The results are shown in Table 2. Table 2 value IC50 (μM) or% of Inhibition Cpd # Aureolysin Aureolysin MMP-1 MMP-2 MMP-8 MMP-9 type I type II 1 0.26 0.43 0.00092 0.00044 0.00033 0.00046 3 9.9 14 0.00076 0.00071 0.00053 0.0016 5 27 41 0.011 0.0077 0.0039 0.030 7 2.2 3.5 0.0033 0.0032 0.0017 0.0051 9 1 .5 2.4 0.0031 0.0018 0.0017 0.0041 1 1 1.3 2.4 0.0028 0.0019 0.0014 0.0036 12 1 .3 2.2 2.3 2.7 1.4 4.6 13 0.33 0.69 0.0056 0.0011 0.0016 0.0019 value IC50 (μM) or% inhibition Cpd # Aureolysin Aureolysin MMP-1 MMP-2 MMP-8 MMP-9 type I type II 14 1 .0 2.5 0.29 0.0046 0.021 0.010 0% @ 0% @ 0% @ 0% @ 15 2.8 2.3 2.7μM 2.7μM 2.7μM 2.7μM 16 0.057 0.093 6.2 2.1 1 .6 2.0 17 1 .2 2.3 0.017 0.0024 0.0044 0.0059 2 . Protease activity in the washes of the skin of Eczema patients: A method has been developed to evaluate protease activity in skin washes. Skin washes from patients with acute eczema can be obtained by aspirating sterile physiological saline solution 0.5 ml onto the surface of the skin using a disposable, sterile plastic Pasteur pipette. The skin area (~ lcm) is defined by a sterile open plastic cylinder. Samples were transferred to 0.05 ml of 0.55 M MOPS buffer pH 7.0, 55 mM calcium chloride and 0.2% Brij 35, mixed, centrifuged to remove debris and frozen at -70 ° C pending analysis. f Zymographic analyzes of the protease content of the samples can be completed by mixing with volume 0.2 0.2 M Tris-HCl pH 6.8 containing 37.5% glycerol (v / v) and 2.5% sodium dodecyl sulfate after electrophoresis through of a gelatin zymogram gel (Invitrogen Corporation) according to the manufacturer's instructions. The gels were washed in Triton X-1.00 at 2.5% (w / v) in buffer solution of 25 mM MOPS pH 7 with or without compound 11 (50 μM) and developed overnight at 37 ° C in buffer solution. MOPS 0.1 M pl-I 7 containing 5 mM calcium chloride with or without compound 11 (50 μM). Cleaning zones due to proteolytic activity can be identified by staining with Coomassie Brilliant Blue R after decolorization in 40% methanol (v / v) / 10% acetic acid (v / v). The proteolytic activity can be attributed to aureolysin or metzincins by an appropriate method known to a person skilled in the art for example by means of molecular weight analysis with confirmation by Western blotting. Representative gels obtained during 6 washed skin samples taken from acute AD sites are shown in Figure 1. Legend to Figure 1: (A) Zymographic analysis of 6 washed samples from the skin of patients with acute AD; (B) Zymographic analysis of the same washed skin samples incubated with 50 μM of compound 11. For (A) and (B), line 1 = size markers (kDa); line 2 = sample washed from the skin 7; line 3 = sample 14; line 4 = sample 17; line 5 = sample 37; line 6 = sample 40; line 7 = sample 48; line 8 = 4 ng of purified aureolysin. Protease activity in washed skin samples was also measured by incubation (9 μl) in 90 mM MOPS pH 7.0, 4.5 mM calcium chloride, 0.045% Brij 35, Mca-Pro-Leu-Gly-Leu-Dap (Dnp) 10 μM α-Arg-amide (Bachem) and 2% dimethylsulphoxide vehicle (v / v) with or without compound 11 (50 μM) at 37 ° C. Samples were incubated at 37 ° C in an optimal plate reader POLARstar (BMG Labtech Ltd.) and fluorescence readings (excitation 320 nm / emission 405 nm) taken every 15 rain for 6 h. The activity was expressed as the ratio of increasing in fluorescence as a function of time. Table 3 shows the results obtained. Table 3 Protease activity with% inhibition of activity of Protein Activity Number Sample 50μ of compound 11 protease 8n 8mPuest ° (FlU / mip / μl) (FlU / mm / μl) 5 0 264 