WO2010061185A2

WO2010061185A2 - Therapeutic target

Info

Publication number: WO2010061185A2
Application number: PCT/GB2009/002753
Authority: WO
Inventors: Kirsty Sarah Hewitson; Claudiu Trandafir Supuran
Original assignee: Union Life Sciences Ltd
Priority date: 2008-11-25
Filing date: 2009-11-25
Publication date: 2010-06-03
Also published as: WO2010061185A3; GB0821537D0

Abstract

The invention relates to methods and materials for treating and\or preventing dandruff and the like which comprise the use of carbonic anhydrase inhibitors, and in particular inhibitors of the Malassezia globosa carbonic anhydrase. The invention further provides inhibitors and compositions for use in the method, and assays for providing further such inhibitors.

Description

Therapeutic target

Technical field

The present invention relates generally to methods and materials for use in the treatment and/or prevention of dandruff and symptoms of dandruff by the use of agents which inhibit the activity of Malassezia globosa. The invention further relates generally to processes for identifying agents to achieve such inhibition.

Background art

Dandruff (historically termed Pityriasis capitis) is a commonly occurring phenomenon, found in more than 50% of the worldwide population and presents as an excessive shedding of dead skin cells from the scalp. For individuals without dandruff, skin cells typically mature and shed in approximately one month. However, for those with dandruff, the shedding rate is accelerated and may take only 2-7days. Dandruff can be triggered by several factors, including an increase in sebum production, irritation by organisms (particular of the Malassezia sp) and individual susceptibility (hereditary factors).

Malassezia (previously known as Pityrosporum) are yeasts, which are found to naturally occur on the skin and scalp of most individuals. There are several recognised species including M. globosa, M. furfur and M. restricta.

Until recently, M. fufur was thought to be responsible for the onset of dandruff, but instead, the scalp specific M. globosa was found to be the causative agent. Malassezia are dependent on external lipids for growth and hence secrete lipases to breakdown triglycerides that occur in the sebum of human skin. Byproducts of this breakdown include unsaturated fatty acids, such as oleic acid, which penetrate the stratum corneum (the top layer of the epidermis), resulting in an inflammatory response. In susceptible individuals, this results in rapid shedding of the stratum corneum and is presented as dandruff.

Several strategies and treatments are available for dandruff, the majority of which aim to target growth of the causative fungi. Active ingredients include:

Zinc pyrithione, also known as zinc pyridinethione, (see Figure 1), present in Head & Shoulders™, Pantene ProV™ and Clinic All Clear™. Its effect is most likely mediated through disruption of fungal membrane activities. At low concentrations, fungi are capable of overcoming the effect of zinc pyrithione.

Ketoconazole (see Figure 1), present in Nizoral™ shampoo. Other azoles have also been used in anti-dandruff treatments and interfere with the synthesis of ergosterol, a key component of fungal cell walls. Since mammals utilise cholesterol, instead of ergosterol, they are not sensitive to ketoconazole and it is specific for fungi. In general, those shampoos that contain ketoconazole have found to be more effective as an anti-dandruff treatment than those containing zinc pyrithione.

Selenium sulphide, present in Selsun Blue™. It appears to suppress the amount of sebum produced by the scalp.

Coal tar, present in Neutrogen T-Gel™ and Denorex™. May cause dying of light coloured hair, smells badly and has been criticised for potential carcinogenic effects.

In addition, US2002168327 relates to lipophilic agent or lipid precursors for treating and/or preventing dandruff by strengthening the scalp.

US2003165449 relates to low molecular weight compounds, non-polymeric compounds and compositions containing these compounds which inhibit M. globosa lipase enzymes or reduce M. globosa lipase enzyme activity. These are claimed for use in compositions for treating dandruff. As noted above, the lipolytic activity of M. globosa is believed to be a key element in the cause of dandruff.

Nevertheless it can be seen that the provision of further methods or materials which have utility in treating dandruff and\or other M. globosa associated conditions would provide a contribution to the art.

Summary of the invention

The present inventors have provided a novel strategy for the treatment of dandruff. The invention is based on the use of inhibitors of the carbonic anhydrase (CA) of M. globosa, as characterised herein by the present inventors. In various aspects the invention provides for the use of such inhibitors in dandruff treatments, as well as assays for providing further such inhibitors, plus other methods and materials relating to CA of M. globosa. Such assays may utilise purified polypeptides based on the CA of M. globosa having SEQ ID No 1 or a variant thereof, plus also nucleic acids, vectors and host cells and related methods and processes of using these.

The inhibitors and topically applied compositions comprising them described herein may also be used in other methods for controlling or inhibiting the CA of M. globosa. For example the treatment of seborrhoeic dermatitis and tinea versicolor.

Brief description of the Figures

Figure 1 shows two currently used ingredients in anti-dandruff shampoos. Zinc pyrithione (ZPT) and ketoconazole (KETO).

Figure 2 shows the structures of some known CA inhibitors. Acetazolamide (1), ethoxzolamide (2), methazolamide (3), dichlorophenamide (4), brinzolamide (5), dorzolamide (6), indisulam (7), topiramate (8) and zonisamide (9).

Figure 3 shows the initial BLAST results of the E. coli carbonic anhydrase Il sequence against the M. globosa database. Two identical sequences were identified, XP_001730815.1 and EDP43601.1 (SEQ ID NO. 1).

Figure 4 shows a CLUSTAL W alignment of carbonic anhydrase sequences from M. globosa (XP_001730815.1 and EDP43601.1), E.coli CA Il (P61517)[35%] and Cryptococcus neoformans CAN 2 (Q314V7)[33%]. Note: (indicates primary accession number), [indicates percentage identity with M. globosa carbonic anhydrase, XPJ301730815.1 and EDP43601.1]. • Indicates the conserved Zn ion binding residues.

Figure 5 shows (A) homology model of the Malassezia globosa carbonic anhydrase (XPJD01730815.1/EDP43601.1) based on its sequence and structure alignment with E.coli CA Il (PDB no. 1 i6o), (B) the conserved active site Zn binding residues, Cys47, Asp49, His 103 and Cys106 (M. globosa carbonic anhydrase numbering). Figure 6 shows an SDS-PAGE analysis of purified GST-MG-CA. Lane M contains appropriate molecular weight (MW) markers, whilst Lane 1 contains the purified GST-MG- CA (MW = 53 kDa, of which GST is 26 kDa and MG-CA is 27 kDa).

Sequences

SEQ ID NO. 1 is the amino acid sequence of XP_001730815.1 and EDP43601.1 , proposed to be the M. globosa carbonic anhydrase.

SEQ ID NO. 2 is the amino acid sequence of E. coli CA Il (P61517), which shares 35% sequence identity with M. globosa carbonic anhydrase (SEQ ID NO. 1).

SEQ ID NO. 3 is the amino acid sequence of C. neoformans CAN 2 (Q314V7), which shares 33% sequence identity with M. globosa carbonic anhydrase (SEQ ID NO. 1).

SEQ ID NO. 4 is the amino acid sequence of E. coli CA I (P0ABE9), which shares 22% sequence identity with M. globosa carbonic anhydrase (SEQ ID NO. 1).

SEQ ID NO. 5 is the amino acid sequence of C. neoformans CAN 1 (Q30E70), which shares 34% sequence identity with M. globosa carbonic anhydrase (SEQ ID NO. 1).

SEQ ID NO. 6 is the amino acid sequence of Candida albicans carbonic anhydrase (Q5AJ71), which shares 34% sequence identity with M. globosa carbonic anhydrase (SEQ ID NO. 1).

