GB2463568A - Formulation that has activity against propionibacteria - Google Patents

Abstract

Antimicrobial and/or anti-acne formulation containing (a) a dialkyl sulphosuccinate (DAS) or derivative thereof and (b) a compound of formula (I) or derivative thereof:wherein each of R1and R2is independently selected froth methyl and ethyl and R3is hydrogen. The formulation may be used to treat conditions such as skin and skin structure conditions, in particular acne. It may in particular be used as an antibacterial agent, more particularly against propionibacteria.

Description

Formulations

Field of the invention

This invention relates to antimicrobial and anti-acne formulations, and to the use of certain combinations of compounds as antimicrobial and anti-acne agents.

Background to the invention

Bisphenols of formula (I) below: >[R3O3k Formula (I) wherein each of R1 and R2 is independently selected from methyl and ethyl and R3 is hydrogen, are known antioxidants.

Where R' and R2 are both methyl, the compound of formula (I) is known as "Antioxidant 2246" or "AU 2246", the chemical names for which include 2,2'- methylene-bis(4-methyl-6-tert-butylphenol); bis(2-hydroxy-3 -tert-butyl-5 -methylphenyl)methane; and 6,6'-di-tert-butyl-2,2'-methylene-di-p-cresol. AO 2246 has been used as a preservative/stabiliser in for example plastics and elastomeric materials, cosmetics, insect repellants and antimicrobial compositions. In the polymer industry it is used as a thermostabiliser, an antioxidant and a free radical polymerisation inhibitor, for example in plastics films intended for the wrapping of foodstuffs.

Where R1 and R2 are both ethyl, the compound of formula (T) is known as "Antioxidant 425" or "AO 425", or as 2,2'-methylene-bis(4-ethyl-6-tert-butylpheno 1).

It too is known for use as an antioxidant and stabiliser.

AO 2246 and other structurally similar bisphenols have also been described for use in modulating cellular immune reactions; for the treatment of hyperlipidemia and arteriosclerosis; to lower blood cholesterol levels and suppress macrophage foaming; as anti-tumour agents; to adjust levels of reactive oxygen species in chronic wounds; and as antiviral agents. They have also been linked with the odour, colour and preservation of smoke-cured meats.

Kawada eta! inJAntibactAntfungAgents, Vol. 9, No. 9: 429-433 also describe the use of certain alkylphenol dimers and o!igomers -although not specifically AO 2246 or AO 425 -as antimicrobial agents, for example against S. aureus. Kim et al in J Antibiot, 1995, 49: 3 1-36 describe the use of a compound they believe to be AO 2246, isolated from the fungus Aspergillusfuinigatus, as an inhibitor of the enzyme acyl-CoA: cholesterol acyltransferase (ACAT). They also report that the compound they isolated has anti-tumour activity, but no in vitro antimicrobial activity against a range of test bacteria. JP-50-140627 however discloses the use of AO 2246 as a disinfectant or antiseptic, and its activity against S. aureus and Bacillus subtilis.

Dialkyl su!phosuccinates (DAS5) are known anionic surfactants. In particular diocty! sulphosuccinate (DOS) and its salts, more particularly sodium DOS, are used as wetting, solubilising, dispersing and emulsifying agents, for example in household cleaning products. The sodium salt is also known for use as a mild contact laxative.

Synonyms for DOS include bis(2-ethylhexyl)sulphonate; 2-sulpho-succinic acid dioctyl ester; 1,4-bis(n-octyl)sulphobutanedioate and 1,4-bis(octyloxy)-1,4-dioxo-2-butanesulphonic acid, and its salts are commercially available under a range of trade names such as Docusate and Duosol.

Dioctyl sodium sulphosuccinate is also known for use as a stabiliser and/or surfactant in formulations containing other active substances, including in anti-acne formulations containing peroxides such as benzoyl peroxide and/or antibiotics such as erythromycin, and in anti-ageing formulations containing retinoids. In WO-86/07258 and WO-2008/146030, DASs, in particular dioctyl sodium sulphosuccinate, are also proposed for use in the topical treatment of bacterial infections and acne.

It has now been found that a DAS can be combined with AO 2246 or certain related antioxidants to provide a formulation having good antimicrobial activity, in particular versus propionibacteria such as Prop/on/bacterium acnes, the bacterium implicated in inflammatory acne. As a result, novel antimicrobial and/or anti-acne formulations can be prepared, in particular for topical application.

Statements of the invention

According to a first aspect of the present invention there is provided an antimicrobial and/or anti-acne formulation containing (a) a dialkyl suiphosuccinate (DAS) or derivative thereof and (b) a compound of formula (I) or derivative thereof: Formula (I) wherein each of R' and R2 is independently selected from methyl and ethyl and R3 is hydrogen, and wherein the formulation is suitable for topical application to the skin. j

The formulation is suitably an antibacterial formulation. It is ideally suitable for topical application to human skin. A formulation which is "suitable for" topical application may also be adapted for topical application.

The term "dialkyl sulphosuccinate" or "DAS" as used herein means a dialkyl ester of sulphosuccinic acid, in which the sulphonic acid group is present as -S(O)2-OH.

Derivatives of dialkyl sulphosuccinates can include salts such as metal salts, ammonium salts (in particular the NH4 salt) and any form of the compound in which the sulphonic acid group is present as the sulphonate -S(O)2-0.

The two alkyl groups of the DAS may be for example C1 to C18 ailcyl groups, in particular C2 to C12 or C2 to Cio alkyl groups, such as C6 to C12 or C6 to Cio and in particular C8. Thus the DAS is suitably a dioctyl sulphosuccinate. The alkyl groups may be substituted or unsubstituted, preferably the latter. Tf substituted, they may for example be mono-, di-or tn-substituted, and may suitably include one or more substituents selected from amido groups and ethers. They may be either straight chain or branched. They may include one or more unsaturated carbon-carbon bonds. In an embodiment of the invention, the alkyl groups of the DAS are substituted with ether groups, as in a dialketh sulphosuccinate such as a dilaureth sulphosuccinate. In another embodiment, the alkyl groups are not substituted with ether groups. In an embodiment, the ailcyl groups do not include unsaturated carbon-carbon bonds.

The DAS or derivative is conveniently used in the form of a salt in which the sulphonic acid group is present as the sulphonate -S(O)2-0, such as in particular a metal salt or ammonium salt. Suitable metal salts include the alkali metal salts (for example the sodium or potassium salts, in particular the former) and the alkaline earth metal salts (for example the calcium salt). In an embodiment, the DAS is used in the form of its sodium salt, for example dioctyl sodium suiphosuccinate (also known as dioctyl sulphosuccinate sodium or docusate sodium).

The DAS or derivative is suitably pharmaceutically acceptable, which term includes suitable for veterinary use.

Suitable DASs for use in the present invention may for example be selected from di-dodecyl (dilauryl) sulphosuccinates; dilaureth sulphosuccinates; diamyl sulphosuccinates; dicapryl sulphosuccinates; dinonyt suiphosuccinates; dioctyl sulphosuccinates; diheptyl sulphosuccinates; dicyclohexyl suiphosuccinates; dihexyl sulphosuccinates; diisobutyt sulphosuccinates; and mixtures thereof These may be used for example in the form of their sodium salts, or in cases of their ammonium (in particular NH4) salts such as ammonium dinonyl sulphosuccinate or ammonium lauryl sulphosuccinate.

In the compound of formula (I), R' and R2 are suitably the same. In an embodiment of the invention, the compound of formula (I) is selected from Antioxidant 2246 (AO 2246, in which both R' and R2 are methyl); Antioxidant 425 (AO 425, in which both R' and R2 are ethyl); and mixtures thereof In another embodiment, the compound of formula (T) is AO 2246.

The compound () or derivative, in particular AO 2246, is ideally used in the form of the isolated compound (whether naturally or synthetically derived, preferably the latter) rather than as part of a plant extract containing a number of different materials.

It is thus suitably used in a substantially pure form, for instance containing 1% w/w or less, preferably 0.5 or 0.2 or 0.1 or 0.05% w/wor less, of impurities.

A "derivative" of either a DAS or a compound of formula (I) may be a pharmaceutically acceptable (which term includes acceptable for veterinary use) derivative. Tt may be for example a salt, ester, complex or solvate or a so-called "prodrug" form or protected form which reverts to an active form of the relevant compound at an appropriate time on or after administration. In the case of a DAS, a derivative may be a free acid -ie a sulphosuccinnic acid -from which the corresponding suiphosuccinate can be derived. In an embodiment, however, the DAS is present in the form of the sulphosuccinate. In an embodiment, the compound (I) is present in the form of a single, underivatised molecule of formula (I).