ci Too 7 0 529 0271 487 14 0 661 00761 885 17 3 42 0123 964 18 0 276 00674 756 21 0 203 00302 851 24 0 000122 N / A 26 0 376 00554 853 30 0 226 0 100 36 0 366 00173 953 37 0 692 0126 818 39 0 0564 0 100 4 400 0 0771166 0103 856 43 0 994 00814 918 48 0 921 00287 969 Both assays show significant inhibition of protease activity in washed samples of the skin of patients with acute AD by compound 11. Compound 12 was also tested for the inhibition of protease activity in a set of 8 washed skin samples from AD patients. Table 4 below shows that there was significant inhibition of protease activity in the washed samples of the skin Table 4 3. Actividd of Staphylococcus aureus protease in culture. Inhibition of the metalloprotease activity compound (aureolisma) in culture supernatants from Staphylococcus to ureus 8325-4 was determined in mixtures (0.1 ml) containing buffer solution of 90 mM MOPS pH 6.8, 4.5 mM calcium chloride, Bp 35 0.045%, Mca-Pro-leu-Gly-leu-Dap (Dnp) -la-Arg-amide 10 μM (Bachem), 4 μl of culture supernatant S. a ureus and 2% dimethyl sulfoxide vehicle (v / v) with or without inhibitor. The culture supernatant of S. aureus was prepared by inoculating 5 ml of tryptic soy broth containing 10% skimmed milk with S. to ureus 8325-4 and incubated at 37 ° C for 6-8 h while shaking. The culture was then centrifuged to remove the cells and the stored supernatant at -70 ° C during subsequent use. The reactions were incubated at 37 ° C in a reader of optimal plate POLARstar (BMG Labtech Ltd.) and fluorescence readings (excitation 320 nm / emission 405 nm) are taken every 15 min for 6 h. the activity was expressed as the ratio of increasing in fluorescence as a function of time. The concentration of the compound that induces a 50% decrease in enzyme activity under test conditions (the IC50 value) was determined by adjusting the curve (XLfit, IDBS Ltd). Table 5 shows the results obtained. These values are similar to the values obtained from newly purified aureolysin (Table 2) that suggest that the metalloprotease activity in the supernatants is due to the activity of aureolysin. Table 5 Compound value IC5o (μM) 0.57 3 20 5 44 7 4.5 9 2.9 11 3.7 12 4.3 13 069 14 36 15 33 16 0.095 17 23 In a second test to measure the activity of S protease. a ureus directly, S. a ureus ATCC 27733 or 8325-4 was grown on 10% (v / v) skimmed milk agar plates containing 2% (v / v) DMSO with or without the compound. The compounds dissolved in DMSO were incorporated into the solid medium immediately before casting. The plates were incubated at 37 ° C for 24-48 hours and the proteolytic activity was determined by measuring the evacuation zone around the individual colonies. An example of this test is shown in Figure 2. Legend to Figure 2. The graph shows the inhibition of proteolytic activity by compound 3 in a milk agar plate assay. The results show evacuation zones of milk proteins. 4. Activation of aureolysin-mediated protease The ability of aureolysin to activate endogenous proteases can be tested by incubating the target protease with aureolysin in a suitable buffer solution containing calcium chloride, sodium chloride and Brij 35 at 37 ° C. This is exemplified immediately by the pro-urokinase activation demonstrated directly by the enzyme assay using a chromogenic substrate in the presence of EDTA to inhibit aureolysin activity (Narasaki et al., J Biol Chem. 240: 14278-87, 2005) and by the activation of proMMP-1 demonstrated by measuring the production of% of the segmentation product of chain length to (I) of collagen using an appropriate image system. The protease content of the samples can also be determined using zymography when mixing with 0.2 volume of 0.2 M Tris-HCl pH 6.8 containing 37.5% (v / v) glycerol and 2.5% (w / v) SDS after electrophoresis through a gelatin zymogram gel (Invitrogen Corporation) according to the manufacturer's instructions. Evacuation zones due to proteolytic activity are identified by staining with Coomassie Brilliant Blue R after decolorization in 40% methanol (v / v) / 10% acetic acid (v / v). 