SEQ ID NO. 7 is the amino acid sequence of human carbonic anhydrase Il (P00918), which shares no significant sequence identity with M. globosa carbonic anhydrase (SEQ ID NO. 1).

SEQ ID NO. 8 is the DNA sequence encoding the proposed M. globosa carbonic anhydrase (XP_001730815.1/EDP43601) of SEQ ID NO. 1. Detailed disclosure of the invention

In one aspect there is provided a method of treating and\or preventing dandruff in a subject which method comprises use of a composition comprising an inhibitor of the M. globosa carbonic anhydrase.

In further aspects there is provided a method of treating and\or preventing dermatitis and tinea versicolor in a subject which method comprises use of a composition comprising an inhibitor of the M. globosa carbonic anhydrase.

Thus the invention provides, inter alia, methods of treating and\or preventing dandruff and\or dermatitis and\or tinea versicolor in a subject which methods comprise use of a composition comprising an inhibitor of the M. globosa carbonic anhydrase.

In other aspects of the present invention there are provided:

An inhibitor of the M. globosa carbonic anhydrase for use in a therapeutic or cosmetic method as described above e.g. methods of treating and\or preventing dandruff and\or dermatitis and\or tinea versicolor in a subject, and

Use of an inhibitor of the M. globosa carbonic anhydrase in the preparation of a therapeutic or cosmetic composition for the method as described above e.g. for treating and\or preventing dandruff and\or dermatitis and\or tinea versicolor in a subject.

Therapeutic or cosmetic composition comprising inhibitors of the Malassezia globosa carbonic anhydrase are also provided.

As described below compositions may optionally comprise other therapeutically effective or cosmetically effective active ingredients e.g. those which have activity against Malassezia spp. Example additional active ingredients include one or more of zinc pyrithione, ketoconazole and\or selenium sulphide.

Preferably the composition is a topically applied one such as a shampoo, conditioner or skin lotion. A preferred composition is a shampoo composition. In such a composition the CA inhibitor may be typically contained in an amount of 0.001-10 wt% based on the total weight of the shampoo composition.

The methods of the present invention may thus comprise applying an effective amount of the composition to hair or skin that has preferably been wetted with water, and then rinsed off. Such effective amounts generally range from about 1 g to about 50 g, preferably from about 1 g to about 20 g. Application to the hair typically includes working the composition through the hair such that most or all of the hair is contacted with the composition.

Thus a method for treating dandruff may comprise the steps of: a) wetting the hair and/or skin with water, b) applying an effective amount of the shampoo composition to the hair and/or skin, and c) rinsing the composition from the hair and/or skin using water. These steps can be repeated as many times as desired to achieve the desired dandruff treating benefit.

Example inhibitors and methods of providing them are described in more detail hereinafter. The inhibitor for use in any of the aspects of the invention may be sourced from known CA inhibitors (e.g. any one or more of the sulphonamide, sulphamide or sulphamate derivatives, such as described below) or may be provided using the screens of the present invention.

In other aspects the invention provides methods for identifying inhibitors of the M. globosa carbonic anhydrase.

Some of these aspects and embodiments will now be discussed in more detail:

Carbonic anhydrases

The carbonic anhydrases (CAs) are a ubiquitous family of metalloenzymes, present in both prokaryotes and eukaryotes, which catalyse the conversion of carbon dioxide to bicarbonate:

These enzymes play important roles in numerous biological processes including pH and CO₂ homeostasis, transport of CO₂ and bicarbonate, fatty acid metabolism, bone resorption and electrolytic secretion in a variety of tissues and organs.

As well as being present in vertebrates, CAs can also be found in a whole range of organisms including bacteria, fungi, algae, plants, archaea and marine diatoms.

The CA from M. glohosa, which has been cloned and used in screening assays by the present inventors, is described in more detail below and has SEQ ID No. 1. A homology model is shown in Figure 5.

Inhibitors of carbonic anhydrases and previous uses thereof

Several human isozymes of CA (see Example 1) have previously been targeted for therapeutic use. Glaucoma is a consequence of damage to the optic nerve that leads to progressive degeneration of sight and, if left untreated, to blindness. The principal cause of glaucoma is increased intraocular pressure as a result of liquid aqueous humour production by the ciliary body. CA II, abundant in the ciliary body, has been shown to be responsible for bicarbonate secretion, a key component of the aqueous humour. Inhibitors of the CAs are commonly used for the treatment of glaucoma. This includes both the water soluble sulphonamides, Brinzolamide (tradename Azopt, Alcon) and Dorzolamide (tradename Trusopt, Merck), as well as acetazolamide, a systemic CA inhibitor (see Figure 2). Although acetazolamide exhibits minimal toxicity, when taken orally undesirable side effects are often observed with this molecule following long term use due to its action as a global CA inhibitor. This includes dizziness, nausea, fatigue, loss of libido, depression and sensations of numbness/tingling in the extremities.

The CAs are also highly abundant in the kidney, where the relevant isoforms influence a number of critical functions including acid-base homeostasis, NH₄ ⁺ ouput and bicarbonate reabsorption. Consequently, CA inhibitors, such as acetazolamide, methazolamide, ethoxzolamide and dichlorophenamide (Figure 2), have been utilised as diuretics. Inhibition of the CA isoforms residing in the proximal tubule causes a reduction in H⁺ secretion, an accompanying increase in NaVK⁺ secretion and ultimately an increase in urine volume. CA isozymes have also attracted attention as novel anti-cancer targets, where a number of them, most notably CA IX, are upregulated under conditions of low oxygen concentration. Tumour progression under hypoxic conditions appears to be associated with a malignant phenotype and is facilitated by their adaptation to the hypoxic environment. One of these mechanisms of adaptation involves a decrease in the extracellular pH value. Although this is partly achieved through the production of lactic acid, as a consequence of high glycolytic rates, increased expression of CA IX under hypoxia leads to an increased production of H⁺ ions. Some of the effects of a decrease in pH include upregulation of angiogenic factors, increased invasion and impaired immune functions. A decrease in sensitivity to several known chemotherapy reagents is also known, presumably because the change in pH alters the solubility profile of these compounds.

lndisulam (Figure 2), a sulphonamide derivative, is currently being evaluated in Phase Il trials (Eisai Co., Ltd) after demonstrating high anti tumour activity in various cancer models. Part of its mechanism of action is thought to be via CA inhibition.

Several studies have also suggested that particular CA isoforms may be targets for obesity treatment. The mitochondrial enzymes (CA VA and CA VB), together with CA II, are involved in de novo lipogenesis i.e. the synthesis of endogenous fatty acids from carbohydrates in mammals. Two anti-epileptic drugs, zonisamide and topiramate (Figure 2), both demonstrated weight loss as side effects in obese individuals during clinical trials. Both of these molecules contain sulphonamide functionalities and have been shown to inhibit several different CA isoforms. Crystallographic studies have also been completed for CA Il with topiramate and show this molecule bound to the active site of the enzyme. It has thus been hypothesised that the weight loss characteristics associated with these compounds is at least partly mediated via inhibition of those CA isoforms involved in lipogenesis. It should be noted, however, that treatments of this nature would not address the issue of high fat diets, but rather the conversion of excessive carbohydrates to fat, which is presumed to contribute only marginally to obesity.

