GB2463566A - Formulations active against propionibacteria - Google Patents

Abstract

An antibacterial and/or anti-acne formulation containing (a) salicylic acid or a derivative thereof and (b) a compound of formula (1) or derivative thereof:wherein each of R1and R2is independently selected from methyl and ethyl and R3ishydrogen. The formulation is particularly active against propionibacteria, and may therefore be used to treat acne.

Description

Formulations

Field of the invention

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

Background to the invention

Salicylic acid (2-hydroxybenzoic acid) is well known for use as an anti-acne agent. It is a keratolytic which is widely used to prevent pores becoming blocked (Wailer JM et al, "Keratolytic' properties of benzoyl peroxide and retinoic acid resemble salicylic acid in man", Skin Pharmacol Physiol, 2006;19: 283-9). It thereby prevents comedogenesis (formation of blackheads and whiteheads) and trapping of the bacterium Propionibacterium acnes within obstructed follicles, which could otherwise result in visible inflammation.

The keratolytic action of salicylic acid is concentration dependent and it is often necessary to combine it with other actives to increase efficacy, especially against inflammatory lesions (see for example Touitou E et al, "Efficacy and tolerability of clindamycin phosphate and salicylic acid gel in the treatment of mild to moderate acne vulgaris", JEurAcadDermatol Venereol 2008;22: 629-3 1). There is therefore a need to find alternative anti-acne formulations in which the activity of salicylic acid can be boosted by the presence of a second active ingredient, ideally one which preferentially targets P. acnes. Salicylic acid alone has very weak antibacterial activity that is almost certainly insufficient to manifest against P. acnes on skin as typically formulated in concentrations of 2% w/v or less.

Moreover since acne is an extremely common condition and also a cause of significant distress to those suffering from it, there is always a need to find new and preferably more effective anti-acne treatments.

It has now surprisingly been found that certain classes of antioxidant, in particular that known as Antioxidant 2246, can be effective as antibacterial agents against propionibacteria, the bacteria implicated in inflammatory acne. Tt has also been found that combinations of salicylic acid with such antioxidants can provide improved antibacterial and anti-acne formulations.

Statements of the invention

According to a first aspect of the present invention there is provided an antibacterial and/or anti-acne formulation containing (a) salicylic acid or a derivative thereof and (b) a compound of formula (I) or derivative thereof: >LjR3o3: Formula () wherein each of R1 and R2 is independently selected from methyl and ethyl and R3 is hydrogen.

This formulation is preferably suitable for topical application to, and/or contact with, the skin, in particular human skin. The salicylic acid or derivative and the compound (I) or derivative are therefore preferably contained in a pharmaceutically acceptable vehicle which can safely be applied to, and/or contacted with, the skin. A formulation which is "suitable for" topical application may also be adapted for topical application.

Suitable vehicles for topical formulations will be well known to those skilled in the art of preparing topical skin care or pharmaceutical preparations. The salicylic acid or derivative and the compound (I) 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.

The salicylic acid may be used in the form of a derivative such as in particular a salt.

A salicylate salt may be for example a metal salt or ammonium salt. Suitable metal salts include the alkali metal salts (for example the sodium and potassium salts, in particular the former) and the alkaline earth metal salts (for example the calcium and magnesium salts). A metal salicylate may be also selected from bismuth salicylate, bismuth subsalicylate and transition metal salts such as zinc, copper or titanium salts.

Other salicylic acid derivatives include salicylic acid esters, in particular alkyl esters, more particularly Cl -C20 or Cl-cl 0 or Cl -C6 alkyl esters such as in particular methyl salicylate ("wintergreen"). Further derivatives include benzyl salicylate and betaine salicylate. In an embodiment, the salicylic acid derivative may be thiosalicylic acid.

In an embodiment of the invention, the salicylic acid is present in the form of the free acid, although dependent on the pH of the formulation the acid may be present in dissociated form, typically as salicylate anions and protons.

The salicylic acid (or any derivative thereof used in a formulation according to the invention) may be either naturally or synthetically derived. Tt may for example be obtained from a natural source such as willow herb.