4 (i). Activation of pro-uPA by aureolysin and its inhibition by Compound 13 The ability of aureolysin to activate urokinase-type plasminogen activator (uPA) was tested by incubating individual chain pro-uPA (American Diagnostica Inc.) with aureolysin at both pH physiological (7.5) and at pH 5.6, the natural pH of the stratum corneum (Ohman, H and Vahlquist, A Acta. Derm. Venereol 74: 375-9, 1994). The incubation mixtures contained 1.4 μM (75 μg / ml) pro-uPA, 0.1 M Tris-HCl pH 7.5 or 0.1 M MES buffer (sodium) pH 5.6, 0.1 M sodium chloride, 5 mM calcium chloride, 0.05% Brij 35 and aureolysin in a final volume of 10 μl (Table 6, Expt. 1). In a second experiment activation at pH 5.6 was tested in the presence and absence of Compound 13 (20 μM) in a final volume of 20 μl (Table 6, Expt. 2). All samples were incubated at 37 ° C for 2.5 h and then capped by the addition of 24 volumes of 60 mM Tris-HCl pH 8.8, 50 mM sodium chloride, 2.5 mM EDTA, 0.01% Tween 80; this buffer also contained 0.83 μM of Compound 13 when it was added to the vehicle control samples in Expt. 2. The urokinase activity was measured by incubating the samples of the stopped mixture for 0.5 h at 37 ° C in 0.1 ml of the same buffer solution containing 0.5 mM S-2444 (Chromogenix Instrumentation Laboratory SpA). The reactions were capped with an equal volume of 0.5 M acetic acid and the product measured at 405 nm. The controls were incubated in the absence of pro-uPA that showed no activity in this assay. Table 6: Aureolysin (μg / ml) uPA activity (O.Sh/μg) Expt. 1 MONTH pl-l 5.6 Tris- MCI pl-l 7.5 0 0.14 0.19 0.1 2.31 0.65 0.3 3.76 1.43 1 4.68 3.29 Expt. 2 Vehicle only compound Compound 13 (20μM) 0 0.13 0.1 1 0.1 2.37 0.36 0.3 4.15 0.52 0.8 5.29 0.96 The data in Table 6 show that: a) Pro ^ uPA activated with aureolysin at both pH 5.6 and 7.5; that b) this activation is more effective at the natural pH of the stratum corneum; and c) that Compound 13 at 20 μM (~ 30x IC50) inhibits this activity by > 82% 4 (ii). Inhibition of the pro-uPA activation by Compound 12 The dose response ratio for the inhibition of pro-uPA activation by Compound 12 was determined in reaction mixtures (20 μl) containing 1.5 μM (78 μg) / ml) pro-uPA, buffer solution of 0.1 M MES (sodium) pH 5.6, 0.1 M sodium chloride, 5 mM calcium chloride, 0.05% Brij 35, aureolysin (75 ng / ml) and 2% DMSO ( v / v) ± of Compound 12. The mixtures were incubated at 37 ° C for 2.5 h and stopped by dilution in 7 volumes of 60 mM Tris-HCl pH 8.8, 50 mM sodium chloride, 2.5 mM EDTA, 0.01 Tween 80 %. Under these conditions the extension of pro-uPA cleavage as determined by the uPA activity was directly proportional to the concentration of aureolysin in the assay. The activity of uPA was measured using S-2444 as described in 4 (i) above. The concentration of the compound that induces a 50% decrease in uPA activity (the IC50 value) was determined by adjusting the curve (XLfit, IDBS Ltd) which is 2.4 μM which is in good agreement with the value in Table 2 determined using a fluorogenic peptide substrate for determining aureolysin activity.