Thus several CA inhibitors have already been identified, some with applied therapeutic uses. All CA inhibitors typically consist of a Zn binding group, normally represented by either a sulphonamide, sulphamate or sulphamide moiety. Example sulphonamide, sulphamate or sulphamide compounds are set out in the following section.

As generally described herein, sulphonamide derivatives, sulphamate derivatives and sulphamide derivatives are those compounds having an -S(O)₂NH₂ group and the pharmaceutically acceptable salts, hydrates and solvates thereof. This group is generally linked to a carbon atom, an oxygen atom (sulphamate derivatives) or a nitrogen atom (sulphamide derivatives).

In one embodiment, the CA inhibitor is a compound of the following general formula (1), or a pharmaceutically acceptable salt, hydrate or solvate thereof:

in which:

-L₁- is independently a single covalent bond,

wherein:

is independently C_1-3 alkylene; and is independently a single covalent bond,

and wherein:

-A is independently C_6-10 carboaryl, C_5-10 heteroaryl, non-aromatic C_3-7 carbocyclyl, or non-aromatic C₅.i₂ heterocyclyl, and is optionally substituted.

In one embodiment, the CA inhibitor is a compound of the following general formula (2), or a pharmaceutically acceptable salt, hydrate or solvate thereof:

wherein:

-Lr is independently a single covalent bond, CH NH O o L L

wherein:

-LA- is independently C_1-3 alkylene; and

-LB- is independently a single covalent bond,

and wherein:

-B- is independently C_6-i₀ carboarylene, C_5-10 heteroarylene or non-aromatic C_5-12 heterocyc-di-yl, and optionally substituted, for example, with one or more groups -R¹;

wherein:

each -R¹ is independently

or

wherein: each -R²- is independently a single covalent bond, C_1-4 alkylene optionally substituted with =0, or C_1-4 haloalkylene optionally substituted with =0; each -R³ is independently -H or -R^b; each -R^b is independently C_1-4 alkyl or C_1-4 haloalkyl, each optionally substituted with =0;

and wherein:

-L₂- is independently a single covalent bond or C_1-4 alkylene;

or wherein:

X-L₂- is independently:

wherein:

-Y- is independently selected from a single covalent bond and C_1-4 alkylene optionally substituted with =O;

-Z- is independently selected from a single covalent bond and C_1-4 alkylene optionally substituted with =0;

and wherein:

-X is independently -H, -F, -Cl, -Br, -I₁ -R^a, -NH₂, -NHR^a, -NR^a ₂, -COOH, -COOR^a, - OH, -0R^a, or -D,

wherein:

-R^a is independently C_1-4 alkyl or C_1-4 haloalkyl; and -D is independently C_6-I0 carboaryl, C_5-10 heteroaryl or non-aromatic C₅-i₂ heterocyclyl, and is optionally substituted, for example, with one or more groups independently selected from C_1-4 alkyl, -NH₂, -F, -Cl, -Br, -I, C_1-4 haloalkyl, and -E;

wherein:

-E is independently phenyl, C₅ heteroaryl, or non-aromatic C₅ heterocyclyl, and is optionally substituted, for example, with one or more groups selected from C_1-4 alkyl and C_1-4 haloalkyl.

The terms "haloalkyl" and "haloalkene" as used herein refer to alkyl and alkylene groups respectively in which one or more hydrogen atoms is replaced by -F, -Cl, -Br or -I. For example, "C_1-4 haloalkyl" includes, amongst other groups, -CH₂F, -CHF₂, -CF₃, -CH₂-CHF- CH₃, and -CF₂-CF₂-CF₂-CF₃. When cyclic groups are defined in terms of their size, the nomenclature "C_x._y" is used. The subscript refers to the number of ring atoms, rather than the number of carbon ring atoms. For example, C₅ heteroaryl includes pyrrole but not pyridine.

Cyclic groups referred to herein are not limited to monocyclic groups. They may be bicyclic, tricyclic, spiro, etc.

The group -L1-

In one embodiment, -L₁- is independently a single covalent bond.

In one embodiment, -L₁- is independently a single covalent bond,

In one embodiment, -L₁- is independently

The group -B-

In one embodiment, -B- is independently phenylene or C₅ heteroarylene, and is optionally substituted.

In one embodiment, -B- is independently phenylene, and is optionally substituted.

In one embodiment, -B- is independently 1,4-phenylene, 1 ,3-phenylene or 1 ,2-phenylene, and is optionally substituted.

In one embodiment, -B- is independently 1,4-phenylene, and is optionally substituted. (That is, the bonds to the groups denoted -L₁- and -L₂- are positioned para to one another.)

In one embodiment, -B- is independently 1 ,4-phenylene.

In one embodiment, -B- is independently the group -B¹-:

In one embodiment, -B- is independently 1 ,3-phenylene, and is optionally substituted.

In one embodiment, -B- is independently 1,2-phenylene, and is optionally substituted.

In one embodiment, -B- is independently C₅ heteroarylene, and is optionally substituted.

In one embodiment, -B- is independently C₅ heteroarylene, wherein the C₅ heteroarylene has from one to three heteroatoms, each independently selected from N, O, and S.

In one embodiment, -B- is independently C₅ heteroarylene, wherein the C₅ heteroarylene has exactly three heteroatoms selected from N, O, and S.

In one embodiment, -B- is independently C₅ heteroarylene, wherein the C₅ heteroarylene has exactly three heteroatoms selected from N and S.

In one embodiment, -B- is independently C₅ heteroarylene, wherein the C₅ heteroarylene has exactly three heteroatoms, two of which are N and one of which is S.

In one embodiment, -B- is independently thiadiazol-di-yl, and is optionally substituted.

In one embodiment, -B- is independently [1 ,3,4]-thiadiazol-2,5-di-yl, and is optionally substituted.

In one embodiment, -B- is independently the group -B²-:

In one embodiment, -B- is independently 1 ,4-phenylene or [1,3,4]-thiadiazol-2,5-di-yl, and is optionally substituted.

Optional substituents on the group -B-

As discussed above, -B- is optionally substituted, for example, with one or more groups -R¹.

In one embodiment, -B- is unsubstituted.

In one embodiment, -B- is optionally substituted with n groups -R¹, wherein n is 0, 1 , 2, or 3.

In one embodiment, -B- is optionally substituted with n groups -R¹, wherein n is 0, 1 , or 2.

In one embodiment, -B- is optionally substituted with n groups -R¹, wherein n is 0 or 1.

In one embodiment, -B- is optionally substituted with n groups -R¹, wherein n is 1.

In one embodiment, -B- is optionally substituted with n groups -R¹, wherein n is 2.

The groups -R¹

In one embodiment: each -R¹ is independently selected from -SO₂NH₂, -F, -Cl, -Br, -I, -R^b, =0, -R²-O-R³, =NR³, and -R²-N(R³)₂, wherein: each -R²- is independently a single covalent bond, C_1-4 alkylene optionally substituted with =0, or C_1-4 haloalkylene optionally substituted with =0; each -R³ is independently -H or -R^b; each -R^b is independently C_1-4 alkyl or C_1-4 haloalkyl, each optionally substituted with =0.

In one embodiment, each -R¹ is independently selected from -SO₂NH₂, -F, -Cl, -Br, -I, C_1-4 alkyl, C_1-4 haloalkyl, -R²-O-R³, and -NHR³ In one embodiment, -R²- is independently a single covalent bond, C_1-4 alkylene, or C_1-4 haloalkylene.

In one embodiment, each -R²- is independently a single covalent bond or C_1-4 alkylene optionally substituted with =0.

