GB2442316A - Antimicrobial formulation containing a pyridine thiol and a quinone - Google Patents

Abstract

An antimicrobial formulation contains a pyridine thiol and a quinone. Preferably the quinone is a benzoquinone, a hydroquinone or a mixture thereof; in particular an alkyl-substituted benzo / hydroquinone (e.g. TBHQ, TBBQ). The pyridine thiol may be a pyrithione (e.g. copper or zinc pyrithione). The formulations may be used to treat microbial infections, for example skin and skin structure conditions such as acne. The formulation may be especially useful against propionibacteria (e.g. propionibacterium acnes).

Description

* 2442316 Formulations

Field of the invention

This invention relates to antimicrobial formulations, and to the use of certain combinations of compounds as antimicrobial agents, in particular for the treatment of acne.

Background to the invention

Pyrithione, also known as l-hydroxy-2(IIf)-pyridinethione or 2-pyridinethiol-l-oxide, and commonly known as 2-mercaptopyridine-N-oxide, is a type of pyridine thiol and is known for use as a bactericide and fungicide. It is typically used in the form of a metal salt (strictly, a chelated complex) such as zinc pyrithione. In particular the zinc salt is known as an antiseborrheic, an antifungal and an antibacterial agent, as well as for the treatment of scalp conditions such as dandruff and as an anti-acne agent. JP- 61197508, for example, describes an anti-dandruff shampoo containing zinc pyrithione and a hydroquinone glycoside to enhance its anti-seborrheic activity.

Pyrithione has also been used as a cosmetic preservative, for inhibiting mould growth on fabrics in commercial laundries and as a preservative (persistence agent) for topical antiseptic formulations. (See Guthery, E. et a!, Am. J. Infect. Control 33(1), 2005, 15-22.) It has now surprisingly been found that when a pyridine thiol is combined with a quinone, especially certain types of benzo-or hydroquinone, a synergistic effect can be observed on their combined level of antimicrobial activity, in particular versus propionibacteria and more particularly versus Propionibacterium acnes, the bacterium implicated in inflammatory acne. As a result, novel antimicrobial formulations can be prepared, in particular for topical application, either with improved efficacy and/or containing lower levels of at least one of the active ingredients than would previously have been thought necessary.

Certain quinones, in particular t-butyl hydroquinone (TBHQ), are known for use as antioxidants and in certain contexts have also been used as antimicrobial agents.

TBHQ for example has been used as a preservative to stabilise foodstuffs, cosmetics and even adhesives. It has also been recognised as an antimycotic (DE-44 34 312).

Other quinones of various types have been disclosed for use as antimicrobial agents - see JP-2003-267910, JP-09-255547, JP-04-21 1646, JP-04-21 1644, US-6,228,891, DE- 199 11 680 and GB-I,133,897 -and as preservatives (JP-02202804 and GB-865,808).

However the antimicrobial, and in particular anti-propionibacterial, synergy between a quinone and a pyridine thiol has not to our knowledge been recognised in the past, particularly in the context of topical antimicrobial treatments.

Statements of the invention

According to a first aspect of the present invention there is provided an antimicrobial formulation containing a pyridine thiol (in particular a pyrithione or pyrithione derivative) and a quinone.

This formulation is preferably suitable for topical application to, and/or contact with, the skin, in particular human skin. The pyridine thiol and the quinone are therefore preferably contained in a pharmaceutically acceptable vehicle which can safely be applied to, and/or contacted with, the skin and/or other epithelia.

The formulation may be suitable for topical application to areas such as the flares, eyes, ears, scalp or vagina. Suitability for application to the skin, nares and tissue within the ears may be most preferred. In an embodiment, the formulation is suitable for topical application to the skin. In an embodiment, it is suitable for topical application to the nares and tissue within the ears.

A formulation which is "suitable for" topical application may also be adapted for topical application. .

Suitable vehicles will be well known to those skilled in the art of preparing topical skin care or pharmaceutical preparations. The vehicle will typically be a fluid, which term includes a cream, paste, gel, lotion, foam, ointment or other viscous or semi-viscous fluid, as well as less viscous fluids such as might be used in sprays (for example for nasal use), drops (eg, ear drops) or aerosols. The pyridine thiol and the quinone 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 pyridine thiol and the quinone may, whether separately or together, be carried in or on a delivery vehicle which is suitable for targeting or controlling its release at the intended site of administration. Such vehicles include liposomes and other encapsulating entities, for example niosomes, aspasomes, microsponges, microemulsions, hydrogels and solid lipid nanoparticles.

In the context of the present invention, the term "quinone" means cyclohexadiene-l,4-dione (a benzoquinone), or any similar compound containing two or more C0 groups in an unsaturated ring. A quinone may be present in the form of a hydroquinone (hydroxyquinone), in which one or more of the C=O groups is instead present as a C-OH group, or as a radical in which one or more of the C0 groups is present as C-0.

It may be present as a mixture of two or more of these forms, for instance as an equilibrium mixture of a benzoquinone and its corresponding hydroquinone.

The two C=O groups or C-OH groups of a benzo-or hydroquinone may be positioned ortho, meta or parato one another. When positioned ortho to one another, this is known as a cyclohexadiene-l,2-dione or o- benzoquinone or, in the case of the corresponding hydroquinone, a catechol. When positioned meta to one another, this is known as a cyclohexadiene-1,3-dione or an m-benzoquinone or, in the case of the corresponding hydroquinone, a resorcinol. When positioned para to one another, this is known as a cyclohexadiene-I,4-dione or ap-benzoquinone or, in the case of the para-substituted HO-Ph-OH, simply as ap-hydroquinone.

Preferably the two C=O groups or C-OH groups are positioned either ortho or para to one another, more preferably para as in p-benzoquinone (cyclohexadiene-1,4-dione) or the corresponding para-substituted hydroquinone HO-Ph -OH.

The quinone may for instance be a benzoquinone (by which is meant an optionally substituted cyclohexadiene dione, typically a cyclohexadiene-I,4-dione or cyclohexadiene-I,2-dione) or its corresponding hydroquinone, by which is meant a compound having an optionally substituted unsaturated 6-membered carbon ring, typically a phenyl ring, substituted with two or more -OH groups. It may be a mixture of a benzoquinone and its corresponding hydroquinone. More preferably the quinone is an optionally substituted hydroquinone. As mentioned above, a hydroquinone may be present in the form of a radical in which one or more of the C-OH groups exists as C-Os.

Such compounds may be substituted with one or more groups such as those selected from alkyl groups (in particular C1 to C6 or C1 to C4 alkyl groups, for instance methyl, ethyl, isopropyl or t-butyl groups); alkoxyl groups (in particular C1 to C6 or C1 to C4 alkoxyl groups such as methoxyl or ethoxyl); halogens such as fluoro, chioro or bromo, in particular chioro; nitro groups -NO2; and amino groups -NR2 (where each R is independently either hydrogen or hydrocarbyl, suitably either hydrogen or C1 to C6 alkyl, more suitably either hydrogen or C1 to C4 alkyl, for example either hydrogen, methyl or ethyl), in particular NI-I2. In particular the substituent(s) may be selected from alkyl, alkoxyl and halo groups, more particularly from alkyl and halo groups, most particularly from alkyl groups. The quinone may include up to four such substituents, but in particular may be mono-or di-substituted with such groups, In certain cases it may be preferred for the quinone not to be unsubstituted hydroquinone. In some cases it may be preferred for the quinone not to be More preferably the quinone is either an alkyl-or halo-substituted benzoquinone or an alkyl-or halo-substituted hydroquinone, or a mixture of an alkyl-or halo-substituted benzoquinone and its corresponding hydroquinone. Yet more preferably it is an alkyl-or halo-substituted hydroquinone. Still more preferably it is either an alkyl-substituted benzoquinone or an alkyl-substituted hydroquinone, or a mixture of an alkyl-substituted benzoquinone and its corresponding hydroquinone. In particular it may be .1 A hydroquinone may be substituted with one or more alkyl groups. An alkyl group may be either a straight or a branched chain alkyl group, of which the latter may be preferred, especially where the number of carbon atoms is 3 or greater. It may be or contain cycloalkyl moieties. It may contain for instance from I to 12 carbon atoms, preferably from I to 10, more preferably from 1 to 8. Particularly preferred alkyl groups are those selected from C1 to C6 alkyl groups, more preferably C, to C5 alkyl groups, yet more preferably C1 to C4 alkyl groups, for instance methyl, ethyl, iso-propyl or t-butyl groups.