Formula (I) above embraces in particular the compound known as "Antioxidant 2246" or AO 2246", in which both R' and R2 are methyl. Synonyms for AO 2246 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. It 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. j

Where R' and R2 are both ethyl, the compound of formula (I) is known as "Antioxidant 425" or "AO 425", or as 2,2'-methylene-bis(4-ethyt-6-tert-butylphenol).

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 et at inJAntibactAntifungAgents, Vol. 9, No. 9: 429-433 also describe the use of certain alkyiphenol dimers and oligomers -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.

To our knowledge, however, compounds of formula (I) have not previously been known to have activity against propionibacteria, nor have they been known for use as anti-acne agents.

In the compound of formula (I), R' and R2 may be either methyl or ethyl, most preferably methyl. R' and R2 are suitably the same.

In an embodiment of the invention, the compound of formula (I) is selected from AO 2246, AO 425 and mixtures thereof In another embodiment, it is AO 2246.

The compound (I) 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% w/w or less, in cases 0.05% w/w or less, of impurities.

A "derivative" of either salicylic acid or a compound of formula () may be a pharmaceutically acceptable (which term includes acceptable for veterinary use) derivative. It may be for example a salt, ester, complex or solvate or a so-called "pro drug" form or protected form which reverts to an active form of the relevant compound at an appropriate time on or after administration. In an embodiment, however, the salicylic acid is present in the form of the free acid (which may be present in dissociated form, as discussed above). In an embodiment, the compound (I) is present in the form of a single, underivatised molecule of the formula (I) defined above.

A formulation according to the invention may contain a mixture of two or more salicylic acid derivatives, or a mixture of salicylic acid with one or more derivatives thereof It may contain a mixture of two or more compounds of formula (I) or derivatives thereof.

In a formulation according to the invention, the combination of the salicylic acid or derivative and the compound (I) or derivative is present as an active (ie antibacterially active) agent. The combination is suitably 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 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 for the treatment of chronic wounds as described in US-2005/0065 182; or as an antiviral agent; or as an antifungal agent.

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

Antibacterial activity encompasses activity against both Gram-positive and Gram-negative bacteria. It may be growth inhibitory activity or more preferably bactericidal (ie lethal to the relevant organism). It may comprise activity against sessile and/or planktonic bacteria. In the context of this invention, activity against a particular species of bacterium may be taken to mean activity against at least one, preferably two or more, strains of that species.

Antibacterial activity may be or include the ability to disrupt and/or suppress biofilm formation by the relevant organism. The bio film may in particular be formed by Propionibacterium acnes. 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 biofilm, and/or on a biofilm already formed by the organism. Thus, it may involve reducing the amount of a previously formed biofllm, and/or impairing such a biofllm.

It may involve killing or inhibiting sessile 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 biofllm 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 antibacterial formulation according to the present invention may be active against Gram-positive and/or Gram-negative bacteria, in particular Gram-positive bacteria.

More particularly, it may be active against one or more bacteria associated with acne, in particular propionibacteria such as P. acnes and in some instances P. granulosum.

The formulation is preferably active against bacteria, 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. Tn 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.

Antibacterial activity may be measured in conventional manner, for instance using the tests described in the examples below. Generally tests for activity involve treating a culture of the relevant micro-organism with the candidate antibacterial compound, incubating the treated culture under conditions which would ordinarily support growth of the organism, and assessing the level of growth, if any, which can occur in the presence of the candidate compound.

The concentration of the salicylic acid or derivative in a formulation according to the invention might suitably be 0.1 or 0.2 or 0.5% w/v or greater, for example 1 or 2% w/v or greater. Its concentration might be up to 5% w/v, or up to 3 or 2 or 1% w/v. In an embodiment of the invention, the concentration of the salicylic acid or derivative is from 0.5 to 5% w/v, or from 0.5 to 3% w/v, or from 0.5 to 2% w/v, such as about 2% w/v. Tn cases, for example in chemical peels for use against acne, its concentration might be up to 10 or 20 or even 30% w/v.