In one embodiment, each -R²- is independently a single covalent bond, -CH₂-, -CH₂CH₂- or -CH₂CH₂CH₂-.

In one embodiment, each -R²- is independently a single covalent bond.

In one embodiment, each -R³ is independently -H or C_1-4 alkyl optionally substituted with =0.

In one embodiment, each -R³ is independently -H, -CH₃, Or -CH₂CH₃.

In one embodiment, each -R^b is independently C_1-4 alkyl or C_1-4 haloalkyl.

In one embodiment, each -R^b is independently -CH₃ or -CF₃.

In one embodiment, each -R¹ is independently selected from -SO₂NH₂, -F, -Cl, -Br, -I₁ C_1-4 alkyl, and C_1-4 haloalkyl.

In one embodiment, each -R¹ is independently selected from -SO₂NH₂, -F, -Cl, -Br, -I, - CH₃, -CF₃, -OCH₃, -OCH₂CH₃, -CH₂CH₂CH₂OCH₃, -NH₂, -NHCH₃, and -NHCH₂CH₃.

In one embodiment, each -R¹ is independently selected from -SO₂NH₂, -F, -Cl, -Br, -I, - CH₃, -CF₃, -OCH₃, -OCH₂CH₃, -CH₂CH₂CH₂OCH₃, -NHCH₂CH₃, =0, and =NH.

In one embodiment, -R¹ is independently -OCH₃.

The group -U-

In one embodiment, -L₂- is independently a single covalent bond or C_1-4 alkylene. In one embodiment, -L₂- is independently a single covalent bond.

In one embodiment, -L₂- is C_1-4 alkylene.

In one embodiment, -L₂- is independently -CH₂-, -CH₂CH₂-, or -CH₂CH₂CH₂-.

In one embodiment, X-L₂- is independently X-Y-SO₂-Z-, X-Y-NH-Z-, X-Y-SO₂-NH-Z-, or X- Y-NH-SO₂-Z-, wherein -Y- is independently selected from a single covalent bond and C_1-4 alkylene optionally substituted with =0, and -Z- is independently selected from a single covalent bond and C_1-4 alkylene optionally substituted with =0.

In one embodiment, X-L₂- is independently X-Y-NH-Z-, X-Y-SO₂-NH-Z-, Or X-Y-NH-SO₂- Z-.

In one embodiment, X-L₂- is independently X-Y-SO₂-NH-Z- Or X-Y-NH-SO₂-Z-.

In one embodiment, -Y- is independently selected from a single covalent bond and C_1-4 alkylene optionally substituted with =O.

In one embodiment, -Y- is independently a single covalent bond, -CH₂- or -CH₂CH₂-.

In one embodiment, -Y- is independently a single covalent bond.

In one embodiment, -Z- is independently selected from a single covalent bond and C_1-4 alkylene optionally substituted with =0.

In one embodiment, -Z- is independently a single covalent bond, -CH₂-, -CH₂CH₂-, or - C(=0)-.

In one embodiment, -Z- is independently a single covalent bond, -CH₂-, Or -CH₂CH₂-.

In one embodiment, X-L₂- is independently X-NH-, X-SO₂NH-, X-SO₂-NH-CH₂-, X-SO₂- NH-CH₂-CH₂-, X-NH-SO₂-, or X-CH₂-NH-C(=O)-. The group -X

-X is independently -H, -F, -Cl, -Br, -I, -R^a,-NH₂, -NHR^a, -NR^a ₂, -COOH, -COOR^a, -OH, 0R^a, or -D.

In one embodiment, -R^a is independently -CH₃ or -CF₃.

In one embodiment, -R^a is independently -CH₃.

In one embodiment, -X is independently -H, -Br, -CH₃, -CF₃, -NH₂, -COOH, -OH, or -D.

In one embodiment, -X is independently -F, -Cl, -Br, -I, -NH₂, -COOH, or -OH.

In one embodiment, -X is independently -Br, -CH₃, -CF₃, -NH₂, -COOH, or -OH.

In one embodiment, -X is independently -D.

In one embodiment, X-L₂- is independently selected from:

The group -D

In one embodiment, -D is independently C_6--_I0 carboaryl, C_5-10 heteroaryl or non-aromatic C_5-I2 heterocyclyl, and is optionally substituted.

In one embodiment, -D is independently phenyl, C₅.i₀ heteroaryl or non-aromatic C₅ heterocyclyl, and is optionally substituted. In one embodiment, -D is independently phenyl, C₅ heteroaryl, C₉ heteroaryl or non-aromatic C₅ heterocyclyl, and is optionally substituted.

In one embodiment, -D is independently phenyl, C₅ heteroaryl, or C₉ heteroaryl, and is optionally substituted.

In one embodiment, -D is independently phenyl, pyrimidinyl, indolyl, and pyrrolidinyl, and is optionally substituted.

In one embodiment, -D is independently phenyl, and is optionally substituted.

Optional substituents on the group -D

In one embodiment, -D is optionally substituted, for example, with one or more groups independently selected from C_1-4 alkyl, -NH₂, -F, -Cl, -Br, -I, C_1-4 haloalkyl, and -E.

In one embodiment, -D is independently unsubstituted.

In one embodiment, -D is optionally substituted with one or more substituents independently selected from -NH₂, -F, -Cl, -Br, -I, -CH₃ or -CF₃.

In one embodiment, -D is optionally substituted with one or more substituents independently selected from -NH₂, -F, -Cl, -Br, -I and -CH₃.

In one embodiment, -D is optionally substituted with exactly one substituent, which is independently selected from -NH₂, -F, -Cl, -Br, -I, -CH₃ or -CF₃.

In one embodiment, -D is optionally substituted with exactly one substituent, which is -NH₂.

In one embodiment, -D is substituted with exactly one substituent, which is -NH₂.

In one embodiment, -D is independently phenyl, and is para-substituted with -NH₂. In one embodiment, -D is independently phenyl, and is substituted with exactly one substituent which is -NH₂ positioned para- to the bond connecting the group -D to the group -L₂-.

In one embodiment, -D is independently substituted with -E.

The group -E

In one embodiment, -E is independently phenyl, C₅ heteroaryl, or non-aromatic C₅ heterocyclyl, and is optionally substituted, for example, with one or more groups selected from C_1-4 alkyl and C_1-4 haloalkyl.

In one embodiment, -E is independently phenyl, C₅ heteroaryl, or non-aromatic C₅ heterocyclyl, and is optionally substituted, for example, with one or more groups selected from C_1-4 alkyl.

In one embodiment, -E is independently phenyl or C₅ heteroaryl, and is optionally substituted, for example, with one or more groups selected from C_1-4 alkyl or C_1-4 haloalkyl.

In one embodiment, -E is independently phenyl, and is optionally substituted.

In one embodiment, -E is optionally substituted with exactly one substituent, which is -CH₃.

In one embodiment, -E is substituted with exactly one substituent, which is -CH₃.

In one embodiment, -E is independently phenyl, and is para-substituted with -CH₃.

In one embodiment, -E is substituted with exactly one substituent, which is -CH₃ and is positioned para- to the bond connecting the group -E to the group -D.