In a formulation according to the invention, the combination of the DAS or derivative and the compound (I) or derivative is present as an active (ie antimicrobially active, preferably antibacterially active) agent. The combination may be present as an anti-acne agent (ie as an agent which is active against acne (which includes against a symptom and/or a cause of acne and/or against one or more micro-organisms associated with acne)). Suitably, the DAS or derivative is not used purely or even primarily as a stabiliser for another substance such as an active ingredient, or as a surface active agent (surfactant), or as a wetting or solubilising or dispersing or emulsifying agent, or as a processing aid. Suitably, the compound (I) or derivative is not used purely or even primarily as an antioxidant; or as a stabiliser for another substance such as an active ingredient, a lipid or a UV screening agent; or as a preservative in a food product or in a product containing another substance such as an active ingredient; or as a free radical polymerisation inhibitor; or for modulating cellular immune reactions; or for reducing serum lipid (including cholesterol) levels; or for suppressing macrophage foaming; or as an anti-hyperlipidemia agent; or as an anti-arteriosclerosis agent; or as an anti-tumour or anti-cancer agent; or as an antiviral agent; or as an antifungal agent; or for the treatment of chronic wounds as described in US-2005/0065 182.

It is possible that the antimicrobial and/or anti-acne activity of a combination of a DAS or DAS derivative and a compound (I) or derivative thereof may be at least partly due to the formation of a reaction product which itself has antimicrobial and/or anti-acne activity. The invention may thus embrace an antimicrobial and/or anti-acne formulation containing a reaction product formed between a DAS or derivative thereof and a compound (I) or derivative thereof, in particular between a DOS such as dioctyl sodium suiphosuccinate and AO 2246; this reaction product may be formed in situ immediately prior to, or at the point of, use.

Antimicrobial activity may in particular be antifungal and/or antibacterial activity, more particularly antibacterial activity. Antibacterial activity encompasses activity against both Gram-positive and Gram-negative bacteria. Antimicrobial activity may be growth inhibitory activity or more preferably biocidal (ie lethal to the relevant organism). It may comprise activity against sessile and/or planktonic micro-organisms, in particular bacteria. It may be or include the ability to disrupt and/or suppress biofllm formation by the relevant organism. In the context of this invention, activity against a particular species of micro-organism may be taken to mean activity against at least one, preferably two or more, strains of that species.

In the present context, the disruption of biofilm formation embraces any negative effect on the ability of an organsim to form, maintain or exist in a biofllm, and/or on a bioflim already formed by the organism. Thus, it may involve reducing the amount of a previously formed bioflim, and/or impairing such a bioflim. Tt may involve killing or inhibiting sessile micro-organisms, in particular bacteria, within a biofilm.

Suppression of biofilm formation embraces any degree of impairment (including complete prevention) of the ability of an organism to form, or more typically to co-aggregate with, a biofilm. It thus embraces total or partial impairment, including reducing the amount and/or strength of biofilm which the organism is able to form and/or the speed with which it is able to do so. It may involve preventing or reducing the growth or the rate of growth of an existing biofilm formed by the organism.

An antimicrobial formulation according to the present invention may in particular be active against one or more of the bacteria referred to herein.

The formulation is preferably active against micro-organisms, in particular propionibacteria, which are wholly or partially resistant to one or more antibiotics, for instance those which are in common clinical use. For example it is ideally active against one or more macrolide-lincosamide-streptogramin (MLS) resistant and/or macrolide-lincosamide-streptogramin-ketolide (MLSK) resistant strains of bacteria. In particular it may be active against one or more erythromycin-resistant, clindamycin-resistant and/or tetracycline-resistant strains of bacteria, for example P. acnes strains, the term tetracycline here referring to the class of antibiotics including for example minocycline and doxycycline as well as the specific antibiotic known as tetracycline.

Preferably the DAS or derivative is antimicrobially active, at least against propionibacteria, in the presence of at least one of, preferably both of, lipid and salt (sodium chloride) -these are species which can be present at the surface of the skin and hence performance in this context can be indicative of suitability for use in topical skin treatment formulations. The same comments apply to the compound (I) or derivative.

The concentration of the DAS or derivative in a formulation according to the invention may be 0.05 or 0.1 or 0.2 or 0.5% w/v or greater, or 1 or 1.5% w/v or greater, or 2 or 3 or 3.5 or 4% w/v or greater. Its concentration may be up to 15% w/v, or up to 10 or 9 or 8 or 7 or 6 or 5% w/v. For example, its concentration might be in the range from 0.1 to 10% w/v, or from 1 or 2 or 3 or 3.5 to 10% w/v or from 2 or 3 or 3.5 to 8% w/v, or from 3 or 3.5 to 7% w/v or from 4 to 6% w/v, such as about 5% w/v. In particular when the formulation is for use against a skin or skin structure condition such as acne, the concentration of the DAS or derivative may for example be from 1 to 10% w/v, or from 2 or 3 or 3.5 to 8% w/v. In cases the concentration may be 3.5% w/w or greater, or 4 or 4.5 or 5% w/w or greater.

The concentration of the compound (I) or derivative in the formulation may be 0.1 or 0.2% w/v or greater, or O.S% w/v or greater. Its concentration may be up to 5% w/v, or up to 2% w/v, such as from 0.5 to 2% w/v or from 1 to 2% w/v, for example about 1.75% w/v. Tn cases, for instance when the formulation is for use in the disinfection of a tissue surface such as the skin, the concentration of the compound (T) or derivative may be for example from 0.5 to 5% w/v or from 1 or 2 to 5% w/v.

The weight ratio of the DAS or derivative in the formulation to the compound (I) or derivative may be up to 50:1, for example up to 30:1, or up to 25:1 or 20:1 or 15:1 or 10:1. In cases it may be up to 5:1 or 2.5:1 or 2:1. This ratio may be 1:20 or greater, for example 1:10 or 1:5 or greater. In cases, in particular when the formulation is intended for application to the skin, the ratio may be 1:1 or greater, or 2:1 or 2.5:1 or greater; it may for instance be from 1:1 to 5:1 orfrom2:1 to 3:1.

A formulation according to the invention may be in non-solid form; it may thus take the form of a fluid. A fluid may for example be a lotion, cream, ointment, varnish, foam, paste, gel or other viscous or semi-viscous fluid, or a less viscous fluid such as might be used in sprays or drops. The formulation may take the form of a solution or suspension, for instance for use as a disinfectant. It may in particular take the form of a lotion, cream, ointment, varnish, foam, paste or gel, more particularly a gel. It may comprise a formulation which is, or may be, applied to a carrier such as a sponge, swab, pad, brush, tissue, cloth, wipe, skin patch, skin adhesive or other material designed for application to a tissue surface, to facilitate its topical administration. It may be intended for pharmaceutical (which includes veterinary but is preferably human) use, and/or for cosmetic or other non-medical care purposes. It may be usable to cleanse hair or nails so as to reduce microbial levels.

The vehicle in which the DAS or derivative and the compound (T) or derivative are contained may be any vehicle or mixture of vehicles, and is ideally suitable for topical application; the type chosen will depend on the intended mode and site of application.

Many such vehicles are known and commercially available. In the context of formulations for topical application to the skin, examples may for instance be found in Williams' Transdermal and Topical Drug Delivery, Pharmaceutical Press, 2003 and other similar reference books. See also Date, AA et al, Skin Pharmacol Physiol, 2006, 19(1): 2-16 for a review of topical drug delivery strategies.

The DAS or derivative and the compound () or derivative may each independently be present in the form of a solution or suspension, the term "suspension" including emulsions, micellar systems and other multi-phase dispersions. Either or both of the DAS or derivative and the compound (T) or derivative may, whether separately or together, be carried in or on a delivery vehicle which is suitable for targeting or controlling its release at an intended site and/or time of administration. Such a vehicle may for instance target the skin or hair follicles. It may delay or otherwise control release of the formulation over a particular time period. Such vehicles include liposomes and other encapsulating entities, for example niosomes, aspasomes, cubosomes, microsponges, microemulsions, hydrogels and solid lipid nanoparticles.

Particularly suitable liposomes, for topical application to the skin, are those made from stratum corneum lipids, eg ceramides, fatty acids or cholesterol.