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

Preferably the weight ratio of the salicylic acid or derivative in the formulation to the compound (I) or derivative is up to 50:1, for example up to 25:1 or 10:1 or 5:1 or 4:1.

This ratio may be 1:50 or greater, for example 1:25 or 1:10 or 1:5 or 1:4 or greater. Tt may for example be in the range from 1:4 to 4:1.

A formulation according to the invention may take the form of a fluid. It may for example take the form of a lotion, cream, ointment, varnish, foam, paste, gel or other viscous or semi-viscous fluid, or of a less viscous fluid such as might be used in sprays or drops, or any other physical form known for topical administration. It may take the form of a solution or suspension, for instance for use as a disinfectant. It may comprise a formulation which is, or may be, applied to a carrier such as a sponge, swab, brush, tissue, cloth, wipe, skin patch or dressing (which includes a bandage, plaster, skin adhesive or other material designed for application to a tissue surface) to facilitate its topical administration. Tt may be intended for pharmaceutical (which includes veterinary but is preferably human) use, and/or for cosmetic or other non-medical care purposes (for example, for general hygiene or skin cleansing or for improving the appearance of the skin).

The vehicle in which the salicylic acid or derivative and the compound (I) or derivative are contained may be any vehicle or mixture of vehicles which is 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' Transderinal and Topical Drug Delivery, Pharmaceutical Press, 2003, and other similar reference books. See also Date, AA et al, Skin Pharinacol Physiol, 2006, 19(1): 2-16 for a review of topical drug delivery strategies.

The salicylic acid or derivative and the compound (I) 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 compounds 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 some cases a polar vehicle may be preferred. Where the formulation is intended for use on the skin, the vehicle may be primarily non-aqueous, although in the case of an anti-acne treatment an aqueous vehicle may be used. The vehicle is suitably volatile.

In cases it may be alcohol-based or silicon-based. By way of example, a lotion or gel formulation according to the invention, in particular one intended for application to the skin, may contain a mixture of water, an alcohol such as ethanol or phenoxyethanol and a glycol such as propylene glycol.

Generally speaking a formulation according to the invention may contain standard excipients and other additives known for use in pharmaceutical formulations. For example, where the formulation is intended for topical application to the skin, examples of suitable excipients and additives include emollients, perfumes, antioxidants, preservatives, stabilisers, gelling agents and surfactants; others may be found in Williams' Transdermal and Topical Drug Delivery (see above). For the treatment of acne, however, it may be preferred for the formulation not to contain an emollient.

Such a formulation may further contain additional active agents such as antimicrobial (in particular 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 healing, additional antimicrobial 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 salicylic acid or derivative and the compound (T) or derivative to be the only active agents in the formulation, or at least to be the only antibacterially active agents and/or the only anti-acne active agents.

In the case where the formulation is intended for application to an area or surface as a disinfectant or antiseptic, it may take the form of a solution or suspension of the salicylic acid or derivative and the compound (I) or derivative in an appropriate fluid vehicle such as an alcohol or a water/alcohol mix. Again conventional excipients and other additives may be included, as may one or more additional antimicrobial (in particular antibacterial) agents.

In particular when the formulation is for use in controlling the transmission of a microbial infection, it may be in the form of a skin wash (for example a hand wash), or of a surface disinfectant such as a spray, or of a cleansing fluid for example for use in disinfecting surgical instruments. It may be carried in or on a cloth, wipe, brush or other cleaning utensil, or a substrate such as a preparation surface or implement or a packaging material; in such cases an item may be impregnated with, or coated with, the formulation.

Thus a formulation according to the invention may be suitable for, more preferably adapted for, use in an area or on a surface other than living tissue, for instance to treat work surfaces or instruments. In these cases the excipients, vehicles and/or other additives included with the salicylic acid or derivative and the compound (I) or derivative may be different to those included in a topical skin care formulation, but again may be conventional as known for use in such contexts.