Some particularly preferred combinations

In one embodiment, the CA inhibitor is a compound of the following general formula (3), or a pharmaceutically acceptable salt, hydrate or solvate thereof:

wherein:

-B- is independently phenylene, and is optionally substituted with n groups R¹;

and wherein:

n is independently 0, 1 , 2, or 3;

and wherein:

each -R¹ is independently selected from -SO₂NH₂, -F, -Cl, -Br, -I, C_1-4 alkyl, C_1-4 haloalkyl, -R²-O-R³, and -NHR³;

wherein:

each -R²- is independently a single covalent bond or C_1-4 alkylene optionally substituted with =0; each -R³ is independently -H or C_1-4 alkyl optionally substituted with =0;

and wherein:

-L₁- is independently a single covalent bond, -CH₂-, -O-, or -NH-; -L₂- is independently a single covalent bond or C_1-4 alkylene;

and wherein:

-X is independently -F, -Cl, -Br, -I, -NH₂, -COOH, or -OH.

In one embodiment, -L₁- is a single covalent bond. In one embodiment, each -R¹ is independently selected from -SO₂NH₂, -F, -Cl, -Br, -I₁ C_1-4 alkyl, and C_1-4 haloalkyl.

In one embodiment, n is independently O, 1 or 2.

In one embodiment, -L₂- is a single covalent bond. In one embodiment, -L₂- is C_1-4 alkylene.

In one embodiment, the CA inhibitor is a compound of the following general formula (4), or a pharmaceutically acceptable salt, hydrate or solvate thereof:

wherein:

-B- is independently 1 ,4-phenylene or [1 ,3,4]-thiadiazol-2,5-di-yl, and is optionally substituted with n groups R¹;

and wherein:

n is independently 0, 1 , 2, or 3;

and wherein:

each -R¹ is a group independently selected from

C_1-4 alkyl, C_1-4 haloalkyl, -R²-O-R³, and -NHR³;

wherein:

each -R²- is independently a single covalent bond or C_1-4 alkylene optionally substituted with =0, and each -R³ is independently -H or C_1-4 alkyl optionally substituted with =0;

and wherein: X-L₂- is independently:

wherein:

-Y- is independently selected from a single covalent bond and C_1-4 alkylene optionally substituted with =O,

and wherein:

-X is independently -D;

wherein:

-D is independently C₆-_Io carboaryl, C_5--_I0 heteroaryl or non-aromatic C_5-12 heterocyclyl, optionally substituted with one or more groups independently selected from C_1-4 alkyl, -NH₂, -F, -Cl, -Br, -I, C_1-4 haloalkyl, and -E;

wherein:

-E is independently phenyl, C₅ heteroaryl, or non-aromatic C₅ heterocyclyl, and is optionally substituted, for example, with one or more groups selected from C_1-4 alkyl.

In one embodiment, n is independently O or 1.

In one embodiment, -R¹ is independently -OCH₃.

In one embodiment, -Y- is independently a single covalent bond, -CH₂-, or -CH₂CH₂-.

In one embodiment, -Y- is independently a single covalent bond. In one embodiment, -Z- is independently a single covalent bond, -CH₂-, -CH₂CH₂-, or - CH(=O)-.

In one embodiment, X-L₂- is independently X-NH-, X-SO₂NH-, X-SO₂-NH-CH₂-, X-SO₂- NH-CH₂-CH₂-, X-NH-SO₂-, Or X-CH₂-NH-C(O)-.

In one embodiment, -D is independently phenyl, C_5-10 heteroaryl or non-aromatic C₅ heterocyclyl, and is optionally substituted.

In one embodiment, -D is independently phenyl, C₅ heteroaryl, C₉ heteroaryl or C₅ heterocyclyl, and is optionally substituted.

In one embodiment, -D is independently phenyl, pyrimidinyl, indolyl, or pyrrolidinyl, and is optionally substituted.

In one embodiment, -D is independently phenyl, and is optionally substituted.

In one embodiment, -D is optionally substituted with exactly one substituent, which is independently selected from -NH₂, -F, -Cl, -Br, -I and -CH₃.

In one embodiment, -D is independently phenyl, and is para-substituted with NH₂. In one embodiment, -D is independently phenyl, and is substituted with exactly one substituent which is -NH₂ positioned para- to the bond connecting the group -D to the group -L₂-.

In one embodiment, X-L₂- is independently selected from:

Combinations

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the chemical groups represented by the variables (e.g., -A, -B-, -B¹-, -B²-, -D, -E, -X, -Y-, -Z-, - L₁-, -L₂-, -L_A-, -LB-, -R¹, -R²-, -R³, -R^a, -R^b, n, etc.) are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace compounds that are stable compounds (i.e., compounds that can be isolated, characterised, and tested for biological activity). In addition, all sub-combinations of the chemical groups listed in the embodiments describing such variables are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination of chemical groups was individually and explicitly disclosed herein. Specific compounds

In one embodiment, the CA inhibitor is selected from any of the compounds shown in Table 2 hereinafter, and pharmaceutically acceptable salts, hydrates and solvates thereof.

Representative members of the genus of compounds have been tested in the assays or models shown in the Examples, particularly Examples 5 to 8.

Preferred compounds for use in the present invention are aryl/heterocyclic sulphonamides.

Where the compound is a substituted benzene sulphonamide, preferably it comprises at least one para-substituent.

Where the compound is a substituted benzene sulphonamide, preferably the substituent is hydrophilic and\or charged (e.g. amino, carboxylate, halogenated) rather than a more hydrophobic group (e.g. alkyl).

The invention explicitly embraces the use of previously described CA inhibitors including acetazolamide (1), ethoxzolamide (2), methazolamide (3), dichlorophenamide (4), brinzolamide (5), dorzolamide (6), indisulam (7), topiramate (8) and zonisamide (9) (Figure 2). Other previously characterised compounds include sulpiride (compound 33 herein) and benzolamide (compound 28 herein). As described in the Examples below dichlorophenamide was amongst the most potent of these (346 nM) although sulpiride (32OnM), indisulam (113nM) and benzolamide (482 nM) were also highly potent.

Based on the findings of Examples 5 to 8, preferred derivatives include, but are not limited to: 4-(2-Aminoethyl) benzenesulfonamide (compound 13); aminobenzolamide (compound 29); 4-(sulfanilyl-amidoethyl)-benzene sulphonamide (compound 30); 4- (sulfanilylaminomethyl)-benzene sulphonamide (compound 31); 4-(2-amino-pyrimidin-4- ylamino)-benzene sulphonamide (compound 32); 5-(trifluoromethyl)aniline - 2,4 - disulphonamide (compound 24); 3-chloroaniline-4,6-disulphonamide (compound 23).

Preferred compounds are any of those having a Ki of less than 100 μM, 10 μM, 1 μM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 450 nM, 400 nM, 350 nM, 300 nM, 25OnM, 200nm, 150nm, 100 nM. The Ki in each case may also be above 50 nM. Each genus of compounds having a Ki in the respective range can be readily inferred from Table 2 below.

Pharmaceutically acceptable active derivatives of such inhibitors and their use are within the scope of the present invention. Examples of such derivatives include, but are not limited to, salts, solvates, amides, esters, ethers, N-oxides, chemically protected forms, and prodrugs thereof.

Preferred compounds may include hydrophobic moieties. This may be particularly advantageous for shampoo formulations so that the compound itself is adapted to remain on the scalp or hair during rinsing.

The use of any of these compounds, including previously characterised CA inhibitors, in any aspect of the invention e.g. the treatment of dandruff (or other M. globosa related skin conditions such as seborrhoeic dermatitis and tinea versicolor) is expressly embraced by the present invention. It should be noted that although concerns have been raised previously with the use of human CA inhibitors as potential systemic drugs, in the context of the present invention it is anticipated that inhibitors will usually be applied topically - for example to the scalp.