The ability of Compound 12 to inhibit the activation of pro-uPA at a higher aureolysin concentration was determined in reaction mixtures (20 μl) containing 1.5 μM (78 μg / ml) pro-uPA, buffer solution of 0.1 M MES (sodium) pH 5.6, 0.1 M sodium chloride, 5 mM calcium chloride, 0.05% Brij 35, aureolysin (1 μg / ml) and 2% (v / v) DMSO ± the Compound 12. The mixtures were incubated at 37 ° C for 2.5 h and capped by dilution in 20 volumes of 60 mM Tris-HCl pH 8.8, 50 mM sodium chloride, 2.5 mM EDTA, 0.01% Tween 80 and the activity of uPA was measured using S-2444 as described in 4 (i) above. The results in Table 7 confirm that, as expected, Compound 12 (at lOx and 130x the IC50 value) which inhibits pro-uPA activation mediated by aureolysin in a dose-dependent manner. Compound 12 (317 μM) had no effect on uPA activity in this assay. Table 7: Aureolysin Compound 12 UPA activity (%) of (μg / ml) (μM) (A O.dh / μg) Relative activity 0 0 0.17 4 1 0 4.2 100 1 25 1.6 37 1 317 0.3 8 The inhibition of pro-uPA activation by inhibiting aureolysin activity on the surface of the Skin is expected to reduce pro-inflammatory management in patients with AD. 4 (iii). Activation of proMMP-1 by aureolysin The ability of aureolysin to activate fibroblast collagenase (MMP-1) was tested by incubating the human rheumatoid synovial fibroblast collagenase proenzyme (Calbiochem) at pH 7.5 with aureolysin both with and without addition of MMP activating aminophenylmercuric acetate (APMA). The activation mixtures in the buffer solution of TCNB (0.1 M Tris-HCl pH 7.5, 10 mM calcium chloride, 0.1 M sodium chloride, 0.05% Brij 35) contained proMMP-1 (25 μg / ml) and aureolysin (3 μg / ml) and / or APMA 1 mM as indicated. The mixtures were incubated at 37 ° C / 1.25 h and cooled by dilution in ice water of TCNB. The collagenase activity in the samples was then determined by incubation at 25 ° C in TCNB containing (pro) MMP-I 0.1 μg / ml and porcine type I collagen 0.16 mg / ml (MD Biosciences). The portions of each mixture were removed at volume time intervals 0.2 buffer solution loaded with 5x gel (0.2 M Tris-HCl pH 6.8 / 37.5% glycerol (v / v) / 2.5% SDS (w / v) / 2 5% (v / v) mercaptoethanol) and heated at 95 ° C for 2.5 min. Collagen cleavage was quantified after the SDS-PAGE gel analysis (NuPAGE at 4-12% Bis-Tris (MES), Invitrogen Corp) when estimating the product band density of / 4 of chain length to (I) using an FC AlphaEase ™ program run image system FluorChem ™ 8800 (Alpha lnnotech Corp.) Segmentation proportions were estimated from the linear portion of the curves and the relative proportion for the untreated control was calculated. The data is shown in Table 8 below. Consistent with the Bed that aureolysin is not the same as a collagenase, control incubations containing aureolysin alone ± APMA showed no activity in this assay. Table 8: VA product of chain length a1 (arbitrary units) MMP-1 + Time (h) MMP-1 + MMP-1 single MMP-1 + APMA aureolysin + aureolysin APMA 0 0 0 0 0 0.5 1314 3387 1709 9652 1 1589 4871 3726 13867 1.5 1558 6404 5115 17022 2 1807 81 15 8742 18227 3 3896 11474 12718 18435 4 6378 13850 16882 19988 5 8645 17154 17558 2021 1 6 10050 19387 19436 21388 Proportion 1 2 3 8 relative These data show that aureolysin not only activates proMMP-1 to an extent comparable to a recognized MMP activator such as APMA but that it has the ability to MMP-1"superactive" when used in combination with APMA. The inhibition of aureolysin, therefore, inhibits the activation of proMMP-1 when the two enzymes are in the same site, for example on the skin of patients with AD colonized with S. to ureus. 5. Keratinocyte activation mediated by aureolysin Keratinocytes produce IL-8, a proinflammatory chemokine. Many bacterial products cause the activation of keratinocytes. Aureolysin can be evaluated for its effects on IL-8 production by keratinocytes. The epidermal keratinocytes in human skin (TCS Cellworks) are maintained as per instructions. The proliferating cultures are trypsinized, harvested, treated with a trypsin inhibitor and resuspended in growth medium at approximately 50,000 cells / well, to provide confluent monolayers in 96-well plates. The cells are incubated overnight at 37 ° C in 5% C02 to allow recovery, the spent medium aspirated from the cavities and replaced with fresh growth medium. The cells are incubated at 37 ° C in 5% C02 for an additional 24 or 48 hours with aureolysin or control buffer solution. Supernatants are removed from each well and the concentration of IL-8 is determined using an ELISA human development kit IL-8 of R & D systems.