Generally speaking a formulation according to the invention may contain one or more agents which enhance the activity of another active agent present in the formulation, or reduce a side effect of such an active, or improve patient compliance on administration of the formulation. It may contain one or more agents which facilitate penetration of an active agent into microbial biofllms. It may contain one or more agents which control the site and/or rate of release of an active agent following administration.

A formulation according to the invention may be incorporated into, and hence applied in the form of, another product such as a cosmetic, a skin care preparation (for example a skin cleanser, toner or moisturiser, a cleansing preparation (for example a hand wash or facial wash), 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 formulation may be, or be incorporated into, a leave on or a wash off skin treatment product. The invention thus provides, according to a second aspect, a product which incorporates an antibacterial or anti-acne formulation according to the first aspect.

A formulation according to the invention may be marketed with an indication that it has antibacterial or anti-acne activity, or enhanced antibacterial or anti-acne activity, in particular against propionibacteria. 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) providing the indication in a commercial which is aired for instance on the radio, television or internet. The antibacterial 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 salicylic acid or derivative and the compound (I) or derivative, in particular to the presence of the compound (T) or derivative. The antibacterial or anti-acne activity of the formulation may be assessed during or after its preparation. The activity may be assessed both before and after incorporation of the salicylic acid or derivative and/or the compound (I) or derivative, for example so as to confirm that either or preferably both contribute to the antibacterial or anti-acne activity of the formulation.

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 antibacterial or anti-acne formulation, such as a formulation according to the first aspect, the kit comprising a source of salicylic acid or a 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. The two compounds may each be present in a suitable respective vehicle.

According to one embodiment, the formulation or kit of the invention may contain both salicylic acid or a 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 antibacterial or anti-acne formulation, which method involves mixing together salicylic acid or a derivative thereof and a compound of formula (I) or derivative thereof, preferably together with a pharmaceutically acceptable vehicle.

According to a fifth aspect of the invention there is provided a formulation (preferably a formulation according to the first aspect of the invention) containing salicylic acid or a pharmaceutically acceptable derivative thereof and 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 (in particular either caused or transmitted by) bacterial activity.

The activity may in particular be propionibacterial activity. The condition may be a skin or skin structure condition, in particular 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. It will typically involve use of the salicylic acid or derivative and the compound (I) or derivative as a bactericidal or anti-acne combination. Treatment may involve use of the formulation to treat a condition which is caused, transmitted and/or exacerbated by (in particular caused or transmitted by) bacterial bioflim formation.

In general the treatment may be administered orally, transdermally or topically. It is preferably administered topically.

In an embodiment of the fifth aspect of the invention, the formulation is for use against one or more bacteria associated with skin or skin structure conditions. It may be 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. In particular it 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 salicylic acid or a derivative thereof 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-O 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 oniy a single mechanism.

According to the fifth aspect of the invention, the formulation of salicylic acid 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 salicylic acid or a pharmaceutically acceptable derivative thereof and a compound of formula (I) or pharmaceutically acceptable derivative thereof in the treatment of a bacterial condition, including acne, the two compounds being administered either simultaneously or sequentially.

A sixth aspect of the invention provides a formulation (preferably a formulation according to the first aspect of the invention) containing salicylic acid or a pharmaceutically acceptable derivative thereof and a compound of formula (I) or pharmaceutically acceptable derivative thereof, 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.

According to a seventh aspect, the invention provides the use of salicylic acid or a pharmaceutically acceptable derivative thereof and a compound of formula (I) or pharmaceutically acceptable derivative thereof, 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) bacterial activity. The condition may be selected from those listed above in connection with the first to the fifth aspects of the invention. It may in particular be acne and/or acne lesions. The salicylic acid or derivative and the compound (I) or derivative will typically be used as an antibacterial and/or anti-acne combination in the manufacture of the medicament.

An eighth aspect provides the use of salicylic acid or a pharmaceutically acceptable derivative thereof and a compound of formula (I) or pharmaceutically acceptable derivative thereof, 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 salicylic acid or a derivative thereof and a compound of formula (I) or derivative thereof, as a combined antibacterial agent, or as a combined anti-acne agent, in the manufacture of an antibacterial 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 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 bacterium, a combination of salicylic acid or a derivative thereof and a compound of formula (I) or derivative thereof Again the two compounds may be applied simultaneously or sequentially. They are suitably applied in a formulation of the type described above. They may in particular be applied to an area or surface which is infected with the relevant bacterium.