A substituent such as an alkyl or halo group may be attached to a carbon atom of the cyclohexyl ring or to an oxygen atom (thus replacing the hydrogen atom of a hydroxyl group on the cyclohexyl ring). Preferably it is attached to a carbon atom.

The hydroquinone may be substituted with up to six alkyl groups, more preferably up to four alkyl groups, but in particular may be a mono-or di-alkyl hydroquinone, preferably the former.

The hydroquinone may be substituted with one butyl group, which is preferably present at the 2 position; it may however be substituted with more than one butyl group, for instance two or three or four. A butyl group is preferably a t-butyl group.

The hydroquinone may be substituted with two butyl groups, which preferably occupy the 2 and 5 positions. Again the butyl groups are preferably t-butyl groups.

Instead or in addition, the hydroquinone may be substituted with one hexyl group, which is preferably an 0-substituted hexyl group for instance replacing the hydrogen atom of a hydroxyl group. The hydroquinone may however be substituted with more than one hexyl group, for instance two or three or even four. A hexyl group may be a straight chain hexyl group.

Instead or in addition, the hydroquinone may be substituted with one methyl group, which is preferably present at the 2 position; it may however be substituted with more than one methyl group, for instance two or three or four or even five. It may for instance be substituted with three methyl groups, which are preferably present at the 2, 3 and 5 positions.

Instead or in addition, the hydroquinone may be substituted with one propyl group, suitably an iso-propyl group, which is preferably present at the 2 position. The hydroquinone may however be substituted with more than one propyl group, for instance two or three or four. A propyl group is again suitably an iso-propyl group.

Instead or in addition, the hydroquinone may be substituted with one ethyl group, which is preferably present at the 2 position. It may however be substituted with more than one ethyl group, for instance two, three, four or even five.

Instead or in addition, the hydroquinone may be substituted with one, two, three or even four pentyl (preferably t-amyl) groups.

In particular the hydroquinone may be substituted with three methyl groups and one hexyl group, the hexyl group preferably replacing the hydrogen atom of a hydroxyl group and the three methyl groups preferably occupying the 2, 3 and 5 positions.

In particular the hydroquinone may be substituted with one methyl and one iso-propyl group, which preferably occupy the 5 and the 2 positions respectively.

In particular the hydroquinone may be substituted with just one t-butyl group, which is preferably present at the 2 position.

The hydroquinone may be substituted with up to six halo groups, more preferably up to four halo groups, but in particular may be a mono- or di-halo hydroquinone, preferably the former. A halo group may be for example either fluoro, chioro, bromo or iodo, suitably either chioro or bromo, more suitably chioro.

A substituted hydroquinone may thus be selected from the group consisting of t-butyl hydroquinone (TBHQ); 2,5-di-t-butyl hydroquinone; 2-methyl-p-hydroquinone; 2-ethyl-p-hydroquinone; 2-chloro-p-hydroquinone; and mixtures thereof. It may be selected from the group consisting of TBHQ, 2-methyl-p-hydroquinone, 2-ethyl-p-hydroquinone, 2-chloro-p-hydroquinone and mixtures thereof. It may in particular be a t-butyl hydroquinone (TBHQ), which is a para-hydroquinone substituted at the 2 position with a t-butyl group.

In certain cases it may be preferred for the hydroquinone not to be a resorcinol, in particular unsubstituted resorcinol. In some cases it may be preferred for the hydroquinone not to be a catechol, in particular unsubstituted catechol (pyrocatechol).

In some cases it may be preferred for the hydroquinone not to be an alkyl-substituted resorcinol or catechol, in particular an alkyl-substituted resorcinol, more particularly a C6 to C9 alkyl-substituted resorcinol, most particularly n-hexylresorcinol.

An alkyl-substituted benzoquinone may be substituted with one or more alkyl groups, an alkyl group being as defined above. Such alkyl groups will be attached to carbon atoms of the cyclohexyl ring.

A benzoquinone may be substituted with up to four alkyl groups, but in particular may be a mono-or di-alkyl benzoquinone, preferably the former.

Such a benzoquinone is preferably substituted with one methyl group, which is preferably present at either the 2 or the 5 position; it may be substituted with more than one methyl group, for instance two or three or even four.

Instead or in addition, the benzoquinone is preferably substituted with one propyl group, which is preferably present at the 2 position; it may be substituted with more than one propyl group, for instance two or three or even four. A propyl group is preferably an iso-propyl group.

In particular the benzoquinone may be substituted with one methyl and one iso-propyl group, which preferably occupy the 5 and 2 positions respectively.

The benzoquinone may be substituted with one butyl group (for instance at the 2 position), or with more than one (for instance two, three or four) butyl groups. A butyl group is preferably a t-butyl group.

The benzoquinone may be substituted with two butyl groups, either or preferably both of which is a t-butyl group. These may for instance occupy the 2 and 5 positions, in particular where the benzoquinone is a para-benzoquinone. They may alternatively occupy the 3 and 5 positions, in particular where the benzoquinone is an ortho-benzoquinone.

Instead or in addition, the benzoquinone is preferably substituted with one ethyl group, which is preferably present at the 2 position; it may however be substituted with more than one ethyl group, for instance two or three or even four.

Instead or in addition, the benzoquinone may be substituted with one, two, three or even four pentyl (preferably t-amyl) groups.

Instead or in addition, the benzoquinone may be substituted with one, two, three or even four hexyl groups.

The benzoquinone may be substituted with up to four halo groups, but in particular may be a mono-or di-halo benzoquinone, preferably the former. A halo group may be is as defined above.

A substituted benzoquinone may thus be selected from the group consisting of thymoquinone (which is a para-benzoquinone substituted at the 2 position with an iso- propyl group and at the 5 position with a methyl group); 2,5-di-t-butyl-l,4- benzoquinone; 2-t-butyl-p-benzoquinone; 2-methyl-p-benzoquinone; 2-chloro-p-benzoquinone; and mixtures thereof. It may be selected from the group consisting of 2-t-butyl-p-benzoquinone, 2-methyl-p-benzoquinone, 2-chloro-p-benzoqujnone and mixtures thereof. More preferably the benzoquinone is 2-t-butyl-p-benzoquinone.

In a formulation according to the invention, it may be particularly preferred for the quinone to be selected from TI3HQ (2-t-butyl-p-hydroquinone), 2-t-butyl-p-benzoquinone, 2-ethyl-p-hydroquinone, 2-methyl-p-hydroquinone and mixtures thereof. Most preferred may be TBHQ, 2-t-butyl-p-benzoquinone (also known simply as t-butyl benzoquinone, or TBBQ) and mixtures thereof.