In an embodiment of the invention, the formulation containing the DAS or derivative and the compound (I) or derivative also contains: (i) a polyoxyalkylene-based solubilising agent; (ii) an organic solvent; and (iii) a thickening agent. It will typically also contain (iv) water. It has surprisingly been found that this particular combination of ingredients can yield a stable formulation even at relatively high concentrations of the DAS or derivative (for example 3.5 or 4 or 4.5 or 5% w/v or greater).

In an embodiment, the formulation is in the form of a gel. It may for example have a viscosity of from 25,000 to 300,000 cps, or from 50,000 to 200,000 cps.

In a formulation of the type defined above, the solubilising agent (i) is polyoxyalkylene-based, for example polyoxyethylene (polyethylene glycol, PEG)-based. Tt may comprise a derivative of a polyoxyalkylene, for example an ether or more particularly an ester. It may for example be selected from (a) polyalkoxylated esters, other than polyalkoxylated sorbitan esters and (b) polyalkoxylated alkyl ethers.

More particularly it may be selected from (a) polyethoxylated esters, other than polyethoxylated sorbitan esters and (b) polyethoxylated alkyl ethers. It is suitably nomornc.

A polyalkoxylated ester may be a polyalkoxylated fatty acid ester. The fatty acid component may be a Cio to C20 group or a C12 to C18 group, for example stearic acid (C18) or 12-hydroxystearic acid. It may in particular be a hydroxyl-substituted fatty acid group. Suitable such solubilising agents are commercially available as MyrjTM 45 (PEG 400-monostearate) and Solutol� HS 15 (PEG-15-hydroxystearate, also known as PEG 660 12-hydroxystearate and Macrogol-15-hydroxystearate).

Solutol� HS 15 is a nonionic solubilising agent available from BASF AG. It consists primarily of polyglycol mono-and di-esters of 1 2-hydroxystearic acid (the lipophilic part of the molecule) and of about 30% of free polyethylene glycol (the hydrophilic part). It is prepared by reacting 15 moles of ethylene oxide with one mole of 12-hydroxystearic acid.

A polyalkoxylated ester may be a polyalkoxylated glyceryl ester (also known as a polyalkoxylated glyceride), which is a glyceride ester of a polyalkylene glycol (typically PEG). II may contain mono-, di-or tri-glycerides, partial glycerides or mixtures thereof. It may for example be formed by esterification of a polya[kylene glycol with a glyceride oil of an appropriate chain length. The glyceride components may for example contain from 6 to 20 or from 8 to 18 carbon atoms. Suitable such solubilising agents are commercially available as GlyceroxTM and LabrasolTM.

GlyceroxTM esters, for example, are PEG-based products which are commercially available from Croda Inc in a number of grades reflecting the molecular weight of the PEG, for example PEG-6, PEG-7, PEG-8 (L8) and PEG-iS (L15). Of these, the PEG- 6 version may be preferred. GlyceroxTM esters are sold for use as emollients, for example in bath products, skin cleansers and shampoos, and as solubilisers, superfatting agents, dispersing agents and emulsifiers.

A glyceride solubilising agent may for example be GlyceroxTM 767 (polyoxyethylene (6) glyceryl monocaprylate/caprate, a mixture of caprylic and capric glycerides of PEG-6) or GlyceroxTM HE, which is a PEG-7 glyceryl cocoate.

A polyalkoxylated alkyl ether may be a polyalkoxylated ether of a fatty alcohol such as a C12 to C20 or C14 to C20 alcohol, for example cetyl (C16), stearyl (C18) or oleyl (C18:i).

It may in particular be a polyethoxylated alkyl ether, also known as a macrogol ether.

Suitable such solubilising agents are commercially available as BrijTM 97 (PEG-monooleyl ether) and CremophorTM A 25 (a mixture of stearic and cetyl ethers).

The solubilising agent (i) may in particular be a polyethoxylated ester, more particularly a fatty acid ester or a polyethoxylated glyceride. Solutol� HS 15, LabrasolTM and GlyceroxTM 767, in particular the first two, have been found to be suitable solubilising agents for use in the present invention. In general, the polyalkylene glycol element of a solubilising agent used in the present invention may have any appropriate molecular weight.

The organic solvent (ii) is suitably an alcohol, for example methanol, ethanol, isopropanol or phenoxyethanol. It may be selected from methanol, ethanol, isopropanol and mixtures thereof, preferably from methanol, ethanol and mixtures thereof. Tn an embodiment of the invention the organic solvent is ethanol.

The thickening agent (iii) may be any thickening agent suitable for topical application.

It is suitably a gelling agent. It may in particular be a cellulose-based thickening agent such as ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose or a carboxymethyl cellulose. Again such agents may be used in the form of a (preferably pharmaceutically acceptable) salt such as for instance the sodium salt. The thickening agent may be a polymeric thickening agent such as a carbomer, which will typically be a homopolymer of acrylic acid, cross-linked with an allyl ether. A preferred thickening agent is hydroxyethyl cellulose.

The solubitising agent (i) may be present at a concentration of 10% w/w or greater, or 15% w/w or greater. It may be present at a concentration of up to 50% w/w, or up to or 30 or 25% w/w. its concentration may for example be from 10 to 30% w/w or from 15 to 25% w/w, such as about 20% w/w. The organic solvent (ii) may be present at a concentration of 10% w/w or greater, or 15 or 17% w/w or greater. It may be present at a concentration of up to 30 % w/w, or up to 25 % w/w. Its concentration may for example be from 10 to 30% w/w or from 15 to 25 or 30% w/w, such as about 20% w/w. The thickening agent (iii) may be present at a concentration of 0.2% w/w or greater, or 0.5% w/w or greater. It may be present at a concentration of up to 5% w/w, or up to 3% w/w. Its concentration may for example be from 0.5 to 5% w/w or from 0.5 to 3% w/w, such as about 1% w/w.

Generally speaking a formulation according to the invention may contain standard excipients and other additives known for use in pharmaceutical or veterinary formulations. Tn particular when intended for application to the skin, it may further contain additional active agents such as antimicrobial (in particular antibacterial or antifungal, more particularly antibacterial) agents. For example, it may contain one or more agents selected from anti-acne agents, keratolytics, comedolytics, agents capable of normalising keratinocyte and/or sebocyte function, anti-inflammatories, anti- proliferatives, antibiotics, anti-androgens, sebostatic!sebosuppressive agents, anti-pruritics, immunomodulators, agents which promote wound heating, additional antimicrobiat agents and mixtures thereof it may instead or in addition contain one or more agents selected from sunscreens, moisturisers, emollients and mixtures thereof An additional antimicrobial agent may for example be selected from the group consisting of biocides, disinfectants, antiseptics, antibiotics, bacteriophages, enzymes, anti-adhesins, immunoglobulins and mixtures thereof it is preferably active as a bactericide, in particular against propionibacteria. It may however be preferred for the DAS or derivative and the compound (I) or derivative to be the only active agents in the formulation, or at least to be the onty antimicrobially, antibacterially or antifungally active agents and/or the onty anti-acne active agents.

A formutation according to the invention may be incorporated into, and hence apptied in the form of, another product such as a cosmetic, a skin or hair care preparation (for example a skin cleanser, toner or moisturiser, or a shampoo, conditioner, styling mousse or get or hair spray), a deodorant or anti-perspirant, a cleansing preparation (for example a hand wash for use by surgeons prior to treating patients), a pharmaceutical (which includes veterinary) preparation, a cosmeceutical preparation, a toiletry product (for instance a bath or shower additive or a soap), or a laundry or other fabric treatment product. The invention thus provides, according to a second aspect, a product which incorporates an antimicrobial and/or anti-acne formulation according to the first aspect.

A formulation according to the invention may be marketed with an indication that it has antimicrobial or anti-acne activity, or enhanced antimicrobial or anti-acne activity, for example against one or more of the pathogens referred to herein. The marketing of such a formulation may for example include an activity selected from (a) enclosing the formulation in a container or package that comprises the relevant indication; (b) packaging the formulation with a package insert that comprises the indication; (c) providing the indication in a publication that describes the formulation; and (d) is providing the indication in a commercial which is aired for instance on the radio, television or internet. The antimicrobial or anti-acne activity of the formulation may be attributed, in such an indication, at least partly to the presence of either or both of the DAS or derivative and the compound (I) or derivative, in particular to the presence of the compound (I) or derivative. The antimicrobial or anti-acne activity of the formulation may be assessed during or after its preparation, for instance against one or more of the pathogens referred to herein. The activity may be assessed both before and after incorporation of the DAS or derivative and/or the compound () or derivative, for example so as to confirm that either or preferably both contribute to the relevant activity.