In this context, "controlling the growth" of a bacterium 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 bacterium in or on the area or surface being treated. It may embrace reducing the risk of transmission of the bacterium 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 bacterium or to bacterium may also embrace the disruption and/or suppression of biofllm formation by the bacterium, as described above.

Again the salicylic acid or derivative and the compound (I) or derivative may be applied to living human or animal tissue, in particular the skin. Thus the method of the eleventh aspect of the invention 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) bacterial activity, the method involving administering to the patient a therapeutically (which term includes prophylactically) effective amount of an antibacterial formulation containing (a) salicylic acid or a pharmaceutically acceptable derivative thereof and (b) a compound of formula (I) or pharmaceutically acceptable derivative thereof The bacterial 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 antibacterially effective amount.

According to a twelfth aspect there is provided a method of treatment of a patient suffering from or at risk of suffering from acne or acne lesions, the method involving administering to the patient a therapeutically (which term includes prophylactically) effective amount of an anti-acne formulation containing (a) salicylic acid or a pharmaceutically acceptable derivative thereof and (b) a compound of formula (I) or pharmaceutically acceptable derivative thereof A thirteenth 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 containing (a) salicylic acid or a pharmaceutically acceptable derivative thereof and (b) a compound of formula (T) or pharmaceutically acceptable derivative thereof.

In accordance with the eleventh to the thirteenth aspects of the invention, the formulation is suitably administered to a human patient. The patient is suitably suffering froni the relevant condition, in particular acne. The methods of the eleventh to the thirteenth aspects of the invention preferably involve applying a formulation according to the first aspect.

A fourteenth aspect of the invention provides the use of salicylic acid or a derivative thereof in an antibacterial or anti-acne formulation, in combination with a compound of formula (T) or derivative thereof, for the purpose of increasing the antibacterial 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 antibacterial or anti-acne activity. For example whilst we do not wish to be bound by this theory, it is believed that salicylic acid may facilitate the skin penetration of compounds such as AO 2246 by reducing the cohesiveness of, and hence loosening, the cells of the stratum corneum.

An increase in antibacterial 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 salicylic acid or derivative. Ideally the increase is as compared to the sum of the activities of the salicylic acid 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 (T) 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 salicylic acid 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 antibacterial or anti-acne activity. Preferably the salicylic acid or derivative is used without any reduction in antibacterial or anti-acne activity compared to the level exhibited by the formulation prior to addition of the salicylic acid or derivative. More preferably it is used to give an increase in antibacterial or anti-acne activity, in particular in vivo. 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 antibacterial 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 fifteenth aspect of the invention provides the use of a compound of formula (I) or or derivative thereof in an antibacterial or anti-acne formulation, in combination with salicylic acid or a derivative thereof, for the purpose of increasing the antibacterial and/or anti-acne activity of the formulation and/or of reducing the amount of the salicylic acid or derivative in the formulation without or without undue loss of antibacterial or anti-acne activity. The above comments regarding the fourteenth aspect of the invention apply mutatis mutandis to the fifteenth 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 antibacterial 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. This is a very common, complex and multi-factorial skin disease in which P. acnes and other Propionibacterium 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, such as the two P. acnes strains designated PRP-002 and PRP-O 10 which are resistant respectively to macrolides- lincosamides-streptogramins (MLS) and tetracycline and to macrolides-lincosamides-streptogramins-ketolides (MLSK) -in other words, PRP-002 is resistant to erythromycin, clindamycin and tetracycline and PRP-010 to erythromycin, clindamycin and tetracycline. In addition, a number of strains of P. granulosum, another bacterium involved in acne, were 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 following tests were carried out to assess antibacterial 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 tl 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 tg/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 MIC assay, a 5 p1 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 MTC 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) Agar dilution MIC assay This is a standard international method for quantitatively assessing the antimicrobial activity of a compound in a solid medium. The test compound was prepared to 40x the highest concentration required (eg 10 mg/mi for a final concentration of 250 tg'ml) and a series of doubling dilutions were performed in a suitable solvent. A set amount of these antimicrobial stock solutions was then added to molten agar medium (ca. 55°C), mixed thoroughly, poured into sterile Petri dishes and allowed to cool/set.