In the present context a quinone, in particular a hydroquinone or benzoquinone, and more particularly an alkyl-substituted hydroquinone or benzoquinone, may be present in the form of a dimer, oligomer or polymer, the monomer unit of which is a quinone as defined above.

A quinone used in the formulation of the invention, in particular thymoquinone, dithymoquinone or thymohydroquinone, is ideally used in the form of the isolated quinone (whether naturally or synthetically derived, preferably the latter) rather than as part of a plant extract containing a number of different materials.

The quinone may be of the type which is active as an antioxidant.

A formulation according to the invention may contain more than one quinone.

The pyridine thiol used in the formulation of the invention may for example be a 2-pyridine thiol, 3-pyridine thiol or 4-pyridine thiol, in particular a 2-or 4-pyridine thiol.

It may be present in the form of a salt or other derivative, for instance a pyridine thiol oxide or hydroxide. It is preferably a pyrithione (ie, an N-oxide pyridine thiol) or tautomer or derivative thereof.

A pyrithione may be present in the form of a pyrithione derivative, eg, a molecular and/or ionic complex containing the pyrithione group, such as for example a pyrithione salt or a dimer, oligomer or polymer containing a pyrithione or pyrithione salt monomer (for example, dipyrithione, also known as di-2-pyridinedisulphjde-I, 1'-dioxide). It may be present in the form of the pyrithione free base.

Suitable salts of pyridine thiols, in particular pyrithiones, include metal salts such as zinc, selenium, silver, copper and sodium salts. A metal salt of a pyridine thiol, in particular pyrithione, may be a divalent metal salt such as for example a zinc, copper (II), calcium, magnesium or cobalt (II) salt.

A pyridine thiol metal salt, in particular a metal pyrithione salt, is preferably either a zinc or a copper (II) salt, most preferably zinc.

Thus preferably the pyridine thiol is present as pyrithione or more preferably a pyrithione salt, in particular a metal salt/complex such as are mentioned above.

A particularly preferred pyridine thiol is zinc pyrithione (zinc-2-pyridinethiol-l -oxide).

A formulation according to the invention may contain more than one pyridine thiol.

s In a formulation according to the invention, both the pyridine thiol and the quinone are present as active (ie, antimicrobially active) agents. Surprisingly, such agents have been found to act together synergistically to inhibit, and often to prevent, microbial activity. In other words, they have been found to increase one another's activity in a maimer which can be synergistic compared to the sum of the activities of the two io agents individually.

It is possible that the potentiation of one another's antimicrobial activity by a quinone and a pyridine thiol may be at least partly due to the formation of a reaction product having an antimicrobial activity greater than the sum of those of the individual reactants. The invention may thus embrace an antimicrobial formulation containing a is reaction product formed between a quinone and a pyridine thiol, in particular between TBHQ and a pyrithione such as zinc pyrithione; this reaction product may be formed in situ immediately prior to, or at the point of, use.

In cases it may be preferred for a formulation according to the invention not to include a peroxide, in particular benzoyl peroxide. In some cases it may be preferred for the formulation not to include methyl p-hydroxybenzoate.

In cases it may be preferred for a formulation according to the invention not to include a phenol, in particular thymol and/or menthol. It may be preferred for the formulation not to contain any radioactive components, in particular a rare earth ore as referred to in JP-2004155901, and/or for it not to contain tourmaline and/or feldspar as referred to in that document.

In cases it may be preferred for a formulation according to the invention not to include a hydroquinone glycoside, for instance as referred to in JP-61 197508.

In a formulation according to the invention the pyridine thiol and the quinone, and their relative proportions, are preferably such as to yield at least an additive level of antimicrobial activity compared to the activities of the individual compounds alone (this is sometimes referred to as an "indifferent" interaction between the compounds).

More preferably, the compounds and their relative proportions are such as to yield a synergistic effect on antimicrobial activity, by which is meant that the antimicrobial activity of the combination of the two compounds is greater than the sum of the individual antimicrobial activities of the same amounts of the two compounds used individually. An increased level of activity in these contexts may be manifested by a lower concentration of the compound(s) being needed to inhibit and/or to kill the relevant organism, and/or by a larger zone of inhibition in a disc diffusion assay, and/or by a faster rate of microbial inhibition or killing.

Antimicrobial activity may be growth inhibitory activity or more preferably biocidal (ie, lethal to the relevant organism). Antibacterial activity encompasses activity against both Gram-positive and Gramnegative bacteria. An antimicrobial formulation according to the present invention is preferably at least antibacterially active, more preferably against Gram-positive bacteria. It may be active against bacteria associated with skin or skin-borne infections, yet more preferably against propionibacteria and most preferably against strains of Propionibacterium acnes.

Thus the formulation may be active against staphylococci (and in cases other Gram-positive cocci such as enterococci and/or streptococci), for example Staphylococcus aureus, and/or against propionibacteria. In an embodiment of the invention, the formulation is active against one or more bacteria associated with acne, for example propionibacteria; it may be active against one or more strains of P. acnes and/or in some instances against one or more strains ofF. granulosum.

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.

The formulation is preferably active against bacteria, in particular propionibacteria, which are wholly or partially resistant to one or more antibiotics, for instance those

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which are in common clinical use. The formulation is ideally active against macrolide- lincosamide-streptogramin (MLS) resistant and/or macrolide-lincosamide-streptogramin-ketolide (MLSK) resistant strains of bacteria. In particular it may be active against erythromycin-resistant, clindamycin-resistant and/or tetracycline-resistant P. acnes strains of bacteria, 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.

Antimicrobial 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 antimicrobial compound, incubating the treated culture under conditions which would ordinarily support growth of the micro-organism, and assessing the level of growth, if any, which can occur in the presence of the candidate compound.

Preferably the pyridine thiol used in the present invention has a minimum inhibitory concentration (MIC), at least against propionibacteria, of 50 ig/ml or less, preferably or 5 pg/ml or less, such as from 0.1 to 10 jg/ml. Its corresponding minimum biocidal concentration (MBC) is preferably 100 tg/ml or less, preferably 50 or 10 .tg/ml or less, such as from 0.1 to 40 g/ml. Suitably the ratio of its MIC to its MBC is from 0.125 to 1, ideally from 0.5 to 1. More preferably the pyridine thiol also exhibits such characteristics in the presence of at least one of, preferably both of, lipid and salt (sodium chloride), for instance as tested in the examples below -these are species which can be present at the surface of the skin and hence perfonnance in this context can be indicative of suitability for use in topical skin treatment formulations, especially in the context of acne treatment.

Preferably the quinone used in the present invention has a minimum inhibitory concentration (MIC), at least against propionibacteria, of 200 tg/m1 or less, preferably or 70 or 50 or 20 or even 10.tg/ml or less, such as from 0.5 to 50 tg/ml. Its corresponding minimum biocidal concentration (MBC) is preferably 200 tg/m1 or less, preferably 150 or 100 or 50 or 20 or even 10 tg/ml or less, such as from 0.5 to 50 tg/ml. Suitably the ratio of its MIC to its MBC is from 0.125 to 1, ideally from 0.5 to 1. More preferably the quinone also exhibits such characteristics in the presence of at least one of, preferably both of, lipid and salt (sodium chloride).

MIC and MBC values may be measured using conventional assay techniques, for instance as described in the examples below.

The concentration of the pyridine thiol in the formulation might suitably be 0.01 % wlv or greater, preferably 0.05 % w/v or 0.1 % w/v or greater. Its concentration might be up to 5 % w/v, or up to 2.5 or 1 or 0.5 or 0.25 % w/v. Its concentration may for example be in the range from 0.5 to 5 % w/v or from 0.5 to 2.5 % w/v.