The formulation of the invention may be prepared in situ, at or immediately before its point of use, for instance its application to the skin or another surface. Thus according to a third aspect, the present invention provides a kit for preparing an antimicrobial or anti-acne formulation, such as a formulation according to the first aspect, the kit comprising a source of a DAS or derivative thereof and a source of a compound of formula (I) or derivative thereof, together with instructions for combining the two compounds so as to make the formulation at or before the point of intended use, and/or for the co-administration of the two compounds to a surface such as the skin.

According to one embodiment, the formulation or kit of the invention may contain both a DAS or derivative thereof and a compound of formula (I) or derivative thereof, each encapsulated (for instance microencapsulated) in a separate delivery vehicle; this might for instance allow their release, and hence their contact with one another, only at the intended site of administration.

A fourth aspect of the invention provides a method for preparing an antimicrobial or anti-acne formulation, which method involves mixing together a DAS or derivative thereof and a compound of formula (II) or derivative thereof, preferably together with a pharmaceutically acceptable vehicle.

According to a fifth aspect of the invention there is provided a formulation according to the first aspect, for use in the treatment of a condition affecting the human or animal body, which condition is caused by, transmitted by and/or exacerbated by (in particular either caused or transmitted by) microbial activity. The activity may in particular be bacterial activity, more particularly propionibacterial activity. The condition may be for example a skin or skin structure condition such as acne.

In the context of the present invention, treatment of a condition encompasses both therapeutic and prophylactic treatment, of either an infectious or a non-infectious condition, in either a human or animal but in particular a human. It may involve complete or partial eradication of the condition, removal or amelioration of associated symptoms, arresting subsequent development of the condition, and/or prevention of, or reduction of risk of, subsequent occurrence of the condition. Tt will typically involve use of the DAS or derivative and the compound (I) or derivative as a biocidal (in particular bactericidal) or anti-acne combination. It may involve the prophylactic treatment of any area of the body, in particular the skin, against microbial infections.

The treatment of a condition also embraces the prevention, or reduction of risk of, dissemination or transmission of the condition, for example from person to person. It may involve use of the formulation against microbial biofilm formation. Thus, the formulation may be used to treat a condition which is caused, transmitted and/or exacerbated by (in particular caused or transmitted by) microbial biofllm formation.

In an embodiment of the fifth aspect of the invention, the formulation is for use against one or more micro-organisms, in particular bacteria, associated with skin or skin-borne infections. Tt may be for use against Gram-positive bacteria, for example staphylococci and/or propionibacteria, in particular against strains of Propionibacterium acnes. In an embodiment, it is for use against one or more bacteria associated with acne, such as P. acnes and in some instances P. granulosum.

Thus according to an embodiment of the fifth aspect of the invention, the formulation is for use in the treatment of a skin or skin structure condition. Such a condition may be a primary or secondary infection. It may for example be a superficial or uncomplicated skin infection amenable to local therapy. It may be acne or an infection associated with acne. It may be a primary or secondary infection due to S. aureus (including MRSA) or a group A beta haemolytic streptococcus (S. pyogenes), Skin and skin structure conditions which might be treated according to the invention include acne, infected atopic eczema, superficial infected traumatic lesions, wounds, bums, ulcers, folliculitis, mycoses and other primary and secondary skin and skin structure infections. In particular the invented formulation may be for use in treating acne or acne lesions (for instance, to reduce acne-related scarring).

Acne is a multifactorial disease of the pilosebaceous follicles of the face and upper trunk, characterised by a variety of inflamed and non-inflamed lesions such as papules, pustules, nodules and open and closed comedones. Its treatment can therefore encompass the treatment (which embraces prevention or reduction) of any of these symptoms, and references to use as an anti-acne agent may be construed accordingly.

In particular, the treatment of acne encompasses the treatment (including prevention) of lesions and/or scarring associated with acne. It also encompasses the treatment of a propionibacterial infection and/or the inhibition of propionibacterial activity which could cause or be otherwise associated with acne or its symptoms. In general, the present invention will be used for the treatment of symptoms which are directly due to acne rather than for instance infections which may arise as a consequence of treating acne with other actives such as antibiotics, and/or secondary infections caused by opportunistic pathogens, which can arise in skin already affected by acne.

Thus, in general terms the fifth aspect of the invention can provide a formulation containing a DAS or derivative and a compound of formula (I) or derivative thereof, for use in the treatment of acne (ie as an anti-acne agent). A potential advantage to the use of a compound (I) or derivative to treat acne and related infections can be that such compounds, as ACAT inhibitors, can be capable of inhibiting sebum secretion (see EP- 0 699 439). Given the multifactorial nature of acne, a compound capable of targeting two different aspects of its pathogenesis can offer considerable advantages over one that targets only a single mechanism.

In an embodiment, the invented formulation may be for use in the disinfection of skin or other tissue surfaces. Moreover, as described below, it may be used for the disinfection of non-living areas and surfaces. It may in particular be used to counter micro-organisms of the type referred to above.

In certain cases, the invented formulation is not for use in the treatment of a polymicrobial or mixed infection, including in cases an infection which involves a Gram-negative bacterium and/or an infection which involves a fungus. In certain cases, it is not for use against a Gram-negative bacterium. In certain cases, it is not for use against a staphylococcal bacterium, in particular S. aureus.

According to the fifth aspect of the invention, the formulation of DAS or derivative and compound (I) or derivative may be prepared in situ, at or immediately before the point of administration. This aspect of the invention thus pertains to any use of a DAS or pharmaceutically acceptable derivative thereof and a compound of formula (I) or pharmaceutically acceptable derivative thereof in the treatment of a microbial condition, including acne, the two compounds being administered either simultaneously or sequentially.

A sixth aspect of the invention provides a formulation according to the first aspect, for use in the treatment of a sebaceous gland disorder, including a condition which is caused or exacerbated by (in particular caused by) sebaceous gland secretions, for example acne. The formulation may be for use in the inhibition of sebum production, and/or in the inhibition of sebaceous gland secretion. In this context -as indeed for its other uses -the formulation may be administered orally, transdermally or topically, preferably topically.

According to a seventh aspect, the invention provides the use of a formulation according to the first aspect, in the manufacture of a medicament (typically a formulation) for the treatment of a condition which is caused by, transmitted by and/or exacerbated by (in particular either caused or transmitted by) microbial activity. The activity may be bacterial or fungal, in particular bacterial. The condition may in particular be acne. The DAS or derivative and the compound (I) or derivative will typically be used as an antimicrobial and/or anti-acne combination in the manufacture of the medicament.

An eighth aspect provides the use of a formulation according to the first aspect, in the manufacture of a medicament (typically a formulation) for the treatment of a sebaceous gland disorder, including a condition which is caused or exacerbated by (in particular caused by) sebaceous gland secretions, for example acne. Again the medicament may be for use in the inhibition of sebum production, and/or in the inhibition of sebaceous gland secretion.

The invention further provides, according to a ninth aspect, the use together of a DAS or derivative thereof and a compound of formula (I) or derivative thereof, as a combined antimicrobial agent, or as a combined anti-acne agent, in the manufacture of an antimicrobial or anti-acne formulation.

A tenth aspect provides the use of a formulation according to the first aspect for non-therapeutic purposes. In an embodiment of this aspect, the formulation is used as an anti-acne or skin care agent for non-therapeutic purposes, for example for general hygiene or skin cleansing or for cosmetic purposes such as to improve the appearance, feel or smell of the skin.

An eleventh aspect provides a method for controlling the growth of a micro-organism, in particular a bacterium, the method comprising applying, to an area or surface which is infected or suspected to be infected or capable of becoming infected with the organism, a formulation according to the first aspect of the invention. Again the DAS or derivative and the compound (I) or derivative may be applied simultaneously or sequentially. They may in particular be applied to an area or surface which is infected with the relevant organism.