The culture medium was as described above.

A MultipointTM Inoculator (AQS Manufacturing Ltd, UK) was used to inoculate the plates by spotting the inocula onto the surface of the agar, delivering approximately 1 to 2 tl per spot (yielding i05 CFU (colony forming units) per spot). The plate(s) were then incubated under the conditions described above, following which they were examined visually for signs of bacterial growth. The MTC value was ascertained when there was a marked reduction in, or total loss of, growth on the test plate at the lowest concentration as compared to that of the growth on the control plate.

The assays were conducted in triplicate and included a positive control (culture medium, diluting solvent and inoculum).

Exam i'le 1 -activity against P. acnes (MICs) The following experiments all used P. acnes NCTC 737 as the test organism. MIC assays, as described above, were carried out using as the test compounds (a) salicylic acid (SA) and (b) AO 2246, both dissolved in ethanol. Both test compounds were sourced from Sigma Aldrich, UK. A combination of SA and AO 2246, according to the present invention, was also subjected to the MIC assay: the combination contained 50% w/w of each compound. All MIC experiments were conducted in triplicate.

The results are shown in Table 1 below; all are collated from a number of experiments.

Table 1

Assay SA A02246 A02246+SA (ratio 1:1 w/w) Assay SA A02246 A02246+SA (ratio 1:1 w/w) MIC(tg/m1) 1000 3.9 3.9 It can be seen from Table 1 that the combination of salicylic acid and a compound of formula (I) is active as an antibacterial agent against P. acnes NCTC 737, even though the salicylic acid alone appears to have very low activity against the bacterium. This result indicates the likely activity of the invented combinations as anti-acne agents, the propionibacteria being implicated in acne.

The data also show that the inclusion of a compound of formula (I) with salicylic acid, in an anti-acne formulation, may be capable of improving the overall efficacy of the formulation.

Exami'le 2 -activity against other i'ropionibacteria (AO 2246 alone) The activity (MIC by agar dilution) of AO 2246 was determined against a panel of different propionibacterium strains. DMSO was used as the solvent. The MIC experiments were performed in triplicate. The results are shown in Table 2 below, which also indicates the resistance phenotype for each of the test species/strains.

Table 2

Test organism Resistance MIC (pg/mi) _______________________________________ phenotype _____________ Propionibacterium acnes NCTC 737 None 3.9 Propionibacterium granulosum NCTC 11865 None 1.95 P. acnes PRP-002 Tet/MLS 39 P. acnes PRP-003 Tet 3.9 P. acnes PRP-004 Tet 3.9 P. granulosum PRP-005 MLSK 39 P. granulosuin PPR-006 MLS 1.95 Test organism Resistance MIC (pg/mi) phenotype P. acnes PPR-007 Clin 1.95 P. acnes PRP-008 Clin 1.95 P. acnes PRP-O1O MLSK 39 P. acnes PRP-017 MLS 39 P. granulosum PRP-019 MLSK 3*9 P. granulosum PRP-021 MLS 39 P. acnes PRP-023 MLSK 39 P. acnes PRP-026 MLS 3.9 P. acnes PRP-039 Tet/MLS 3.9 P. granulosuin PRP-043 MLS 1.95 P. granulosuin PRP-044 MLS 39 P. acnes PRP-046 None 3.9 P. aciws PRP -053 Tet/MLS 3.9 P. granulosum PRP-055 None 3.9 P. acnes PRP-059 MLS 3.9 P. acnes PRP-068 Ery 3.9 P. acnes PRP-1O1 Tet/MLS 39 P. acnes PRP-102 Tet/MLS 39 [Abbreviations: American Type Culture Collection (ATCC), National Collection of Type Cultures (NCTC), Propionibacterium Panel Number (PRP), Tetracycline (Tet), Erythromycin (Ery), Clindamycin (Clin), Macrolide-Lincosamide-Streptogramin (MLS), Macroliode-Lincosamide-Streptogramin-Ketolide (MLSK).] AO 2246 can be seen to possess an excellent level of activity against the wide range of propionibacterium strains tested. This is not predictable from the prior art on bis-phenol antioxidants of this type. Combinations of compounds of formula (I) and their derivatives with salicylic acid and its derivatives can therefore be expected to be active against a range of propionibacteria, and hence to be of use as anti-acne agents. The results are likely to be of particular clinical value for the antibiotic resistant test strains.