The concentration of the quinone in the formulation might suitably be 0.05 or 0.1 % w/v or greater, preferably 0.3 % wlv or greater. Its concentration might be up to 5 % w/v, preferably up to 3 or 2 % w/v, more preferably up to I % w/v, such as from 0.05 to 2.5 % w/v or from 0.5 to 2.5 % w/v, for example about 0.5 % w/v.

Due to the presence of the other compound, it may be possible for the concentration of either the pyridine thiol or the quinone, at the site of action when the formulation is applied in vivo, to be less than the MBC, or even than the MIC, of that compound alone. For instance the concentration of at least one of the compounds at this point may be 0.8 or less times its MBC or MIC, such as 0.5 or less, 0.25 or less or 0.125 or less.

Preferably the weight ratio of the pyridine thiol in the formulation to that of the quinone is from 500:1 to 1:500, more preferably from 50:1 to 1:50 or from 1:20 to 20:1, yet more preferably from 1:5 to 5:1, most preferably from 1:5 to 1:1, such as (in particular when used against propionibacteria) about 1:4 or more preferably 1:2.

As described above, the formulation of the invention is preferably suitable for, and more preferably adapted for, topical administration to human skin. It may take the form of a lotion, cream, ointment, foam, paste or gel or any other physical form known for topical administration, including for instance a formulation which is, or may be, applied to a carrier such as a sponge, swab, brush, tissue, cloth, wipe, skin patch or dressing to facilitate its topical administration. It may be intended for pharmaceutical (which includes veterinary) use, and/or for cosmetic or other non-medical care purposes (for example, for general hygiene or cleansing).

The vehicle in which the pyridine thiol and the quinone 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 to those skilled in the art and are readily available commercially. Examples may for instance be found in Williams' "Transdermal and Topical Drug Delivery", Pharmaceutical Press, 2003, and other similar reference books. See also Date, A. A. et al, Skin Pharmacol. Physiol., 2006, 19(1): 2-16 for a review of topical drug delivery strategies. Either or both of the active ingredients may bepresent in the form of a Suspension or other type of multi-phase dispersion, as described above.

Also as described above, the vehicle may be such as to target a desired site and/or time of delivery of the formulation. It may for instance target the formulation to the skin or hair follicles, most preferably to the hair follicles. It may delay or otherwise control release of the formulation over a particular time period. Either or both of the pyridine thiol and the quinone may be microencapsulated, for instance in liposomes or ethosomes -particularly suitable liposomes, for topical use, are those made from stratum comeum 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 the vehicle may be alcohol-based or silicon-based.

By way of example, a lotion or gel formulation may contain a mixture of water, an alcohol such as ethanol or phenoxyethanol and a glycol such as propylene glycol.

A formulation according to the invention may contain standard excipients and/or other additives known for use in pharmaceutical or veterinary formulations, in particular topical skin care formulations. Examples include emollients, perfumes, antioxidants, preservatives, stabilisers, gelling agents and surfactants; others may be found in Williams' "Transdermal and Topical Drug Delivery", supra.

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For the treatment of acne, however, it may be preferred for the formulation not to contain an emollient.

The formulation may further contain additional active agents. For example, it may contain one or more additional 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 (in particular antibacterial) agents and mixtures thereof. It may in particular contain one or more agents selected from anti-acne agents, keratolytics, comedolytics, sebostatic/sebosuppressive agents, anti-inflammatories and additional antibacterial agents. It may instead or in addition contain one or more agents selected from sunscreens, moisturisers and mixtures thereof.

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.

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 pyridine thiol and the quinone to be the only active agents in the formulation, or at least to be the only antimicrobially or antibacterially active agents and/or the only anti-acne active agents.

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, a pharmaceutical (which includes veterinary) or cosmeceutical preparation or a toiletry product (for instance a bath or shower additive or a cleansing preparation).

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The invention provides, according to a second aspect, a product which incorporates an antimicrobial formulation according to the first aspect of the invention.

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. Thus according to a third aspect, the present invention provides a kit for preparing an antimicrobial formulation according to the first aspect, the kit comprising a source of a pyridine thiol and a source of a quinone, 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, for instance 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 a pyridine thiol and a quinone, 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 formulation, which method involves mixing together a pyridine thiol and a quinone, 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 a pyridine thiol and a quinone, for use in the treatment of a condition which is caused by, transmitted by and/or exacerbated by (in particular caused and/or transmitted by) microbial, in particular bacterial, activity.

According to an embodiment of this fifth aspect of the invention, the formulation is for use in the treatment of a skin or skin structure condition. It may in particular be for use in the treatment of acne (ie, as an anti-acne agent).

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

V

condition, in either a human or animal but in particular a human. ft may involve complete or partial eradication of the condition, removal or amelioration of associated symptoms, arresting or slowing 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 fonnulation as a microbiocide, in particular as a bactericide and/or bacteriostatic agent, more particularly against propionibacteria and/or Gram-positive cocci such as staphylococci or streptococci. Most particularly it may involve use of the formulation against propionibacteria.

Treatment may involve the prophylactic treatment of any area of the body, in particular the skin or nares or another epithelial or mucosal surface, against micro-organism infections, including against staphylococcal infections such as those associated with MRSA, and in particular against propionibacterial infections.

Skin and skin structure conditions which might be treated according to the invention include acne, eczema, superficial infected traumatic lesions, wounds, bums, ulcers, folliculitis, mycoses and other primary and secondary skin and skin structure infections. In particular the 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, charactensed 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.

The treatment of acne also encompasses the treatment andior reduction of lesions and/or scarring associated with acne.

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.

In another embodiment of the invention, the formulation may be for use as a therapeutic or prophylactic treatment against staphylococci on the skin, or in the nares or ears, which might otherwise cause for example MRSA-associated infections.

According to the fifth aspect of the invention, the formulation of pyridine thiol and s quinone 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 pyridine thiol and a quinone in the treatment of a microbial condition, the two compounds being administered either simultaneously or sequentially.

According to a sixth aspect, the invention provides the use of a pyridine thiol and a quinone 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 caused and/or transmitted by) microbial, especially bacterial, activity. As discussed above, either or preferably both of the pyridine thiol and the quinone may be included in the medicament as an antimicrobial, in particular antibacterial, agent, and/or as an IS anti-acne active agent.

The medicament may be for the treatment of a condition which is caused by, transmitted by and/or exacerbated by Gram-positive cocci such as staphylococci. !t may be for the treatment of a skin or skin structure condition such as acne.

The invention further provides, according to a seventh aspect, the use together of a pyridine thiol and a quinone, as an antimicrobial agent, in particular as a bactericide, and/or as an anti-acne agent, or in the manufacture of an antimicrobial or specifically antibacterial formulation or of an anti-acne formulation.

An eighth aspect provides a method for controlling the growth of a micro-organism, in particular a bacterium such as a propionibacterium or a staphylococcus, the method comprising applying, to an area or surface which is infected or suspected to be infected or capable of becoming infected with the micro-organism, a combination of a pyridine thiol and a quinone. Again the two compounds may be applied simultaneously or sequentially.

In this context, "controlling the growth" of a micro-organism embraces inhibiting or preventing its growth, whether completely or partially, as well as killing or inactivating 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.

The method of the invention may thus be used to treat an existing occurrence of the organism or to prevent, or reduce the risk of, a potential subsequent occurrence.

Again the area or surface to which the pyridine thiol and the quinone are applied will typically be living tissue, in particular human skin. In this case the pyridine thiol and the quinone may be applied for therapeutic purposes or for non-therapeutic (eg, purely cosmetic) purposes. Thus the method of the eighth 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 caused and/or transmitted by) microbial, especially bacterial, activity, for example acne, the method involving administering to the patient a therapeutically or prophylactically effective amount of an antimicrobial formulation containing a pyridine thiol and a quinone.