In this context, "controlling the growth" of a micro-organism embraces inhibiting or preventing its growth, whether completely or partially, as well as killing either completely or partially a culture of the organism. It also embraces reducing the risk of subsequent growth of the organism in or on the area or surface being treated. It may embrace reducing the risk of transmission of the organism from the area or surface being treated to another area or surface and/or living body. The method of the invention may thus be used to treat an existing occurrence of the organism or to prevent a potential subsequent occurrence. Controlling the growth of a micro-organism may also embrace the disruption and/or suppression of bioflim formation by the organism, as described above.

The method of the eleventh aspect encompasses a method of treatment of a human or animal patient suffering from or at risk of suffering from a condition which is caused by, transmitted by and/or exacerbated by (in particular either caused or transmitted by) microbial, in particular bacterial, activity, the method involving administering to the patient a therapeutically (which term includes prophylactically) effective amount of an antimicrobial formulation according to the first aspect. The microbial condition may be any of those referred to above in connection with the first to the ninth aspects of the invention. The formulation is suitably administered in an antimicrobially effective amount. Tt may be administered topically.

A twelfth aspect provides a method of treatment of a patient suffering from or at risk of suffering from a sebaceous gland disorder, the method involving administering to the patient a therapeutically (which term includes prophylactically) effective amount of a formulation according to the first aspect.

In accordance with the eleventh and twelfth aspects of the invention, the formulation is suitably administered to a human patient. The patient is suitably suffering from the relevant condition, in particular acne.

A thirteenth aspect of the invention provides the use of a DAS or derivative thereof in an antimicrobial or anti-acne formulation, in combination with a compound of formula (I) or derivative thereof, for the purpose of increasing the antimicrobial and/or anti-acne activity of the formulation and/or of reducing the amount of the compound (I) or derivative in the formulation without or without undue loss of antimicrobial or anti-acne activity.

An increase in antimicrobial or anti-acne activity may be as compared to that of the compound (I) or derivative alone, at the same concentration as used when combined with the DAS or derivative. Ideally the increase is as compared to the sum of the activities of the DAS or derivative and the compound (I) or derivative individually, again at the same respective concentrations as used when the two are combined. A reduction in the amount of the compound () or derivative in the formulation may be as compared to the amount which would otherwise have been used in the formulation in order to achieve a desired level of activity, in particular in order to have acceptable efficacy in the context of its intended use. The reduction may be manifested by reduced side effects which would otherwise have been observed during use of the formulation, for example local irritation and/or undesirable systemic absorption of the compound (I) or derivative. According to the invention, the DAS or derivative may therefore be used for the dual purposes of reducing an undesired property of a formulation containing a compound of formula (I) or derivative thereof, without or without undue loss of antimicrobial or anti-acne activity. Preferably the DAS or derivative is used without any reduction in antimicrobial or anti-acne activity compared to the level exhibited by the formulation prior to addition of the DAS or derivative. More preferably it is used to give an increase in antimicrobial or anti-acne activity. It may however be used to reduce the amount of the compound (I) or derivative present, and/or its associated side effects, whilst maintaining the antimicrobial or anti-acne activity of the resultant formulation at a level, albeit lower than that which it would otherwise have exhibited, which is still acceptable in the context of its intended use.

A fourteenth aspect of the invention provides the use of a compound of formula (I) or or derivative thereof in an antimicrobial or anti-acne formulation, in combination with a DAS or derivative thereof, for the purpose of increasing the antimicrobial and/or anti-acne activity of the fonnulation and/or of reducing the amount of the DAS or derivative in the formulation without or without undue loss of antimicrobial or anti-acne activity. The above comments regarding the thirteenth aspect of the invention apply mutatis inutandis to the fourteenth aspect.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and do not exclude other moieties, additives, components, integers or steps. Moreover the singular encompasses the plural unless the context otherwise requires: in particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Other features of the invention will become apparent from the following examples. Generally speaking the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims and drawings). Thus features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. Moreover unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

The present invention will now be further described with reference to the following non-limiting examples.

Detailed description

Experimental tests were conducted to determine the antimicrobial and/or anti-acne activity of formulations according to the invention.

Test micro-organisms The principal test micro-organism used was Propionibacteriuin acnes NCTC 737.

This is a propionibacterial strain and is the type strain of the genus; it is fully susceptible to antibiotics. The propionibacteria are clinically significant due to their involvement in acne, in which P. acnes and other Propionibacteriuin spp. (for example P. granulosum) play key roles. They are also opportunistic pathogens in compromised hosts. Thus, activity observed against these micro-organisms is expected to be a good predictor of activity against acne. Other propionibacterial strains were also tested, as described in Example 2. These included certain antibiotic-resistant propionibacteria.

In addition, a strain of P. granulosum, another bacterium involved in acne, was also tested in Example 2. The propionibacteria were cultured and maintained on Wilkins-Chalgren Anaerobe Medium (agar and broth) at pH 6.0; all cultures were incubated anaerobically at 37°C for 72 hours.

The staphylococcal test strain used was Staphylococcus aureus ATCC 29213.

S. aureus and other staphylococci are common causes of a wide range of skin, skin structure and wound infections; S. aureus is also known to exacerbate eczema. The ATCC 29213 strain is known to be susceptible to beta-lactam antibiotics such as methicillin. Activity observed against this micro-organism and the P. acnes strain is expected to be a reasonable qualitative predictor of activity against micro-organisms responsible for skin and skin structure infections. The staphylococci were cultured and maintained on Mueller-Hinton Medium (agar and broth) at pH 7.2; cultures were incubated aerobically at 37°C for 19-20 hours.

Two further test microbial species were used to demonstrate the suitability of formulations according to the invention for the treatment of conditions affecting the oral cavity. The first was Porphyromonas gin givalis, a Gram-negative anaerobic bacterium which is typically associated with periodontal lesions and infections, gingivitis and adult periodontal disease. Activity observed against this micro- organism is expected to be a reasonable qualitative predictor of activity against micro-organisms responsible for periodontal lesions and infections and periodontal disease.

Tests were performed with three different strains of P. gingivalis, namely NCTC 11834, ATCC BAA-308 and ATCC 53978.

Also tested was Streptococcus inutans -this is a Gram-positive, microaerophilic bacterium associated principally with the human oral cavity. Clinically, S. mutans plays a significant role in dental caries and in infective endocarditis. if the bacterium enters the bloodstream, for example after a tooth extraction, it can bind to the endocardium within the heart and if left untreated can prove fatal. Activity observed against this micro-organism is thus expected to be a reasonable qualitative predictor of activity against micro-organisms responsible for dental caries and infective endocarditis. Tests were performed on two different strains of S. mutans, namely ATCC 25175 and ATCC 35668. Tests were also performed on S. sobrinus ATCC 33478 (see below), another bacterium implicated in dental caries.

The P. gin givalis strains were cultured and maintained on Wilkins-Chalgren Anaerobe Medium (agar and broth) at pH 7.0; all cultures were incubated anaerobically at 37°C for 5-7 days. The S. inutans strains were cultured and maintained on Wilkins-Chalgren Anaerobe Medium (agar and broth) at pH 7.0 supplemented with 1 gIL glucose; all cultures were incubated at 37°C for 48 h in an atmosphere containing 5% Co2.

The following test organisms were also used: Test organism Grain ___________ Culture conditions ______ test Growth Temp Atmosphere Time media (°C) (h) Actinornyces naeslundii + WC pH7 37 5% CO2 24 ATCC 12104 Bacillus cereus ATCC + MH 30 Aerobic 24 Clostridiun, dfJIcile ATCC + WC pH7* 37 Anaerobic 48 Clostridiurn sporo genes + WC pH7 37 Anaerobic 24 ATCC 3584 Con nebacteriurn + MH 37 Aerobic 24 rnucifaciens ATCC 700355 Corynebacteriurn stria turn + MH 37 Aerobic 24 NCTC 764 Enterococcusfaecalis ATCC + MH 37 Aerobic 24 Gardnerella vaginalis -MH + 5% 37 5% CO2 48 Test organism Grain ___________ Culture conditions ______ test Growth Temp Atmosphere Time media (°C) (h) ATCC 14018 lysed blood (broth) WC pH7 ____________________________ _______ (agar) ________ ____________ ______ Listeria nionocytogenes ATCC 15313 + MH 37 Aerobic 24 Prevotella nigrescens ATCC 33563 -WCpH7 37 Anaerobic 48 Streptococcus pyogenes + 2 0 4 ATCC 12344 MH 5/0 CO2 WCpH7+ Streptococcus sobrinus + I gIL 37 5% Co2 48 glucose [Abbreviations: American Type Culture Collection (ATCC), Mueller-Hinton (MH), Wilkins-Chalgren (WC), * denotes where agar medium was needed to maintain the organism 5% fresh horse blood was added to the media] Of the above pathogens, Bacillus cereus is associated with food poisoning; Enterococcusfitecalis and Listeria nionocyto genes are both food-borne pathogens, the former being implicated in burn and wound infections. Clostridium sporogenes is associated with food spoilage, whilst Clostridium dfficiIe may also be spread by contaminated food. Corvnebacterium mucifliciens and Corynebacteriuin striatum are closely related to organisms (aerobic diphtheroids of the genus Coiynebacterium) that cause body odour. Prevotella nigrescens and Actinomyces naeslundii are implicated in periodontitis and other anaerobic infections, and Streptococcus sobrinus in dental caries. Streptococcus pyogenes is associated with skin and soft tissue infections.