Catalog: DY208) using the instructions of the manufacturers. The results of the experiments are shown in Table 9. In these experiments, the cells are incubated at 37 ° C in 5% C02 for 48 hours with aureolysin or control buffer solution. Poly IC and lipoteichoic acid (LTA), which stimulates the production of IL-8 in keratinocytes were used as positive controls. Table 9: Keratinocytes IL-8 (pg / ml) ± SE Not stimulated 199 +/- 3.2 Positive control of Poly IC 282 +/- 0.9 Positive control of LTA 454 +/- 61.0 Control of regulatory solution 493 +/- 3.6 Aureolysin (50μg / ml) 687 +/- 0.7 The experiment shows that aureolysin can stimulate the production of IL-8 in keratinocytes (687 pg / ml) above and above the control of aureolysin buffer (493 pg / ml). LTA (454 pg / ml) and a laser extension, Poly IC (282 pg / ml) stimulated IL-8 production compared to unstimulated control (199 pg / ml). 6. Impact of Compounds 11 and 12 on the growth and viability of S. aureus The effect of the compounds on growth and viability of S. A ureus can be estimated by growing the organism in liquid culture after placing it in solid medium to count the viable cells. Alternatively growth can be estimated by turbidometry in 96-well micro-titer plates. The brain heart infusion medium (5 ml, Becton Dickinson and Co.) containing 10% skimmed milk and 1% (v / v) DMSO vehicle ± 50 μM of the compound was inoculated with S. to ureus 8325-4 (approximately 107 cells in tryptic soy broth) and incubated for 16 h at 37 ° C / 220 rpm. The duplicate samples (0.1 ml) were then removed from each culture, diluted in PBS, sprayed on brain heart infusion agar (1.5%) and incubated at 37 ° C. Viable cell counts were determined from the number of colonies as that sample in Table 10. Table 10: Compound Cells / my sd Vehicle 1 .3x109 6.4x108 1 1 1 .6x10q 3.5x108 12 1.3x109 3.5x108 Tryptic soy broth (0.18 ml) containing S. to ureus 8325-4 (approximately 105 cells) was mixed with 20 μl of 20% (v / v) DMSO vehicle ± the compound in the cavities of a flat-bottomed clear polystyrene 96-well micro-title plate. The plate was incubated overnight at 37 ° C / 220 rpm and the absorbance was measured at 620 nm the next day. Growth inhibition was determined by reference to vehicle control and the concentration of actinonin (Sigma) which induces a 50% decrease in the terminal absorbance (IC50 value) was determined by adjusting the curve (XLfit, IDBS Ltd) as shown in Table 11. Table 11: Compound value IC50 (μM) Inhibition at 0.1 mM Actinonipa 2.6 Compound 1 1 - 2% Compound 12 - 0% The data in Tables 10 and 11 show that the Compounds 11 and 12 are not antibacterial, whereas actinonin of the positive control compound (Sigma), a hydroxamate-based peptide des-peptide inhibitor.