However the invention further embraces the use of inhibitors identifiable using the methods described herein.

Specifically, as noted above the present inventors have cloned and expressed the M. globosa carbonic anhydrase (XP_001730815.1/EDP43601). This accession number had previously been putatively attributed to a β-class basidomycete CA but no examination of its properties, or suggestion of its utility in the present context, had been made (see "Evolution of carbonic anhydrases in fungi" Skander Elleuche & Stefanie Poggeler; Curr Genet (2009) 55:211-222.

The amino acid sequence is shown in SEQ ID No. 1. Polypeptides having this sequence (and nucleic acids encoding it) thus have utility in providing assays and other materials suitable for identifying or confirming inhibitors of the enzyme, for example for use in the methods described herein relating to dandruff treatment. Such means for assisting in the screening process can have considerable commercial importance and utility. Use of M. globosa carbonic anhydrase and related materials

Thus aspects of the present invention may utilise an isolated nucleic acid molecule encoding SEQ ID No. 1 , for example a nucleic acid which comprises SEQ ID No. 8.

Nucleic acid as used herein may include cDNA, RNA, genomic DNA and modified nucleic acids or nucleic acid analogs (e.g. peptide nucleic acid). Where a DNA sequence is specified, e.g. with reference to a figure, unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed. Nucleic acid molecules may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of other nucleic acids of the species of origin. Where used herein, the term "isolated" encompasses all of these possibilities. The nucleic acid molecules may be wholly or partially synthetic. In particular they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Alternatively they may have been synthesised directly e.g. using an automated synthesiser.

Nucleic acids may consist or consist essentially of the coding sequence described above.

In aspects of the present invention, there may be utilised nucleic acids which are variants of the sequences discussed above.A variant nucleic acid molecule shares homology with, or is identical to, all or part of the sequences discussed above.

Such variants likewise have utility in providing assays and other materials suitable for identifying or confirming inhibitors of the enzyme.

Generally speaking variants may be:

(i) Novel, naturally occurring, nucleic acids, isolatable from Malassezia globosa, and which may differ slightly between isolates.

(ii) Artificial nucleic acids, which can be prepared by the skilled person in the light of the present disclosure. Such derivatives may be prepared, for instance, by site directed or random mutagenesis, or by direct synthesis. Preferably the variant nucleic acid is generated either directly or indirectly (e.g. via one or more amplification or replication steps) from an original nucleic acid encoding SEQ ID No. 1.

Particularly included are variants which comprise only a distinctive part or fragment (however produced) corresponding to a portion of the sequence provided e.g. the likely active site as shown in Figure 5 and comprising Cys47, Asp49, His103 and Cys106.

Also included are nucleic acids corresponding to those above, but which have been extended at the 3¹ or 5' terminus.

The term 'variant' nucleic acid as used herein encompasses all of these possibilities. When used in the context of polypeptides or proteins it indicates the encoded expression product of the variant nucleic acid.

Some of these aspects relating to variants will now be discussed in more detail.

Homology (similarity or identity) may be assessed using the National Centre of Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST; Altschul et al., 1990) and is available from several sources, including the NCBI (Bethesda, MD) and on the Internet, the sequence analysis programs BLASTP, BLASTN, BLASTX, TBLASTN, TBLASTX. BLAST can be accessed at http://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence identity using this program is available at http://www:ncbi.nim. nih.gov/BLAST/blast help.html.

For comparisons of amino acid sequences of greater than 30 amino acids, the "BLAST 2 Sequences" function in the BLAST program is employed using the BLASTP program with the default BLOSUM62 matrix set to default parameters, (open gap 11 , extension gap 1 penalties).

Homology may be at the nucleotide sequence and/or encoded amino acid sequence level. Preferably, the nucleic acid and/or amino acid sequence shares at least about 65%, or 70%, or 80% identity, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% identity. As is shown in Figures 3-4, and the sequence listings, CAs from other species show only modest amino acid identities with SEQ ID No. 1 (22%-35%). Changes or additions to native or wild-type sequences may be desirable for a number of reasons. For instance they may introduce or remove restriction endonuclease sites or alter codon usage.

Alternatively changes to a sequence may produce a derivative by way of one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide. Leader or other targeting sequences (e.g. membrane or golgi locating sequences) may be added to the expressed protein to determine its location following expression if it is desired to isolate it from a microbial system. In the Examples below SEQ ID No. 1 is expressed as a fusion with GST. Other desirable mutations may be random or site directed mutagenesis e.g. to alter the stability of the encoded polypeptide. Changes may be by way of conservative variation, i.e. substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine, for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. As is well known to those skilled in the art, altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptides conformation. Also included are variants having non-conservative substitutions. As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide's three dimensional structure. In regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e.g. altered stability or specificity.

In another embodiment the nucleotide sequence information provided herein may be used to design probes and primers for probing or amplification. An oligonucleotide for use in probing or PCR may be about 30 or fewer nucleotides in length (e.g. 18, 21 or 24). Generally specific primers are upwards of 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16-24 nucleotides in length may be preferred. Those skilled in the art are well versed in the design of primers for use in processes such as PCR. If required, probing can be done with entire restriction fragments of the gene disclosed herein which may be 100's or even 1000's of nucleotides in length. Small variations may be introduced into the sequence to produce 'consensus' or 'degenerate' primers if required. PCR requires the use of two primers to specifically amplify target nucleic acid, so preferably two nucleic acid molecules with sequences characteristic of the M. globosa CA encoding sequence are employed. Using RACE PCR₁ only one such primer may be needed (see "PCR protocols; A Guide to Methods and Applications", Eds. lnnis et al, Academic Press, New York, (1990)).

Thus a M. globosa CA-encoding sequence may be obtained or obtainable by a method involving use of PCR as follows:

(a) providing a preparation of nucleic acid from M. globosa. Test nucleic acid may be provided from a cell as genomic DNA, cDNA or RNA, or a mixture of any of these, preferably as a library in a suitable vector. If genomic DNA is used the probe may be used to identify untranscribed regions of the gene (e.g. promoters etc.), such as are described hereinafter,

(b) providing a pair of nucleic acid molecule primers useful in (i.e. suitable for) PCR,

(c) contacting nucleic acid in said preparation with said primers under conditions for performance of PCR,

(d) performing PCR and determining the presence or absence of an amplified PCR product.

The expression product of the amplified PCR product may then be assayed for CA activity.

As used hereinafter, unless the context demands otherwise, the term "MG-CA nucleic acid" is intended to cover any of the nucleic acids of the invention described above, including functional variants. Likewise unless the context demands otherwise, the term "MG-CA" is intended to cover any of the polypeptides of the invention described above, including functional variants.

For expression purposes the MG-CA nucleic acid described above may be in the form of a recombinant and preferably replicable vector.

"Vector" is defined to include, inter alia, any construct (e.g. plasmid, cosmid or phage) in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform a prokaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication).

Generally speaking, those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press or Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.

Preferred vectors of choice are those which are pET or pGEX derived, since these vectors readily permit the addition of an affinity tag (His or GST respectively) for purification purposes. Examples of such vectors are commercially available and well known to those skilled in the art - see also Example 3.

Preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial cell. This will generally be a heterologous promoter e.g. one not naturally occurring in M. globosa.

By "promoter" is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA). "Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.The promoter may be an inducible promoter e.g. an IPTG inducible promoter.