Gardnerella vaginalis can cause vaginitis, vaginosis and certain sexually transmitted infections.

The following tests were carried out to assess antimicrobial activity against the test organisms.

(a) Minimum inhibitory concentration MIC) assay This is a standard international method for quantitatively assessing the antimicrobial activity of a compound in a liquid medium. The method used a sterile 96-well microtitre plate, capable of holding about 200 pi of liquid per well. The wells contained liquid culture medium and ranges of decreasing concentrations of the relevant test compound in doubling dilutions (eg 1000, 500, 250, 125.. .tg/ml, etc down to 0.49 jig/ml). The culture media were as described above. The wells were inoculated with a liquid suspension of freshly grown micro-organism and incubated under the conditions described above. After incubation, the microtitre plate was examined visually (with the aid of a light box) for cloudiness in each well, which would indicate microbial growth. The MIC value was recorded as the lowest concentration of test compound required to inhibit microbial growth, ie the lowest concentration for which the liquid in the well remained clear. The assays included both negative (culture medium with no micro-organisms) and positive (culture medium plus diluting solvent plus micro-organism) controls.

Since inhibition does not necessarily indicate killing of microbial cells, merely that growth as visible to the naked eye has been inhibited, it is desirable to conduct a further test (the MBC assay described below) to establish the concentration of the test compound needed to kill the test organism.

(b) Minimum biocidal (bactericidal) concentration (MBC) assay This assay, normally carried out after an MIC assay, determines the minimum concentration of a compound that is lethal to the micro-organism being tested.

Following an M1IC assay, a 5 tl sample was withdrawn from the first microtitre well that showed positive growth and from all the subsequent wells that showed no growth.

These samples were then individually sub-cultured on antibiotic-free agar medium, under the incubation conditions described above. Following incubation they were examined visually for microbial growth. The MBC was taken to be the lowest test compound concentration for which the incubated sample showed no growth.

The ratio of MIC to MBC should ideally be as close to 1 as possible. This facilitates selection of the lowest possible effective concentration of a test compound with a reduced risk of selecting a sub-lethal concentration which could promote resistance or allow the target microbial population to recover.

(c) Disc diffusion assay (DDA) This is an internationally recognised standard method for qualitatively assessing the antimicrobial activity of a compound or combination of compounds. A sterile paper disc was impregnated with a sample of the test compound(s) in a suitable solvent and minutes allowed for the solvents to evaporate (where possible). The disc was then placed on an agar plate onto which the test micro-organism had been inoculated. The plate was then incubated under the conditions described above, following which it was examined visually for signs of microbial growth. If the test compound(s) had antimicrobial activity, a circular zone of no growth would be obtained around the disc.

The diameter of this zone of "inhibition" was measured using a Pr0t0COLTM automated zone sizer (Synbiosis, Cambridge, UK). In general, a greater diameter and/or area of the zone of inhibition indicates a greater antimicrobial activity in the relevant test compound(s), although other factors such as test compound mobility through the agar gel may also influence the result.

(d) Synergy disc diffusion assay (SDDA) This is a variation on the DDA method, in which two compounds are tested together for their combined antimicrobial activity. Two test compounds A and B were placed on a single paper disc and the above described DDA procedure repeated. An increase in diameter of the zone of inhibition, compared to the greater of the zone diameters for the two compounds individually, was taken to indicate potential antimicrobial synergy.

Example I activity against P. acnes (MIc, MBC & (S)DDAs) The following experiments all used P. acnes NCTC 737 as the test organism. MIC and MBC assays, as described above, were carried out using as the test compounds (a) the sodium salt of dioctyl sulphosuccinate (DOSS), dissolved in ethanol and (b) AO 2246, dissolved in DMSO. Both compounds were sourced from Sigma Aldrich, UK.

The two test compounds were then subjected to disc diffusion assays, again as described above, both alone and in combination. For these experiments, 50 tg of the DOSS was loaded onto each disc, and 200 tg of the AO 2246. MIC/MBC experiments were conducted either in duplicatet or in triplicate. (S)DDA experiments were conducted in triplicate. The results are shown in Table 1 below; all are collated from a number of experiments.

Table 1

Assay DOSS A02246 DOSS +A02246 MIC (tg/in1) l5.6 3.9 - MBC (jig/mi) 62.5 15.6 - MIC/MBC ratio 0.25 0.25 - (S)DDA (mm) 23.87 (� 6.36) 9.03 (� 0.62) 41.40 (� 1.36) SDDA increase (mm) --17.54 SDDA area increase (%) --200.95 It can be seen from Table I that the invented combination of a DAS and a compound of formula (I) is highly active as an antibacterial agent against P. acnes NCTC 737.

Furthermore, the data indicate a synergistic antibacterial interaction between the two compounds, the combination showing a significant increase in zone diameter over that exhibited by either compound alone. This indicates the likely activity of such combinations as anti-acne agents, the propionibacteria being implicated in acne.

Exami'le 2 -activity against other ropionibacteria The (S)DDAs of Example 1 were repeated against three other Propionibacterium spp strains, including two with known antibiotic resistance. 50 tg of the DOSS and 200 tg of the AO 2246 were loaded onto each disc. The experiments were conducted in triplicate. The results are shown in Table 2 below; all are collated from a number of experiments. The table also indicates the resistance phenotype for each of the test strains.

Table 2

DDA (S)DDA (S)DDA (S)DDA DDA DOSS + increase area Resistance AO Test organism DOSS AO (mm) increase phenotype (mum) 2 2246 (%) (miii) P. granulosumNCTCll86S None 13.07 10.89 48.66 128549 _________________________ _________ (�0.82) (�1.65) (�0.72) ________ ________ P. acnes PRF-002 Tet/MLS 34.03 0.0 41.82 7 78 50 96 _________________________ _________ (�1.26) (�0.0) (�5.98) ________ ________ P. acnesPRP-010 MLSK 10.17 0.0 39.64 2947 14194 _________________________ _________ (�0.36) (�0.0) (�10.16) ________ ________ [Abbreviations: National Collection of Type Cultures (NCTC), Propionibacterium Panel Number (PRP), Tetracycline (Tet), Macrolide-Lincosamide-Streptogramin (MLS), Macrolide-Lincosamide-Streptogramin-Ketolide (MLSK).] The invented combination of compounds can be seen to possess an excellent level of activity against all three of the propionibacterial strains tested. Again the SDDA data demonstrate a synergistic antimicrobial interaction between the two compounds. This indicates the utility of the combination to treat (including to prevent) infections associated with such bacteria, in particular acne. The results are likely to be of particular clinical value for the antibiotic resistant test strains.

Example 3 -activity against other micro-oranisms The Example 1 (S)DDAs were repeated, for DOSS and AO 2246, against a panel of further test micro-organisms. These included S. aureus, Strep. mittans and P. gingivalis. 200 tg of the DOSS and 400 tg of the AO 2246 were loaded onto each disc. The experiments were conducted in triplicate. The results are shown in Table 3 below; all are collated from a number of experiments.

Table 3

DDA (S)DDA (S)DDA (S)DDA DDA AO DOSS + increase area Test organism DOSS 2246 A 0 (mum) increase (mm) / 2246 (%) muzm, (mm) S.aureusATCC292l3 11.42 0.0 15.13 371 7553 __________________________ (�0.2) (�0.0) (�0.29) ________ ________ Strep. mutansATCC 25175 28.42 0.0 33.38 496 3795 __________________________ (�0.41) (�0.0) (�0.75) ________ ________ P. gingivalisNCTC 11834 48.17 9.92 50.08 1 91 8 09 _________________________ (�0.57) (�0.27) (�0.31) ________ ________ It can be seen from Table 3 that the invented combination is highly active against all three test organisms, and is therefore active against both Gram-positive and Gram-negative bacteria. This indicates its likely utility as an agent in any treatment targeted against these micro-organisms. It appears that again there may well be a positive interaction between the two compounds, in terms of their combined antimicrobial activity in each case.