The method of the eighth aspect of the invention preferably involves applying a formulation according to the first aspect.

A ninth aspect of the invention provides the use of a pyridine thiol in an antimicrobial or anti-acne formulation, in combination with a quinone, for the purpose of increasing the antimicrobial (in particular antibacterial) and/or anti-acne activity of the formulation and/or of reducing the amount of the quinone in the formulation 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 quinone alone, at the same concentration as used when combined with the pyridine thiol. Ideally the increase is as compared to the sum of the activities of the pyridine thiol and the quinone individually, again at the same respective concentrations as used A reduction in the amount of the quinone 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 s would otherwise have been observed during use of the formulation, for example local irritation and/or undesirable systemic absorption of the quinone. According to the invention, the pyridine thiol may therefore be used for the dual purposes of reducing an undesired property of a formulation containing a quinone, without undue loss of antimicrobial or anti-acne activity.

Preferably the pyridine thiol is used without any reduction in antimicrobial or anti-acne activity compared to the level exhibited by the formulation prior to addition of the pyridine thiol. More preferably it is used to give an increase in antimicrobial and/or anti-acne activity. It may however be used to reduce the amount of the quinone 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 tenth aspect of the invention provides the use of a quinone in an antimicrobial or anti-acne formulation, in combination with a pyridine thiol, for the purpose of increasing the antimicrobial (in particular antibacterial) and/or anti-acne activity of the formulation and/or of reducing the amount of the pyridine thiol in the formulation 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 pyridine thiol alone, at the same concentration as used when combined with the quinone. Ideally the increase is as compared to the sum of the activities of the quinone and pyridine thiol individually, again at the same respective concentrations as used A reduction in the amount of the pyridine thiol 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 pyridine thiol.

According to the invention, the quinone may therefore be used for the dual purposes of reducing an undesired property of a formulation containing a pyridine thiol, without undue loss of antimicrobial and/or anti-acne activity.

Preferably the quinone is used without any reduction in antimicrobial or anti-acne activity compared to the level exhibited by the formulation prior to addition of the quinone. More preferably it is used to give an increase in antimicrobial and/or anti-acne activity. It may however be used to reduce the amount of the pyridine thiol 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.

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.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

Other features of the present 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 b 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 S 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 and the accompanying Figure 1, which is an isobologram showing FIC (fractional inhibitoiy concentration) values for mixtures of TBHQ with zinc pyrithione against a propionibacterial strain, as referred to in Example 2 below.

Detailed descriinion Experimental tests were conducted to determine the antimicrobial activities of formulations according to the invention. As a comparison, the antimicrobial activities of formulations containing a quinone or a pyridine thiol alone were also measured.

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

Other propionibacterial strains, including some P. granulosum strains and including some having antibiotic resistance, were also used as test organisms in Example 4.

Propionibacteria are known to be involved in acne, which is a complex, multi-factorial skin disease in which P. acnes and other Propionibacteriupn spp. play key roles.

Activity observed against the chosen test organisms is therefore expected to be a reasonable qualitative predictor of activity against micro-organisms responsible for skin and skin structure infections, in particular acne.

All the propionibacteria were cultured and maintained on Wilkins-Chaigren 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 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 jil of liquid per well. The wells contained liquid culture medium and ranges of decreasing concentrations of the relevant test compound in doubling dilutions (e.g., 1000, 500, 250, 125... igIml, etc..

down to 0.49 j.tg/ml). The culture medium was 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, i.e., the lowest concentration for which the liquid in the well remained clear.

The assays were conducted in duplicate (minimum) and 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 bactericidal concentration (MBC) assay This assay, normally carried out after an MIC assay, detennines the minimum concentration of a compound that is lethal to the micro-organism being tested.

Following an MIC assay, a 5 jil 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 lest compound concentration for which the incubated sample showed no growth.

The ratio of MIC to MBC should ideally be as close to I 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 d?[fusion assay (DDA) This is an internationally recognised standard method for qualitatively assessing the antimicrobial activity of a compound.

A sterile paper disc was impregnated with a sample of the test compound in a suitable solvent and 30 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 is 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 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 Pr0I0COLTM 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, although other factors such as test compound mobility through the agar gel may also influence the result.

(d) Synergy disc d[fusion 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 In practical terms, an increase of greater than 5 mm could be treated as significant.

(e) Supplemented disc diffision assays Either the DDA or the SDDA tests may be carried out using an agar gel supplemented with lipid and/or salt to simulate some of the major components present in human skin and to assess whether these substances might reduce the antimicrobial activity observed for the test compounds. Performance under these conditions can provide a more reliable indication of activity on topical application. The supplements used in Examples 1 and 3 below were lipid (Triolein at I % v/v) and sodium chloride (100 mM.

(f) Fractional inhibitoiy concentration (FIC) assay This assay was used to determine the mode of interaction between two antimicrobial test compounds A and B. It was similar to the MIC assay, utilising a 96-well microtitre plate and liquid culture medium. The test compounds were added together to each well at a range of concentrations starting at their respective MIC values and descending in doubling dilutions as with the MIC assay. Typically an 8 x 8 array of wells could be used to combine 8 different concentrations of compound A (from its MIC downwards, including zero) with 8 different concentrations of compound B (ditto).

The wells were inoculated with freshly grown micro-organism and incubated under the conditions described above.

As for the MIC assay, the results were read by the naked eye. A minimum inhibitory concentration was recorded for each combination of A and B. A fractional FIC index (FICI) was then calculated for each compound in that mixture, and these two indices were added together to give an overall FICI indicative of the mode of' interaction.

Thus for each mixture tested, the FIC for compound A (FICA) = MIC for (A + B) / MJC for A alone. Similarly the FIC for compound B (FIC) = MIC for (A + B) / MJC for B alone. The overall FICI = FICA + FICB. * .

An FICI of 0.5 or less was taken to indicate synergy, a value greater than 0.5 to 4.0 an indifferent effect and values greater than 4.0 antagonism (i.e., the two compounds counter one another's activity, leading overall to a diminished antimicrobial effect) (see Odds FC, "Synergy, antagonism, and what the chequerboard puts between them", JAntimicrob Chemot her, 2003; 52:1). These results can be depicted visually on a plot (isobolograrn) of FICA against FICB for the mixtures tested.

Example I -activity a2ainst P. acnes (MIC, MBC and (S)DDA assays) The following experiments all used P. acnes NCTC 737 as the test organism.

MIC, MBC and DDA assays, as described above, were carried out using the test compounds (a) t-butyl-p-hydroquinone (TBHQ), dissolved in ethanol, and (b) zinc pyrithione (ZP), dissolved in DMSO. Both test compounds were sourced from Sigma-Aldrich, UK.

Mixtures of TBHQ and ZP were then subjected to SDDA assays as described above, including in the presence of salt and lipid (SL-(S)DDA measurements). Increases in zone diameter (mm) were measured with respect to the ZP in unsupplemented assays and the TBHQ in supplemented assays, these being the compounds showing the larger zones of inhibition during the previous individual disc diffusion assays.

For the (S)DDA experiments, 200.tg of TBHQ or ZP was loaded onto each disc.

All the (S)DDA experiments were conducted in triplicate.

The MIC and MBC results are shown in Table I below and the (S)DDA results in Tables 2 (unsupplemented) and 3 (supplemented). All results are collated from a number of experiments.