Exami'le 3 -activity against Propionibacterium sir, -AO 425 alone The test compound AO 425 (ex Sigma Aldrich, UK) was tested against P. acnes NCTC 737, using MIC and MBC assays as described above. The compound was dissolved in DMSO, and all the experiments were conducted in triplicate. The results are shown in Table 3 below; all are collated from a number of experiments.

Table 3

MIC (igIm1) 7.8 MBC(tgIm1) 31.25 MIC/MBC ratio 0.25 It can be seen from Table 3 that the AO 425 is also highly active as an antibacterial agent against P. acnes NCTC 737. This again indicates its likely activity as an anti-acne agent, and the utility of a combination of AO 425 and salicylic acid in the treatment of acne.

In contrast, other structurally similar alkyl-bis-phenol compounds were found to be far less active against P. acnes NCTC 737 than AO 2246 and AO 425. 2,2'-ethylidene-bis(4,6-di-tert-butylpheno 1) 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.

Exami'le 4 -activity of other salicylates with AO 2246 vs P. acnes The following experiments all used P. acnes NCTC 737 as the test organism. MIC, MBC and disc diffusion (DDA) assays, as described above, were carried out using as the test compounds (a) bismuth subsalicylate, dissolved in DMSO, (b) copper (IT) salicylate, dissolved in DMSO, (c) thiosalicylic acid, dissolved in ethanol, and (d) AO 2246, dissolved in DMSO. The bismuth subsalicylate and thiosalicylic acid were sourced from Sigma Aldrich, UK and the copper (II) salicylate was produced in-house.

Combinations of each of the salicylates with AO 2246 were then subjected to synergy disc diffusion assays (SDDA5). For the (S)DDA tests, 200 tg of each test compound was loaded onto each disc. All experiments were conducted in triplicate. The results are shown in Table 4 below.

Table 4

Compound or combination MIC MBC Average (S)DDA (pg/mi) (pg/in!) zone diameter __________________ _______ ______ (mm �80) A02246 1.95 3.9 9.24�0.36 Bismuth subsalicylate 3.9 7.8 21.54 � 4.05 Copper (II) salicylate 31.25 62.5 46.01 � 1.44 Thiosalicylic acid 250 >250 0 � 0 AO 2246 + bismuth subsalicylate 1.95 3.9 20.28 � 1.55 AO 2246 + copper (II) salicylate 1.95 3.9 46.75 � 2.1 AO 2246 + thiosalicylic acid 1.95 3.9 8.72 � 0.18 The three combinations according to the invention, of AO 2246 plus salicylic acid derivative, can be seen to be highly active against P. acnes NCTC 737. This result again indicates the likely activity of the invented combinations as anti-acne agents.

Exami'le 5 -topical anti-acne formulations The results from Examples 1 to 4 show that the combination of salicylic acid or derivative and a compound of formula (I) can be an effective antibacterial agent, in particular against the bacteria associated with acne. This can be of use in preparing antibacterial formulations, in particular for topical application to the skin, for either prophylactic or therapeutic use in any context where such bacteria are thought to be involved as possible sources of infection. More specifically, it can be of use in preparing anti-acne formulations, again suitably for topical use.