(Clements, JM et al., Animicrob Agents Chemother, 45: 563-570, 2001), exhibits an IC5o value in this 2.6 μM assay. 7. Impact of Compound 12 on S. aureus protease activities Aureolysin is responsible for the activation of the serine protease glutamyl endopeptidase staphylococcal (V8 protease) and is indirectly responsible for the activation of staphylopain B of staphylococcal cysteine protease (Shaw, L et al., My Crobiology 150: 217-228, 2004). The inhibition of aureolysin would therefore be expected to have an impact on the activity of these proteases despite the fact that none is similarly targeted by a metalloprotease inhibitor. The total impact of the compound on the activity of these staphylococcal proteases can be tested by growing S. aureus in the presence of the compound and analysis of the cellular conditioned medium for protease activity while maintaining the same concentration of that compound. Duplicate samples of brain heart infusion medium (5 ml, Becton Dickinson and Co.) containing 10% skimmed milk and 2% (v / v) DMSO carrier ± Compound 12 were inoculated with S. aureus 8325-4 (approximately 107 cells in broth are tryptic) and incubated for 16 h at 37 ° C / 220 rpm. The cultures were centrifuged to remove bacteria and the culture supernatants were stored at -70 ° C depending on the analysis of protease activity. Enzyme activities were measured in mixtures containing buffer solution of 90 mM MOPS (sodium) pH 7. 0 (0.1 ml), Brij 35 at 0.045%, DMSO vehicle at 2% (v / v) ± Compound 12 and culture supernatant (4 μl). The concentration of the compound used was the same as that which had been used to culture the sample analyzed. Additional additions to the reaction mixtures were as follows. Aureolysin assay: 4.5 mM calcium chloride, 9 μM E-64 (to inhibit the activity of cystatin protease) and 10 μM Mca-Pro-Leu-Gly-leu-Dap (Dnp) -Ala-Arg-amide ( Bachem); V8 protease assay: 10 μM Mca-Leu-Glu-Val-Asp-GIy-Trp-lys (Dnp) -amide (Bachem); cysteine protease assay: 1.8 mM cysteine-HCl (pH adjusted with NaOH), 9 mM EDTA and 0.1 mM Z-Phe-Arg-AMC hydrochloride (Bachem). The proportion of product formation at 37 ° C was monitored during readings taken from 6 h to 15 min intervals using an OPTIMA POLARstar plate reader (BMG LABTECH Ltd) with 320 nm excitation / 405 nm emission for aureolysin and V8 assays and excitation 390 nm / emission 460 nm for the cysteine protease assay. Enzyme activities were determined from the linear proportion of increased fluorescence and converted to percent inhibition by reference to vehicle control samples. The results in Table 12 show that Compound 12 almost completely suppresses the level of V8 protease activity and that it significantly suppresses the level of cysteine protease activity.

Table 12: (%) Inhibition Compound 12 protease V8 (μM) Aureolysin Cysteine protease 317 99 99 87 159 98 97 78 79 94 91 53 40 89 83 27 The use of Compound 12 to inlibe the aureolysin activity on the skin of patients with AD colonized with S. aureus will therefore be expected to have the additional benefit of decreasing the activity of other extracellular staphylococcal proteases. 8. Selectivity profile of Compound 12 Compound 12 has broad spectrum activity with comparable potency against aureolysin and MMPs (Table 2). Their inhibitory activity against other proteases, including those of different catalytic classes, can be determined by using analogs of biochemical assays suitably configured to those previously used for aureolysin. The inhibitory activity of Compounds 11 and 12 against a range of purified enzymes was tested as described below. The inhibitory activity was tested in mixtures of reaction (0.1 ml) containing 2% DMSO vehicle (v / v) ± the compound plus conditions as follows. Protease V8: buffer solution of 90 mM MOPS (sodium) pH 7.0, 4.5 mM calcium chloride, 0.045% Brij 35, 10 μM Mca-Leu-Glu-Val-Asp-Gly-Trp-lys (Dnp) -amide ( Bachem) and V8 (BioCentrum Ltd, 30 ng). Stafopain A and B: 