Exami'le 4 -activity against other micro-organisms (AO 2246 alone) The activity of AO 2246 was tested against a number of bacteria, using MIC and MBC assays as described above. DMSO was used as the solvent, and the experiments were performed in triplicate. The results are shown in Table 4 below.

Table 4

Test organism MIC MBC MIC/MBC ____________________________________ (pg/mi) (pg/mi) ratio Actii'iornyces jiaeslwidiiATCC 12104 1.95 3.9 0.5 Bacillus cereus ATCC 11778 1.95 3.9 0.5 Closti'idium dfJIcile ATCC 7000057 3.9 3.9 1 Closti'idium sporo genes ATCC 3584 3.9 31.25 0.125 Corvnebacterium mucifaciens ATCC 700355 1.95 3.9 0.5 Cori'nebacterium striatuni NCTC 764 <0.49 1.95 <0.25 Enterococcusfaecalis ATCC 29212 1.95 7.8 0.25 Gardnerella vaginalis ATCC 14018 15.6 15.6 1 Listeria monocytogenes ATCC 15313 1.95 7.8 0.25 Porphyroinonas gingivalis NCTC 11834 3.9 3.9 1 Porphyronionas gingivalis ATCC BAA-308 3.9 3.9 1 Porphyroinonas gingivalis ATCC 53978 0.98 1.95 0.5 Prevotella nigrescens ATCC 33563 7.8 >15.6 <0.5 Streptococcus mutans ATCC 25175 15.6 62.5 0.5 Streptococcus mutans ATCC 35668 7.8 31.25 0.25 Streptococcus pyogenes ATCC 12344 0.98 0.98 1 Streptococcus sobrinus ATCC 33478 15.6 31.25 0.5 [Abbreviations: American Type Culture Collection (ATCC), National Collection of Type Cultures (NCTC).] The Table 4 data show that the AO 2246 has excellent activity against the test organisms, which include both Gram-positive and Gram-negative bacteria. This is surprising in view of the prior art on bis-phenol antioxidants of this type.

Combinations of compounds of formula (I) and their derivatives with DASs and their derivatives can therefore be expected to be active against these types of pathogen.

In further experiments, AO 2246 was tested against a wide range of both staphylococcal and propionibacterial strains, including several antibiotic resistant strains, and found to have excellent activity in particular against the propionibacteria.

Exami'le 5 -activity against other micro-oianisms (AO 425) Example 4 was repeated, against a similar range of test organisms, using AO 425 as the test compound. Again DMSO was used as the solvent, and both MIC and MBC experiments were performed in triplicate. The results are shown in Table 5 below.

Table 5

Test organism MIC MBC MIC/MBC (pg/in!) (pg/mi) ratio Propionibacteriuin acnes NCTC 737 7.8 31.25 0.25 Test organism MIC MBC MIG/MBC ____________________________________ (pg/mi) (pg/ma ratio Staphylococcus aureus ATCC 29213 7.8 62.5 0.125 Actinonn'ces naeslundii ATCC 12104 0.98 3.9 0.25 Bacillus cereus ATCC 11778 1.95 3.9 0.5 Clostridium difficile ATCC 7000057 7.8 7.8 1 Clostridium sporo genes ATCC 3584 3.9 7.8 0.5 Gardnerella vaginalisATCC 14018 15.6 31.25 0.5 ListerianionocytogenesATCC 15313 0.98 7.8 0.125 Porphvromonas gingivalis NCTC 11834 1.95 1.95 1 Porphi'roinonas gingivalis ATCC BAA-308 1.95 3.9 0.5 Porphi'romonas gingivalis ATCC 53978 0.98 0.98 1 Prevotella nigrescens ATCC 33563 15.6 >15.6 <1 Streptococcus inutans ATCC 25175 15.6 62.5 0.25 Streptococcus mutans ATCC 35668 7.8 >250 <0.03 Streptococcus sobrinus ATCC 33478 31.25 62.5 0.5 [Abbreviations: American Type Culture Collection (ATCC), National Collection of Type Cultures (NCTC).] The Table 5 data again show excellent antimicrobial activity for the AO 425. Most Gram-positive micro-organisms were found to be susceptible, as were certain Gram-negative bacteria associated with periodontal disease. Combinations of AO 425 with DASs and their derivatives can therefore be expected to be active against these types of pathogen.

With regard to activity against P. acnes NCTC 737, it is of note that other structurally similar alkyl-bis-phenol compounds were found to be far less active against the bacterium than AO 2246 and AO 425. 2,2'-ethylidene-bis(4,6-di-tert-butylphenol) and 4,4'-methylenebis(2,6-di-tert-butylphenol) for example, when tested against this organism, were both found to have MIC and MBC values greater than 250 tg/ml.

When tested against C. mucfaciens ATCC 700355, E.faecalis ATCC 29212 and S. pyogenes ATCC 12344, AO 425 was found to have lower activity than AO 2246.

Thus, it may be preferable to use AO 2246 in the treatment of conditions in which such bacteria are implicated.

Exctm,le 6-activity of other suiphosuccinates with AO 2246 vs P. acnes The following experiments all used P. acnes NCTC 737 as the test organism. MTC and MBC assays, as described above, were carried out using as the test compounds (a) disodium laureth suiphosuccinate, dissolved in distilled water, (b) disodium C-dodecyl sulphosuccinate, dissolved in distilled water, and (c) AO 2246, dissolved in DMSO.

The two DASs were sourced from Evonik Industries AG, Germany. Each of the test compounds was then subjected to disc diffusion assays, again as described above.

Combinations of each of the sulphosuccinates with AO 2246 were also subjected to synergy disc diffusion assays (SDDAs). For the (S)DDA tests, 200 tg of each test compound was loaded onto each disc. All experiments were conducted triplicate. The results are shown in Table 6 below.

Table 6

Compound or MIC MBC Average (S)DDA combination (pg/in!) (pg/mi) zone diameter _______________ _______ ______ (mm �SD) AO 2246 0.98 1.95 7.1 � 0.1 Dis odium laureth sulphosuccinate 62.5 62.5 16.4 � 0.8 Disodiuin C-dodecyl sulphosuccinate 15.6 15.6 26.1�0.9 AO 2246 + disodium laureth suiphosuccinate <0.49 0.98 28.0 � 0.6 AO 2246 + disodiurn C-dodecyl suiphosuccinate 3.9 7.8 27.5 � 1.0 The two combinations according to the invention, of AO 2246 plus dialkyl sulphosuccinate, can be seen to be highly active against P. acnes NCTC 737.

Moreover in the case of the disodium laureth sulphosuccinate, the data indicate a synergistic antibacterial interaction with the AO 2246, the combination showing a significant increase in DDA zone diameter over that exhibited by either compound alone.

Exam,le 7-anti-acne activity A trial was carried out on human subjects to assess the activity, in skin prone to acne, of a formulation according to the invention. The test formulation (A) contained 5% w/v DOSS, 2% w/v salicylic acid and 1.75% w/v AO 2246, in an aqueous gel. The negative control (B) was an alcohol-free, active-free aqueous gel. The positive control (C) was a commercially available anti-acne product (GamierTM "Pure A Intensive Treatment Night Gel", purchased in the EU) which, on assay, was found to contain 2% w/v salicylic acid.

The aqueous gel used as the base for formulation A was a micro-emulsion containing ethanol, propylene glycol, diethylhexyl sodium sulphosuccinate, propylene glycol laurate, PEG-8 caprylic/capric glycerides, hydroxypropylceliulose, sodium hydroxide and the balance water. The gel used as the negative control (B) contained propylene glycol, carbomer, tromethamine, methylparaben, propylparaben and the balance water.

The ingredients of the commercially available positive control (C) were listed as "aqua, alcohol denat., glycerin, BIS-PEG-18 methyl ether dimethyl silane, acrylates/C 10-30 alkyl acrylate crosspolymer, triethanolamine, camphor, Centella Asiatica / Centella Asiatica extract, Cucumis Sativus/Cucumber Fruit extract, Hamamelis Virginiana / Witch Hazel extract, salicylic acid, zinc PCA, hexylene glycol, PEG-60 hydrogenated castor oil, pentylene glycol, propylene glycol, fragrance, benzyl salicylate, hexyl cinnamal, limonene, linalool".