The present invention also provides methods comprising introduction of such a construct into a microbial cell and/or induction of expression of a construct within the cell, by application of a suitable stimulus e.g. an effective exogenous inducer, particularly in thecontext of the provision of compounds for the treatment of dandruff and the like. Thus aspects of the invention may include the provision of a host cell containing or transformed with a heterologous construct according to the present invention, especially a microbial cell.

The term "heterologous" is used broadly in this aspect to indicate that the gene/sequence of nucleotides in question (MG-CA nucleic acid) has been introduced into said cells (or progenitors thereof) using genetic engineering, i.e. by human intervention.

For production of MG-CA, cells may be cultured (typically at 37ºC, with a potential reduction in temperature following induction in the case of IPTG inducible systems). Affinity chromotography may then be used for isolation of the MG-CA with subsequent cleavage of affinity tag (His/GST) if appropriate. Additional purification steps (gel filtration, ion exchange, hydrophobic interaction) may be utilised to obtain homogenous MG-CA material. In the light of the present disclosure these steps will not present an undue burden for those skilled in the art.

Host cells as described above provide for the production and use of full length, or fragments of, the polypeptides disclosed herein, especially active portions thereof. An "active portion" of a polypeptide means a peptide which is less than said full length polypeptide, but which retains an essential biological activity - here CA activity.

A "fragment" of a polypeptide means a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, typically at least about nine to 13 contiguous amino acids and, most preferably, at least about 20 to 30 or more contiguous amino acids.

Fragments of the polypeptides may include one or more epitopes useful for raising antibodies to a portion of any of the amino acid sequences disclosed herein. Preferred epitopes are those to which antibodies are able to bind specifically, which may be taken to be binding a polypeptide or fragment thereof of the invention with an affinity which is at least about 100Ox that of other polypeptides.

Thus purified MG-CA protein, or a fragment or other variant thereof, e.g. produced recombinantly by expression from encoding nucleic acid therefore, may be used to raise antibodies employing techniques which are standard in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and might be screened, preferably using binding of antibody to antigen of interest.

For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, 1992, Nature 357: 80-82). Antibodies may be polyclonal or monoclonal.

Antibodies may be modified in a number of ways. Indeed the term "antibody" should be construed as covering any specific binding substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or synthetic.

As an alternative or supplement to immunising a mammal, antibodies with appropriate binding specificity may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.

Specific binding members such as antibodies and polypeptides comprising antigen binding domains of antibodies that bind and are preferably specific for an MG-CA polypeptide represent further aspects of the present invention, as do their use and methods which employ them.

M. globosa carbonic anhydrase for screening

In one aspect the present invention provides a method of screening for an MG-CA inhibitor (and hence potential dandruff active agent), the method generally comprising comparing under comparable reaction conditions the activity of MG-CA in the presence and absence of the test compound. The MG-CA may be assayed in vitro or in vivo in the context of a recombinant host cell described above.

A typical in vitro method for assaying inhibitory activity of an agent (e.g. for use in treating and\or preventing dandruff, dermatitis or tinea versicolor in a subject) may comprise: (a) providing a recombinant host cell as discussed above which expresses MG-CA, (b) monitoring the activity of the MG-CA in the presence of the agent,

(c) optionally comparing the value obtained in step (b) with a reference value.

In one aspect there is provided a method for identifying a compound capable of treating and\or preventing dandruff, dermatitis or tinea versicolor in a subject, which method comprises:

(a) providing a test compound;

(b) comparing under comparable reaction conditions the activity of a polypeptide, which polypeptide is the MG-CA or a variant or active portion thereof, in the presence and absence of the test compound.

Preferred assays are performed with isolated enzyme, for example such methods may comprise:

(a) providing isolated MG-CA

(b) incubating the MG-CA in the presence of the agent,

(c) measuring the activity of the MG-CA,

(d) optionally comparing the value obtained in step (c) with a reference value (e.g. the activity of the MGrCA in the absence of agent).

"Isolated" enzyme in this context embraces use of a synthetically prepared, recombinant form or naturally occurring M. glohosa CA which is in a purified, partially purified, substantially isolated state, or part of a crude extract.

In a particularly preferred embodiment the method for identifying the inhibitor of the MG- CA comprises e.g. for use as a compound capable of treating and\or preventing dandruff, dermatitis or tinea versicolor in a subject, which method comprises:

(a) providing isolated MG-CA,

(b) contacting the MG-CA with a test agent under conditions suitable for measuring the activity of the MG-CA,

(c) determining the activity of the MG-CA in the presence and absence of the test agent, wherein the test compound is an inhibitor if the activity of the M. globosa carbonic anhydrase polypeptide is greater in the absence of the test compound than in the presence of the test compound.

In one embodiment the method comprises: (a) providing a test compound; (b) providing a polypeptide, which polypeptide is the MG-CA or a variant or active portion thereof, in isolated form (e.g. in isolated form from a host cell comprising a recombinant vector including nucleic acid encoding said polypeptide);

(c) incubating the polypeptide in the presence of the test compound,

(d) measuring the activity of the polypeptide,

(e) optionally comparing the value obtained in step (d) with a reference value.

As noted above MG-CA catalyses:

CO₂ + H₂O → HCO₃- + H⁺

Preferably activity is measured by the carbonic anhydrase CO₂ hydration reaction.

Preferably activity is measured by change in pH of a solution resulting from said reaction in which the MG-CA is incubated.

Preferably the change in pH is measured optically e.g. using an indicator such as Phenol red.

Change in kinetic parameters of MG-CA caused by putative inhibitors can be assessed in similar fashion. Variables will include CO₂ concentration, enzyme concentration, reaction time, inhibitor concentration, inhibitor incubation time and so on. One of ordinary skill in the art will be able to readily select such variables in the light of the present disclosure and without undue burden.

In one aspect the putative inhibitor may also be tested and compared against CAs from other organisms (discussed in Example 1), such as to establish specificity for MG-CA. Specifically in such a method the method steps are repeated using an enzyme other than the MG-CA and determining the degree to which the test agent inhibits the MG-CA and other enzyme. The enzyme may for example be derived from human, mouse, Plasmodium falciparum, Helicobacter pylori, Candida sp, Cryptococcus neoformans, Mycobacterium tuberculosis, Brucella suis, Malassezia sp or Legionella pneumophilia.

Agents for the assays or screens may be provided from any source, including known enzyme inhibitors (including known CA inhibitors), naturally occurring compounds e.g. plant extracts, or the product of a combinatorial library such as are now well known in the art (see e.g. Newton (1997) Expert Opinion Therapeutic Patents, 7(10): 1183-1194).

Alternatively K| of test compounds may be determined, which is the concentration required to produce half maximum inhibition under selected conditions. Inhibitors may also be tested directly for antifungal activity against M. globosa. A minimum inhibitory concentration (MIC) of each test compound may be determined. This represents the minimum concentration of the compound that inhibits the growth of M. globosa following incubation. Both broth and agar dilution methods may be used to determine MICs. Agar dilution involves the incorporation of different concentrations of the antimicrobial substance into a nutrient agar medium followed by the application of a standardized number of cells to the surface of the agar plate. For broth dilution, often determined in 96-well microtiter plate format, the selected organism is inoculated into a liquid growth medium in the presence of different concentrations of an antimicrobial agent. Growth can be visually assessed after incubation for a defined period of time and the MIC value determined..

Following assay the inhibitor may be provided in isolated form or formulated as a therapeutic or cosmetic composition. Thus the methods of identifying inhibitors may further comprise the step of producing or providing them.