Table I

Test compound MIC MBC MICMBC L () () Test compound MIC MBC MJC/MBC (pg/mi) (pg/rn!) ratio TBHQ 7.8 7.8 ZP 0.98 7.8 0.125 Table 2 (unsupplemented (SDDAs') Test compound DDA SDDA SDDA SDD4 area (mm) with increase increase (%) TBHQ (mm) ___________ (mm) _____ TBHQ 9.27 _________ zP r-8,29 90 12 Table 3 (supplemented (S)DDAs) Test compound SL-DDA SL-SDDA SL-SDDA SL-SDDA (mm) with increase area TBHQ (mm) increase (%) ___________ (mm) ___ TBHQ 53.64 ________ _______ ( 2.77) ______ zp ( 088) ( 1.99) 5 7 22 38 (SL-(S)DDA = (S)DDA carried out in the presence of salt and lipid) These data show that both of the test compounds are active against P. acnes NCTC 737. When the TBHQ is combined with the pyrithione, however, the data demonstrate a synergistic antimicrobial interaction between the two test compounds, with a to significant increase in zone diameter over that exhibited by either compound alone This synergistic interaction is maintained in the presence of salt and lipid.

Example 2 -activity against P acnes (FIC assays.) Mixtures of TBHQ with ZP, containing various relative proportions of the two actives, were then subjected to FIC assays against P. acnes NCTC 737, as described above.

The results were used to prepare FIC isobolograms. All assays were conducted in triplicate.

A representative isobologra.m is shown in Figure 1; the dashed line indicates where overall FICIs (ie, FICTBHQ + FICzp) equal 1, which would indicate a purely indifferent interaction. The figure clearly demonstrates the synergistic activity of the quinone/pyrithione combination against P. acnes NCTC 737, the overall FICI being 0.37.

Example 3-activity against P. acnes (other quinones) A series of quinones was tested with zinc pyrithione (ZP) in a similar manner to thatdescribed in Example 1. The MIC and MBC results are shown in Table 4 below and the (S)DDA results in Tables 5 (unsupplemented assays) and 6 (assays supplemented with salt and lipid). All results are collated from a number of experiments.

For the (S)DDA experiments, 200.*g of each test compound was loaded onto each disc with the exception of 2-t-butyl-p-benzoquinone which was used at 50 g per disc in unsupplemented and 100 tg per disc in supplemented assays. The test compounds were dissolved in DMSO, with the exception ofp-benzoquinone and thymoquinone which were dissolved in ethanol.

Table 4

Test compound MIC MBC MIC/MBC _________________________________ (pg/mi) (pg/mi) ratio 2-t-butyl-p-benzoquinone 7.8 15.6 0.5 2-methyl-p-hydroquinone 62.5 125 0.5 2-methyl-p-ben.zoquinone 15.6 31.25 0.5 2-chloro-p-benzoquinone 62.5 125 0.5 2-chloro-p-hydroquinone 125 >250 <0.5 2-ethyl-p-hydroquinone 7 8 31.25 0.25 Test compound MIC MBC MJC/MBC (pg/mi) (pg/mi) ratio p-benzoquinone 62.5 62 5 1 Thymoquinone 15.6 31.25 05 Table 5 (unsupplemented (S)DDAs Test compound DDA SDDA SDDA SDDA area (mm) with ZP increase increase (%) 21.88 Zinc pynthione (ZP) ___________________________________________ ____________________________________ ( 1.86) 2-t-butyl-p-benzoqurnone 15.76 146.8 ___________________________________ ( 0.47) ( 1 81) __________ 000 3192 2-methyl-p-hydroquinone 10 04 112.8 _________________________________ ( 0.0) ( 6.55) _________ 3450 4140 690 440 2-methyl -p-benzoquinone ( 0.18) ( 1.11) __________ 70 47.68 2-chloro-p-benzoquinone 25 80 374 7 ______________________________________ ( 0 62) ( 6 34) ___________ 0.00 31.72 9.83 1101 2..chloro-p-hydroquinone ( 0.0) ( 1 70) __________ 0.00 33 37 2-ethyl-p-hydroquinone 11.48 132.5 ________________________________ ( 0.0) ( 7.13) _________ 35.43 41 60 6. 18 37.9 p-benzoquinone ( 0.47) ( 1.29) __________ 37.38 5448 1710 112.4 Thymoquinone ( 1.42) ( 1 55) __________ Table 6 (supplemented (S)DDAs) Test compound SL-DDA SL-SDDA SL-SDDA SL-SDDA (mm) with ZP increase (mm) Zinc pynthione (ZP) 3135 mmincreasejj 61.68 2-t-butyl-p-benzoquinone -1 43 -4.58 ____________________________________ ( 0.3 1) ( 2.75) ___________ ____________ 36 13 5730 2-methyl-p-hydroquinone 2117 151 52 _________________________________ ( 2.17) ( 2.90) __________ 44.99 51 60 2-methyl-p-benzoquinone 6.61 31 54 ___________________________________ ( 0.98) ( 1 40) __________ 36.44 4926 2-chloro-p-benzoquinone 12.82 82 74 ______________________________________ ( 3.94) ( 1 87) ___________ Test compound SL-DDA I SL-SDDA SL-SDD4 SL-SDDA (mm) with ZP increase area _________ (mm) (mm) increase (%) 2-chloro-p-hydroquinone 17.50 142.80 2-ethyl-p-hydroquinone ( 10.82) -22.08 190.47 (SL-(S)DDA = (S)DDA carried Out in the presence of salt and lipid) Again these data demonstrate a synergistic antimicrobial interaction when a quinone is combined with zinc pyrithione, there being a significant increase in zone diameter over that exhibited by either compound alone. This synergistic interaction is maintained in nearly all cases in the presence of salt and lipid; furthermore the activity of the quinones alone appears in all cases to be enhanced by the presence of the supplements.

Indeed, in some cases, for example the combination of 2-methyl-p-hydroquinone or 2-ethyl-p-hydroquinone with zinc pyrithione, antimicrobial synergy appears to be far io more marked under the supplemented conditions than the unsupplemented ones, indicating the potential value of such a combination in topical skin treatment formulations, in particular to treat acne.

Example 4-activity aRains: other yropionibacteria The activities of TBHQ and ZP against a panel of different propionibacteria were assessed using the MIC and MBC assays described above The results are shown in Table 7, which also indicates the resistance phenotype for each of the test strains.

Table 7

ResiNtance TBHQ TBHQ ZPMIC ZPMBC Test organism phenotype MIC MBC (pg/mi) (pg/rn!) ________ (pg/rn!) (pg/mi) _______ P. granulosum NCTC 11865 None 3 9 7.8 0.98 7.8 P. acnes PRP-002 Tet/MLS 3.9 7.8 0.49 7.8 P. acnes PRP-003 Tet 7.8 7.8 0 24 15.6 Resistance TBHQ TBHQ ZPMJC ZPMBC Test organism phenotype MIC MBC (pg/mi) (pg/mi) ___________________ _______ (pg/mi) (pg/mi) _______ _______ P. acnes PRP-004 Tet 1.95 7.8 0.49 7.8 P. granulosum PRP-005 MLSK 62.5 62.5 0.98 7.8 P. granulosuin PRP-006 MLS 7.8 7.8 0.49 7.8 P. acnesPRP-007 Clin 3.9 7.8 1.95 15.6 P. acnes PRP-008 Clin 3.9 7.8 0.98 7.8 P. acnes PRP-010 MLSK 3.9 15.6 3.9 15.6 P. acnesPRP-017 MLS 3.9 7.8 0.98 15.6 P. granulosum PRP-019 MLSK 31.25 31.25 1.95 3.9 P. granulosum PRP02 1 MLS 15.6 31.25 0.49 3.9 P. acnes PRP-023 MLSK 3.9 7.8 3.9 7.8 P. acnes PRP-026 MLS 3.9 7.8 9.9 31.25 P. acnes PRP-039 Tet/MLS 3.9 7.8 0.12 7 8 P. granulosum PR.P-043 MLS 15.6 15.6 0.49 1.95 P. granulosum PRP-044 MLS 15 6 31.25 3.9 7.8 P. acnes PRP-046 None 1.95 7.8 1.95 7.8 P. acnes PRP-053 TetJMLS 3 9 7.8 1.95 15 6 P. granulosum PRP-055 None 3.9 7.8 0.98 7.8 P. acnes PRP-059 MLS 3.9 7.8 3.9 7.8 P. acnes PRP-068 Ery 3.9 7.8 0.49 62.5 P. acnes PRP-101 Te1JMLS 3.9 7.8 0.49 15.6 P. acnesPRp-102 Tet/MLS 7.8 15.6 0.98 3125 [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)J (S)DDA assays were then conducted on the two test compounds individually and in 3' combination, against the same panel of test organisms. The solvents used were ethanol for the TBHQ and DMSO for the ZP, and again 200.ig of each test compound was loaded onto each disc. The results are shown in Table 8.