The detailed compositions of Formulations A and B are shown in Table 6 below.

Table 6

Ingredient (% v/v or,v/v) Formulation A Negative __________________________ _______________ Control (B) Water 34.7 87.97 Ethanol 34 NA Propylene glycol 14 9.99 Diethylhexyl sodium 5 NA sulphosuccinate (DOSS) _______________ ___________ Propylene glycol laurate 3.5 NA PEG 80 caprylic/capric 3.5 NA glycerides __________________ ______________ Ingredient (% v/v or w/v) Formulation A Negative ___________________________ _______________ Control (B) Salicylic acid 2 NA Hydroxypropyl cellulose 1.30 NA Sodium hydroxide 0.25 NA Methylene di-t-butyl-cresol 1.75 NA (AO 2246) _______________ ___________ Carbomer NA 1.0 Tromethamine NA 0.88 Methylparaben NA 0.18 Propylparaben NA 0.02 The trial was a two-centre randomised, blinded controlled parallel group study in 47 male or female subjects aged 16 to 35 years. Twenty-three subjects were treated with the test formulation A, and the remaining 24 subjects were treated with either the negative control (formulation B) or the positive control (formulation C). Treatments (1 g of the relevant formulation) were applied topically by hand to the entire face by the participants at home, once daily over an eight week study period. Once applied, the formulations were left on the skin rather than being rinsed off No concomitant anti-acne medications were permitted and previous treatment was stopped at least four weeks before the baseline visit.

Treatments were randomly assigned to the subjects using a computer-generated code.

Treatment application was double-blind for the placebo and test treatment and observer-blind for the positive control (different tube, overlabelled). Lesion counts were performed at baseline and on study days 15, 29 and 57. Adverse events such as skin irritation were documented by spontaneous reporting. All comparisons were versus baseline (intragroup not intergroup).

The results, in terms of the percentage reduction in lesion counts from the "baseline" at day 1, are summarised in Tables 7 to 10 below. In these tables, * indicates P = 0.01 - 0.05, ** indicates P = 0.001 -0.01, and *** indicates P<0.0001, derived from mean counts.

Table 7 -inflamed spots ii TREA TMENT Percent reduction from baseline ________________ Daj'15 Daj'29 Day57 Formulation A 4.8 18.5* 31.6*** Formulation B 17.1* 16.5* 29.9** Formulation C 12.4* -1.0 20.8 Table 8 -non-inflamed spots TREA TMENT Percent reduction from baseline _______________ Dayl5 Day 29 Day57 Formulation A 16.5* 26.5** 339*** Formulation B 0.5 12.3 9.8 FormulationC -1.2 11.5 -8.7 Table 9 -total number of spots TREA TMENT Percent reduction from baseline _______________ Dayl5 Day 29 Day57 Formulation A 10.7* 22.6** 32.8*** Formulation B 8.6 14.4 19.6* Formulation C 6.3 4.6 7.5 Table 10 -categorical analyses TREA TMENT Percent of participants with �=33% reduction from baseline at week 8 Inflamed spots Non-inflamed spots Total number of _____________________ ______________________ _____________________ spots Formulation A 50.0 50.0 50.0 FormulationB 25.0 8.3 25.0 Formulation C 33.3 20.0 16.7 Of the adverse events reported during the treatment period, all were assessed to be mild and most to be unlikely to be linked to the test formulations. No subjects using formulation A or either control formulation reported any side effects considered to be product related.

The trial results can be summarised as follows.

* The treatment according to the invention, using Formulation A, produced reductions in inflamed spots, non-inflamed spots and total lesions.

* Greatest activity was observed against non-inflamed lesions (otherwise known as open and closed comedones, or blackheads and whiteheads respectively).

This activity was detectable by day 15. Most inflamed spots arise from comedones (sometimes microcomedones which are not visible or palpable).

* The treatment using Formulation A also performed better than the positive control (C), a marketed product containing the same concentration of salicylic acid.

* The formulation of the invention was well tolerated, despite its relatively high concentrations of the anionic surfactant DOSS and the keratolytic salicylic acid. Tt therefore appeared to be suitable for application as a "leave on" product.

Without wishing to be bound by these theories, it is possible that the DAS acts, via its detergent action, to reduce the number of pre-existing comedones and/or to prevent the formation of new comedones. In other words, it may loosen the "mortar" (interstitial lipids) in which the "bricks" (the comeocytes) are set. The DAS appears to retain at least some of its detergent activity after its initial application, and so can continue to have an effect on acne lesions whilst it remains in contact with the skin. Surprisingly, however, that contact does not appear to cause skin irritation.

It is also possible that the DAS, via its known detergent and penetration enhancing effects, may facilitate the follicular penetration of salicylic acid. Again these effects appear to continue after the formulation is first applied to the skin, thus boosting the anti-acne effects of the salicylic acid and providing prolonged activity against acne lesions when the formulation is left in contact with the skin.

At the same time, the DAS, salicylic acid and AO 2246 appear to interact in a way that causes the combination to be less of an irritant than might have been expected from the properties of its individual components. This is likely to be of value in formulating "mild" skin care preparations which can be left in contact with the skin for a prolonged anti-acne effect.

Claims (14)

1. Claims 1. An antimicrobial and/or anti-acne formulation containing (a) a dialkyl sulphosuccinate (DAS) or derivative thereof and (b) a compound of formula (T) or derivative thereof: R3O3J< Formula (I) wherein each of R' and R2 is independently selected from methyl and ethyl and R3 is hydrogen, and wherein the formulation is suitable for topical application to the skin.
2. 2. A formulation according to claim 1, wherein the DAS is a dioctyl suiphosuccinate.
3. 3. A formulation according to claim 1 or claim 2, wherein the compound of formula (I) is AO 2246.
4. 4. A formulation according to any one of the preceding claims, which is in the form of a fluid.
5. 5. An antimicrobial and/or anti-acne formulation which is substantially as herein described.
6. 6. A product containing an antimicrobial or anti-acne formulation according to any one of the preceding claims.
7. 7. A kit for preparing an antimicrobial or anti-acne formulation, the kit comprising a source of a DAS or derivative thereof and a source of a compound of formula (I) or derivative thereof, together with instructions for combining the two compounds so as to make the formulation at or before the point of its intended application, and/or for the co-administration of the two compounds to a surface.
8. 8. A formulation containing (a) a DAS or pharmaceutically acceptable derivative thereof and (b) a compound of formula (I) or pharmaceutically acceptable derivative thereof, for use in the treatment of a condition affecting the human or animal body, which condition is caused by, transmitted by and/or exacerbated by microbial (in particular bacterial) activity.
9. 9. A formulation containing (a) a DAS or pharmaceutically acceptable derivative thereof and (b) a compound of formula (I) or pharmaceutically acceptable derivative thereof, for use according to claim 8, wherein the condition is acne and/or acne lesions.
10. 10. A formulation containing (a) a DAS or pharmaceutically acceptable derivative thereof and (b) a compound of formula (I) or pharmaceutically acceptable derivative thereof, for use according to claim 8 or claim 9, the use being substantially as herein described.
11. 11. Use of (a) a DAS or pharmaceutically acceptable derivative thereof and (b) a compound of formula (I) or pharmaceutically acceptable derivative thereof, in the manufacture of a medicament for the treatment of a condition which is caused by, transmitted by and/or exacerbated by microbial (in particular bacterial) activity.
12. 12. Use of(a) a DAS or derivative thereof and (b) a compound of formula (I) or derivative thereof as an anti-acne or skin care agent for non-therapeutic purposes.
13. 13. A method for controlling the growth of a micro-organism, the method comprising applying, to a non-living area or surface which is infected or suspected to be infected or capable of becoming infected with the organism, a combination of a DAS or a derivative thereof and a compound of formula (I) or derivative thereof
14. 14. A method of treatment of a human or animal patient suffering from or at risk of suffering from a condition which is caused by, transmitted by and/or exacerbated by microbial activity, the method involving administering to the patient a therapeutically (which term includes prophylactically) effective amount of an antimicrobial and/or anti-acne formulation containing (a) a DAS or pharmaceutically acceptable derivative thereof and (b) a compound of formula (I) or pharmaceutically acceptable derivative thereof