90 mM MOPS buffer (sodium) pH 7.0, 1.8mM-l-IC.l cysteine (pH-adjusted with NaOH), Brij 35 0.045%, 0.1 mM Z-Phe-Arg-AMC hydrochloride (Bachem) and stafopain A or B (BioCentrum Ltd, 30 ng). Human Kallikrein 5: Regulating solution with sodium phosphate 0.1 M pH 8.0, Brij 35 0.045%, Boc-Val-Pro-Arg-AMC 0.1 mM (Sigma) and recombinant human kallikrein 5 (R &D Systems Inc; 6ng). Human Kallikrein 7: 72 mM Tris-HCl pH 8.0, Brij 35 0.033%, S-2586 1.2 mM (Chromogenix Instrumentation Laboratory SpA) and recombinant human kallikrein 7 (R & D Systems Inc; 0.6 μg) which had been activated with thermolysin according to the manufacturer's instructions. The enzyme that converts human angiotensin (ACE): buffer solution of MES 45 mM (sodium) pl-l 6.5, Brij 35 to 0.045%, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys ( Dnp) -OH 10 μM (R & D Systems Inc) and recombinant human ACE (R &D Sytsems Inc., 1.3 ng). Human cathepsin D: 0.1 M sodium acetate buffer solution pH 3.5, 0.2 M sodium chloride, 0.045% Brij 35, Mca-Pro-Leu-Gly-Leu-Dap (Dnp) -Ala-Arg-amide 10 μM (Bachem) and recombinant human cathepsin D (R & D Systems Inc; 8ng) activated according to the manufacturer's instructions. Human ADAM17: 25 mM Tris-HCl pH 9.0, 2.5 μM zinc sulfate, Brij 35 at 0.005%, Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Ser-Arg-amide 10 μM (R & D Systems Inc) and recombinant human ADAM17 (R & D Systems Inc; 2.5 ng). All reactions were incubated at 37 ° C / 1 h and maintained with 0.1 ml of 0.5 M acetic acid except for the cathepsin D assay which was closed with 0.1 ml of 0.15 M Tris base. The inhibition percentage at 0.1 mM calculated with reference to vehicle control and, where appropriate, the IC50 values were determined by adjusting the curve (XLfit, IDBS Ltd). The data in Table 13 demonstrate that Compound 12 does not inhibit aspartyl protease cathepsin D, Kallikrein serine proteases 5 and 7 and V8, nor staphypain A and B of cysteine proteases s taphylococcal. Also, Compound 12 does not inhibit ACE (family metalloprotease M2) and is only an extremely weak inhibitor of ADAM17 which indicates that it is not a "shedase" inhibitor; these contrasts markedly with their diastereoisomers, Compound 11, which is a potent inhibitor of ADAM17. Table 13: IC50 value or inhibition at 0.1 mM Enzyme Compound 1 1 Compound 12 ACE 0% ADAM17 0.025μM 79μM Cathepsin D 1% Kallikrein 5 0% Kallikrein 7 0% Stapopain A 5% 0% Stapopain B 1 1% 10% Protease V8 0% 0% All references mentioned in this application, which include patents and patent applications, are hereby incorporated by reference for the fullest possible scope. For all the specification and the claims that follow, unless the context requires otherwise, the word 'comprises' and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of an integer, stage, set group of integers or group of stages but not for the exclusion of any other whole number, stage, group of integers or group of stages. ACE abbreviations enzyme that converts angiotensin AD atopic dermatitis APMA 4-aminophenylmercuric acetate DCC N, N'-dicyclohexylcarbodiimide DCU N, N '-dicyclohexylurea DMAP 4-dimethylaminopi ridine DMSO dimethyl sulfoxide E-64 L- trans-epoxysuccinyl-leucylamide- (4-guanidino) -butane EDAC 7- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride EDC N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide EDTA ethylendia inotetraacetic acid HOBT 1-hydroxybenzotriazole hydrate LTA Lipoteichoic acid MES 4-morpholineethanesulphonic acid MOPS 4-Mo folinpropane sulphonic acid NMM 7-methylmorpholine PBS phosphate-regulated saline TCNB 0.1 M Tris-HCl pH 7.5, 10 mM CaCl2, 0.1 NaCl M, Brij 35 0.05% TFA trifluoroacetic acid THF tetrahydrofuran uPA urokinase-type plasminogen activator

Claims (18)

1. CLAIMS 1. A compound that is an inhibitor of aureolysin, for topical application in the treatment or prevention of an inflammatory condition in the skin that is characterized by colonization with Staphylococcus a ureus.