Table 8

TBHQ DDA ZPDDA TBHQ ZP SDDA SDDA area Test organism (mm) (mm) SDDA (mm) increase increase (%) ________________ _______ _______ _______ (mm) _______ P. granidosum NCTC 11865 11.50 47.84 51.68 3.48 16.70 ________________________ ( 0.93) ( 2.04) ( 3.32) __________ ___________ P. acnes PRP-002 18.12 40.28 46.50 6.22 33.27 _____________________ ( 0.18) ( 1.26) ( 0.47) _________ __________ P. acnes PRP-003 18.12 27.24 42.15 14.91 139.43 ________________________ ( 0.47) ( 4.31) ( 2.89) __________ ___________ P. acnes PRP-004 19.88 31.59 33.66 2.07 13.53 ______________________ ( 1.08) ( 2.07) ( 1.82) _________ __________ P. granulosum PRP-005 10.67 28.79 41.94 13.15 112.21 ______________________ (*0.72) ( 4.04) ( 1.61) _________ ___________ P. granulosum PRP-006 9.42 40.49 704 37.79 ________________________ ( 0.78) ( 0.65) ( 0.62) __________ ___________ P. acnes PRP-007 12.74 16.88 31.27 14.39 243.17 ______________________ ( 0.82) ( 1.79) ( 2.59) _________ __________ P. acnes PRP-008 17.92 28.38 32.41 4.02 30.32 ________________________ (*2.00) ( 1.09) ( 1.40) __________ ___________ P. acnesPRp-OIO 18.54 14. 39 19.16 443.58 ________________________ ( 1.40) ( 2.53) ( 1.35) __________ ___________ P. acnes PRP-017 13.98 14.19 24.96 10.77 209.40 _______________________ ( 0.93) (*1.09) ( 0.18) _________ ___________ P. granulosum PRP-019 10.36 25.27 32.83 7.56 68.78 ________________________ ( 0.47) ( 4.58) ( 3.41) __________ ___________ P. granulosum PRP-02 1 0. 00 40.49 42.77 2.28 11.58 ______________________ ( 0) ( 1.47) ( 1.87) _________ __________ P. acnes PRP-023 18.54 23.30 36.97 13.67 151.76 ______________________ ( 1.77) (*1.08) ( 2.04) _________ __________ P. acnes PR.P-026 13.98 29.41 39.66 10.28 81.65 _______________________ ( 0.82) ( 0.47) ( 2.18) _________ ___________ P. acnes PRP-039 17.71 32. 83 38.42 36.95 ________________________ ( 0.31) ( 3.29) ( 2.87) __________ ___________ P. granulc'sum PRP-043 0.00 29.10 35.00 5.90 44.66 ________________________ ( 0) ( 1.26) ( 0.36) __________ ___________ P. granulosum PRP-044 0.00 28.17 36.14 64.59 ________________________ ( 0) ( 4.66) ( 2.33) __________ ___________ P. acnes PRP-046 20.71 27.75 47.02 19.27 187.10 ______________________ ( 0.36) ( 1.09) ( 4.14) _________ __________ P. acm's PRP-053 17.09 29.93 41.01 11.08 87.74 ________________________ ( 0.82) ( 2.53) ( 2.47) __________ ___________ P. granu/osum PR.P-055 18.02 53.64 53.85 0.21 0.78 ________________________ ( 0.62) ( 1.29) ( 1.00) __________ ___________ * . .1 TBHQ DDA ZP DDA TBHQ + ZP SDDA -SDDA area Test organism (mm) (mm) SDDA (mm) increase increase (%) ________________ ________ _______ _______ (mm) ________ P. acnes PRP-059 19.88 18.74 41.73 22 99 395 86 ______________________ ( 1.89) ( 1.82) ( 1.40) _________ __________ P. acnes PRP-068 16.78 20.82 41.84 21 02 303 85 _______________________ ( 0.93) ( 1.42) ( 3.49) __________ ___________ P. acnes PRP-101 12.84 24.34 36.14 11 80 12046 _____________________ ( 1.26) ( 1.77) ( 1.26) _________ __________ P. acnes PRP-102 14.50 21.33 39.25 17 92 238 61 ________________________ ( 2.25) ( 1.90) ( 3.68) __________ ___________ These data show both TBHQ and ZP to be active against all of the propionibactenal strains tested. Combinations of the two actives, however, appear from the SDDA results to be acting synergistically against most of the test organisms, with significant increases in zone diameters and areas. This potentiation of antibacterial activity is observed against many of the antibiotic resistant strains, a fact likely to be of considerable clinical importance.

Example 5-activity against Propionibacterium spp (copper pvriihi one) Copper (II) pyrithione (CuP) was tested against P. acnes NCTC 737 with a number of quinones, using the same procedure as in Example 3. The MIC and MBC results are shown in Table 9 below and the (S)DDA results in Table 10. All results are collated from a number of experiments.

For the (S)DDA experiments, 200 tg of each test compound was loaded onto each disc, with the exception of 2-t-butyl-p-benzoquinone which was used at 100.ig per disc. The TBI-IQ was dissolved in ethanol and the copper pyrithione, 2-methyl-p.

benzoquinone and 2-chloro-p-benzoquinone in DMSO.

Table 9

Test compound MIC MBC MIC/MBC _________________________________ (pg/mi) (pg/mi) Ratio Copper pyrithione 0 24 0.49 0.5 TBHQ 7.8 7.8 Test compound MIC MBC MIC/MBC _________________________________ (pg/mi) (pg/mi) Ratio 2-t-butyl-p-benzoquinone 7.8 15.6 0.5 2-methyl-p-benzoquinone 15 6 31.25 0.5 2-chloro-p-benzoquinone 62.5 125 0.5

Table 10

Test compound DDA SDDA SDDA SDJJA area ( 0.48) .. .. -r-i..

Copper pyrithione (CuP) or __________________________________ 41.03 * _____________________________ _______________.

TBHQ ( 1.13) ( 2.66) 5.01 18.53 * * 52.61 56.33 2-t-butyl-p-benzoquinone ( 2.81) ( 1.00) 3.72 14 64 * 39.90 48.17 2.methy1pbenzoquinonc* ( 0 95) ( 0 18) 7.14 37.83 2543 47.13 2chlorop.benzoquInone* ( 0.54) ( 0 82) 6.10 31.95 * Denotes data obtained in a separate series of experiments to those involving TBHQ and CuP.