A topical formulation for use in treating acne may for example be prepared by combining salicylic acid, or a pharmaceutically acceptable derivative thereof such as a metal salicylate, with a compound of formula (I) such as AO 2246 or a pharmaceutically acceptable derivative thereof, in a suitable fluid vehicle and optionally together with conventional additives. Such vehicles and additives may be for instance as found in Williams' Transdermal and Topical Drug Deliveiy (see above) and other similar reference books, and/or in Rolland A et al, "Site-specific drug delivery to pilosebaceous structures using polymeric microspheres", Pharm Res 1993; 10: 173 8-44; Mordon S et al, "Site-specific methylene blue delivery to pilosebaceous structures using highly porous nylon microspheres: an experimental evaluation", Lasers Surg Med 2003; 33: 119-25; and Alvarez-Roman R et al, "Skin penetration and distribution of polymeric nanoparticles", J Controlled Release 2004; 99: 53-62.

The formulation may be prepared and administered using known techniques. It may for example take the form of a cream, lotion or in particular a gel. The concentrations of the two active agents may be in the ranges described above, and will be determined based on the intended use of the formulation, its intended mode of administration and the activities of the particular chosen active agents. Suitably, the formulation is administered topically to acne-affected skin.

Claims (18)

1. Claims 1. An antibacterial and/or anti-acne formulation containing (a) salicylic acid or a derivative thereof and (b) a compound of fonTnula (I) or derivative thereof: Formula (I) wherein each of R' and R2 is independently selected from methyl and ethyl and R3 is hydrogen.
2. 2. A formulation according to claim 1, which is suitable for topical application to human skin.
3. 3. A formulation according to claim 1 or claim 2, wherein the salicylic acid derivative is a salicylate.
4. 4. A formulation according to claim 1 or claim 2, wherein the salicylic acid is used in the form of the free acid.
5. 5. A formulation according to any one of the preceding claims, wherein the compound of formula (I) is Antioxidant 2246.
6. 6. A formulation according to any one of the preceding claims, which is in the form of a lotion, cream, ointment, varnish, foam, paste, gel or other viscous or semi-viscous fluid, or of a spray, or of drops, or of a solution or suspension.
7. 7. An antibacterial and/or anti-acne formulation which is substantially as herein described.
8. 8. A product containing an antibacterial or anti-acne formulation according to any one of the preceding claims.
9. 9. A kit for preparing an antibacterial or anti-acne formulation, the kit comprising a source of salicylic acid or a derivative thereof and a source of a compound of formula () 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.
10. 10. A formulation containing (a) salicylic acid or a pharmaceutically acceptable derivative thereof and (b) a compound of fonnula (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 bacterial activity.
11. 11. A formulation containing (a) salicylic acid or a pharmaceutically acceptable derivative thereof and (b) a compound of formula (I) or pharmaceutically acceptable derivative thereof, for use according to claim 10, wherein the bacterial activity is propionibacterial activity.
12. 12. A formulation containing (a) salicylic acid or a pharmaceutically acceptable derivative thereof and (b) a compound of formula (I) or pharmaceutically acceptable derivative thereof, for use according to claim 10 or claim 11, wherein the condition is acne and/or acne lesions.
13. 13. A formulation containing (a) salicylic acid or a pharmaceutically acceptable derivative thereof and (b) a compound of fonnula (I) or pharmaceutically acceptable derivative thereof, for use according to any one of claims 10 to 12, the use being substantially as herein described.
14. 14. Use of(a) salicylic acid or a 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 bacterial activity.
15. 15. Use of (a) salicylic acid or a derivative thereof and (b) a compound of formula (I) or derivative thereof, as an anti-acne or skin care agent for non-therapeutic purposes.
16. 16. A method for controlling the growth of 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 bacterium, a combination of salicylic acid or a derivative thereof and a compound of formula (I) or derivative thereof
17. 17. 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 bacterial activity, the method involving administering to the patient a therapeutically (which term includes prophylactically) effective amount of an antibacterial and/or anti-acne formulation containing (a) salicylic acid or a pharmaceutically acceptable derivative thereof and (b) a compound of formula (I) or pharmaceutically acceptable derivative thereof
18. 18. A method according to claim 17, wherein the condition is acne and/or acne lesions.