2. 2 . A compound that is an aureolysin inhibitor, characterized in that it is for topical application in the treatment or prevention of atopic dermatitis.
3. A compound according to claim 1 or claim 2, characterized in that it has an IC50 value of less than 50 μM in the aureolysin II assay defined herein, and an IC50 value of not less than 0.5 μM in the assay MMP-9 defined herein.
4. 4. A compound according to claim 3, characterized in that the IC50 value in the aureolysin assay is less than 5 μM.
5. 5. A compound, characterized in that it is (S) -N1- ((S) -3,3-dimethyl-l- (methylamino) -l-oxobutan-2-yl) -N4-hydroxy-2-isobutylsuccinamide (Compound 12 ) or a pharmaceutically acceptable salt or solvate thereof.
6. 6. A compound, characterized in that it is (S) -N4-hydroxy-2-isobutyl-Nl- ((S) -1- (methylamino) -l-oxo-4-phenylbutan-2-yl) -succinamide (Compound 16 ) or a pharmaceutically acceptable salt or solvate thereof.
7. 7. A topical pharmaceutical composition that comprises a compound according to any of claims 1 to 6 and a pharmaceutically acceptable carrier or excipient, for use in the treatment or prevention of an inflammatory condition in the skin characterized by colonization with Staphylococcus aureus.
8. 8. A topical pharmaceutical composition, characterized in that it comprises a compound according to any of claims 1 to 6 and a pharmaceutically acceptable carrier or excipient, for use in the treatment of atopic dermatitis.
9. 9. A composition according to claim 7 or claim 8, characterized in that it additionally comprises an additional medicament selected from an antibiotic, an agent that modulates the inflammatory response, which includes spheroidal and non-spheroidal anti-inflammatory agents and an immunosuppressant.
10. 10. Use of an aureolysin inhibitor in the manufacture of a topical medicament for the treatment or prevention of an inflammatory condition in the skin characterized by colonization with S taphylococcus a ureus.
11. 11. The use according to claim 10, characterized in that it is for the treatment or prevention of atopic dermatitis.
12. 12. The use according to claim 10 or claim 11, characterized in that the compound is administered in combination with an additional medicament selected from an antibiotic, an agent that modulates the inflammatory response, which includes steroidal and non-spheroidal anti-inflammatory agents and an immunosuppressant.
13. 13. A method for classifying an agent of use in the treatment or prevention of an inflammatory condition in the skin, characterized by colonization with Staphylococcus aureus, comprising: (i) contacting the agent with aureolysin; and (ii) determine if the aureolysin is inhibited.
14. 14. A method for classifying an agent of use in the treatment or prevention of an inflammatory condition in the skin, which is characterized by colonization with Staphylococcus a ureus, which comprises: (i) obtaining skin wash from patients; (ii) contacting the agent with skin washes; and (iii) determining whether the proteolytic activity is inhibited.
15. 15. A method according to claim 14, characterized in that the determination is by zymography or enzyme assay.
16. 16. A method for the treatment of an injury in skin associated with an inflammatory condition in the skin in a mammal that is characterized by colonization with Staphylococcus a ureus, which comprises (i) obtaining skin washes from the site of the skin lesion and, if the presence of activity of the skin metalloprotease is confirmed, then (ii) topically administering an aureolysin inhibitor to the skin lesion.
17. 17. A method for the treatment of a skin lesion associated with an inflammatory skin condition in a mammal that is characterized by colonization with Staphylococcus a u reus, which comprises (i) determining the presence of Staphylococcyseus at the site of the skin lesion and, if the presence of Staphylococcus aureus is confirmed, then (ii) topically administering an aureolysin inhibitor to the lesion of the skin.
18. 18. A method according to claim 16 or claim 17, characterized in that the aureolysin inhibitor is a compound as defined in any of claims 3 to 6.