These data demonstrate a synergistic antimicrobial interaction when a quinone is combined with copper pyrithione, there being in nearly all cases a significant increase in zone diameter over that exhibited by either compound alone.

Example 6 -topical anti-acne formulations The results from Examples I to 5 show that the combination of a quinone and a pyridine thiol can be an effective antimicrobial agent, in particular against the bacteria associated with acne, with a synergistic impact on the antimicrobial activity of the combination compared to those of the individual compounds alone. This can be of use in preparing antimicrobial 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.

Even in cases where a combination of a quinone and a pyridine thiol has an additive, as opposed to synergistic, antimicrobial activity compared to those of the individual compounds, this can be of considerable benefit when preparing formulations for topical use. One of the compounds may be used to replace a proportion of the other, thus lowering any side effects and/or other undesirable properties of the combination without undue loss of antimicrobial activity.

A topical formulation for use in treating acne may for example be prepared by combining a quinone, in particular an alkyl-substituted hydroquinone such as TBHQ, with a pyridine thiol, in particular a pyrithione such as zinc pyrithione, 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 Delivery", Pharmaceutical Press, 2003 and other similar reference books, and/or in Rolland A et a!, "Site-specific drug delivery to pilosebaceous structures using polymeric microspheres", Pharm. Res. 1993; 10: 1738-44; Mordon S eta!, "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 Re! a!, "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 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.

Claims (45)

1. Claims 1. An antimicrobial formulation containing a pyridine thiol and

   a quinone.
2. 2. A formulation according to claim 1, which is suitable for topical application.
3. 3. A formulation according to claim 2, which is suitable for topical application to the skin.
4. 4. A formulation according to any one of the preceding claims, wherein the quinone is a benzoquinone, a hydroquinone or a mixture thereof.
5. 5. A formulation according to claim 4, wherein the two C0 groups or C-OH groups of the benzoquinone or hydroquinone are positioned para to one another.
6. 6. A formulation according to claim 4 or claim 5, wherein the quinone is an alkyl-substituted benzoquinone, an alkyl-substituted hydroquinone or a mixture thereof.
7. 7. A formulation according to claim 6, wherein the quinone is a mono-or di-alkyl benzoquinone, hydroquinone or mixture thereof.
8. 8. A formulation according to claim 6 or claim 7, wherein the quinone is an alkyl-
9. 9. A formulation according to any one of claims 6 to 8, wherein the quinone is a benzoquinone or hydroquinone substituted with one or more C1 to C4 alkyl groups.
10. 10. A formulation according to any one of the preceding claims, wherein the quinone is selected from the group consisting of TBHQ (2-t-butyl-p-hydroquinone), 2-t-butyl-p-benzoquinone (TBBQ), 2-ethyl-p-hydroquinone, 2-methyl-p-hydroquinone and mixtures thereof.
11. II. A formulation according to claim 10, wherein the quinone is selected from TBHQ, TBBQ and mixtures thereof.
12. 12. A formulation according to any one of the preceding claims, wherein the quinone is active as an antioxidant.
13. 13. A formulation according to any one of the preceding claims, wherein the pyridine thiol is a pyrithione or tautomer or derivative thereof.
14. 14. A formulation according to claim 13, wherein the pyrithione is present in the form of a metal pyrithione salt
15. 15. A formulation according to claim 14, wherein the pyrithione is selected from the group consisting of zinc and copper (H) pyrithiones.
16. 16. A formulation according to claim 15, wherein the pyrithione is zinc pyrithione.
17. 17. A formulation according to any one of the preceding claims, wherein the concentration of the pyridine thiol is 0.05 % w/v or greater.
18. 18. A formulation according to any one of the preceding claims, wherein the is concentration of the quinone is 0.1 % w/v or greater.
19. 19. A formulation according to any one of the preceding claims, wherein the concentration of the pyridine thiol is up to 2.5 % wlv.
20. 20. A formulation according to any one of the preceding claims, wherein the concentration of the quinone is up to 2.5 % w/v.
21. 21. A formulation according to any one of the preceding claims, wherein the weight ratio of the pyridine thiol to the quinone is from 50:1 to 1:50.
22. 22. A formulation according to claim 21, wherein the weight ratio of the pyridine thiol to the quinone is from 1:5 to 1:1.
23. 23. A formulation according to any one of the preceding claims, which additionally contains one or more agents selected from anti-acne agents, keratolytics, comedolytics, sebostatic/sebosuppressive agents, anti-inflammatories, additional antibacterial agents and mixtures thereof.
24. 24. A formulation according to any one of the preceding claims, which is in the form of a cream, paste, gel, ointment, lotion, foam or other viscous or semi-viscous fluid.
25. 25. An antimicrobial formulation substantially as herein described.
26. 26. A product containing an antimicrobial formulation according to any one of the preceding claims.
27. 27. A product according to claim 26, which is a cosmetic, a skin care preparation, a pharmaceutical or cosmeceutical preparation or a toiletry product.
28. 28. A kit for preparing an antimicrobial formulation, the kit comprising a source of a pyridine thiol and a source of a qwnone, 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.
29. 29. A method for preparing an antimicrobial formulation, which method involves mixing together a pyridine thiol and a quinone.
30. 30. A formulation containing a pyridine thiol and a quinone, 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 activity.
31. 31. A formulation containing a pyridine thiol and a quinone, for use according to claim 30, wherein the condition is caused by, transmitted by and/or exacerbated by bacterial activity.
32. 32. A formulation containing a pyridine thiol and a quinone, for use according to claim 30 or claim 31, wherein the condition is a skin or skin structure condition.
33. 33. A formulation containing a pyridine thiol and a quinone, for use according to claim 32, wherein the condition comprises acne and/or acne lesions.
34. 34. A formulation containing a pyridine thiol and a quinone, for use according to claim 31, against one or more bacteria associated with acne.
35. 35. A formulation containing a pyridine thiol and a quinone, for use according to claim 31 or claim 34, wherein the bacteria include one or more propionibacteria.
36. 36. A formulation containing a pyridine thiol and a quinone, for use according to claim 35, wherein the bacteria include one or more erythromycin-resistant, clindamycin-resistant and/or tetracycline-resistant P. acnes strains of bacteria.
37. 37. A formulation containing a pyridine thiol and a quinone, for use according to any one of claims 30 to 36, wherein the formulation is prepared in situ, at or immediately before the point of administration, and/or wherein the pyridine thiol and the quinone are administered either simultaneously or sequentially.
38. 38. Use of a pyridine thiol and a quinone 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.
39. 39. Use according to claim 38, wherein the condition is a skin or skin structure condition.
40. 40. Use according to claim 39, wherein the condition comprises acne and/or acne lesions.
41. 41. 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

    I

    exacerbated by microbial, in particular bacterial, activity, the method involving administering to the patient a therapeutically or prophylactically effective amount of an antimicrobial formulation containing a pyridine thiol and a quinone.
42. 42. A method according to claim 41, wherein the condition is a skin or skin structure condition.
43. 43. A method according to claim 42, wherein the condition comprises acne and/or acne lesions.
44. 44. Use of a pyridine thiol in an antimicrobial or anti-acne formulation, in combination with a quinone, for the purpose of increasing the antimicrobial and/or anti-acne activity of the formulation and/or of reducing the amount of the quinone in the formulation without undue loss of antimicrobial or anti-acne activity.
45. 45. Use of a quinone in an antimicrobial or anti-acne formulation, in combination with a pyridine thiol, for the purpose of increasing the antimicrobial and/or anti-acne activity of the formulation and/or of reducing the amount of the pyridine thiol in the formulation without undue loss of antimicrobial or anti-acne activity.