Any of the inhibitors described above may be described herein for brevity as "MG-CA inhibitors".

Optimisation

Preferred inhibitors may be those which bind to the enzymatically required Zn ion or compete with the Zn ion, the catalytic water molecule and/or substrate for binding at the active site.

When a putative MG-CA inhibitor has been defined, its structure may be modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.

Additionally the three dimensional structure of MG-CA may be modelled with bound inhibitor (see Figure 5). A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The novel compound can be 'docked' with the MG-CA model to further optimise its structure. Software for modelling and docking inhibitors and CA enzymes is well known in the art - see e.g. "Docking of sulfonamides to carbonic anhydrase Il and IV1" Emilio Xavier Espositoa, KeIIi Barana, Ken Kelly and Jeffry D. Madura: Journal of Molecular Graphics and Modelling, Volume 18, Issue 3, June 2000, Pages 283-289; see also http://www.chemcomp.com where a variety of MOE ("Molecular Operating Environment") applications are available.

Product forms

"MG-CA inhibitors" either as explicitly disclosed herein or identified using methods as disclosed herein may be formulated into compositions.

These compositions per se, and their therapeutic or cosmetic uses, form further aspects of the present invention.

Suitable lotions, shampoos and conditioners for application to the scalp are well known to those skilled in the art. In the light of the present invention these product forms may be adapted by addition of the inhibitors of the M. globosa CA to form products of the present invention.

For example published US patent application US2002168327 describes known compositions such as transparent or opaque emulsions, lotions, creams, pastes or gels.

Particularly preferred product forms are shampoos and conditioners, especially shampoos.

Shampoo composition will comprise one or more cleansing surfactants which are cosmetically acceptable and suitable for topical application to the hair. Further surfactants may be present as an additional ingredient if sufficient for cleansing purposes is not provided as emulsifier for any emulsified components in the composition, e.g. emulsified silicones. Examples of suitable cleansing surfactants, anionic surfactants, amphoteric and zwitterionic surfactants, nonionic surfactants are well known in the art and described in US2002168327. Also described therein is the use of cationic deposition polymers, for enhancing conditioning performance of the shampoo. "Deposition polymers" are agents which enhance deposition of a silicone component in the shampoo composition onto the intended site during use, i.e. the hair and/or the scalp.

Shampoos and conditioners may also include aesthetic agents e.g. to opacify or pearlise them to enhance consumer appeal. Examples of suitable aesthetic agents are well known in the art and described in US2002168327.

Conditioners also form one aspect of the present invention. Such conditioners may be formulated as conditioners for the treatment of hair (typically after shampooing) and subsequent rinsing, and will typically comprise one or more cationic surfactants which are cosmetically acceptable and suitable for topical application to the hair. Examples of suitable cationic surfactants and other conditioning agents are well known in the art and described in US2002168327, and include emulsified silicones, used to impart for example wet and dry conditioning benefits to hair such as softness, smooth feel and ease of. compatibility.

Shampoos and conditioners may also include other ingredients including viscosity modifiers, preservatives, colouring agents, polyols such as glycerine and polypropylene glycol, chelating agents such as EDTA, antioxidants, fragrances, and sunscreens. Examples of suitable additional agents are well known in the art and described in US2002168327.

The compositions herein can be prepared by any method known in the art. The exact method will depend on the nature of the composition. The compounds of the present invention can be added to the compositions separately, or for example be combined with other ingredients commonly used in cosmetic compositions or medicaments, or for example dispersed or dissolved in water or oil or a water-in-oil emulsion prior to addition to the composition.

Other active agents

As described in US2003165449, suitable, non-limiting examples of anti-dandruff particulates include: heavy metal salts of pyridinethione, especially zinc pyridinethione, selenium sulfide, particulate sulfur, and mixtures thereof. Preferred are pyridinethione salts. Such anti-dandruff particulate should be physically and chemically compatible with the essential components of the composition, and should not otherwise unduly impair product stability, aesthetics or performance.

Pyridinethione anti-dandruff particulates, especially 1-hydroxy-2-pyridinethione salts, are highly preferred particulate anti-dandruff agents for use in compositions of the present invention. The concentration of pyridinethione anti-dandruff particulate typically ranges from about 0.1% to about 4%, by weight of the composition, preferably from about 0.1% to about 3%, most preferably from about 0.3% to about 2%. Preferred pyridinethione salts include those formed from heavy metals such as zinc, tin, cadmium, magnesium, aluminum and zirconium, preferably zinc, more preferably the zinc salt of 1-hydroxy-2- pyridinethione (known as "zinc pyridinethione" or "ZPT"), most preferably 1-hydroxy-2- pyridinethione salts in platelet particle form, wherein the particles have an average size of up to about 20μ, preferably up to about 5μ , most preferably up to about 2.5μ. Salts formed from other cations, such as sodium, may also be suitable. Pyridinethione anti- dandruff agents are described, for example, in U.S. Pat. No. 2,809,971 ; U.S. Pat. No. 3,236,733; U.S. Pat. No. 3,753,196; U.S. Pat. No. 3,761 ,418; U.S. Pat. No. 4,345,080; U.S. Pat. No. 4,323,683; U.S. Pat. No. 4,379,753; and U.S. Pat. No. 4,470,982.

Selenium sulfide is a particulate anti-dandruff agent suitable for use in the compositions of the present invention, effective concentrations of which range from about 0.1% to about 4%, by weight of the composition, preferably from about 0.3% to about 2.5%, more preferably from about 0.5% to about 1.5%. Selenium sulfide is generally regarded as a compound having one mole of selenium and two moles of sulfur, although it may also be a cyclic structure that conforms to the general formula SexSy, wherein x+y=8. Average particle diameters for the selenium sulfide are typically less than 15μm, as measured by forward laser light scattering device (e.g. Malvern 3600 instrument), preferably less than 10μm. Selenium sulfide compounds are described, for example, in U.S. Pat. No. 2,694,668; U.S. Pat. No. 3,152,046; U.S. Pat. No. 4,089,945; and U.S. Pat. No. 4,885,107.

Another preferred agent for use in combination with the inhibitors of the present invention is ketoconazole. Ketoconazole was originally described by Heeres et al., for instance, in U.S. Pat. No. 4,335,125, in which its principal utility was as an antifungal compound. Ketoconazole was also disclosed by Rosenberg et al. in U.S. Pat. No. 4,569,935 to be useful in the topical treatment of psoriasis and seborrheic dermatitis. Pursuant to this utility, ketoconazole has been marketed in a 2% shampoo formulation. In the compositions of the present invention it may be typically present at 0.3% to 3% ketoconazole.,

Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

The invention will now be further described with reference to the following non-limiting Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.

The disclosure of all references cited herein, inasmuch as it may be used by those skilled in the art to carry out the invention, is hereby specifically incorporated herein by cross- reference.

Examples

The present invention is based on the identification and inhibition of the CA from M. globosa, for the purpose of providing novel treatments for dandruff.

Example 1 - Background to CAs

In humans, sixteen different isoforms have been identified, with varying physiological importance. Accordingly, these isozymes possess different catalytic activities, cellular locations and tissue distributions. In terms of localisation, five are found in the cytoplasm (CA MII₁ CAVII, CAXIII), five are membrane bound (CA IV, CA IX, CA XII, CA XIV, CA XV), two are located in the mitochondria (CA VA and VB) and there is a single secreted isozyme (CA Vl). The function of isozymes VIII, X and Xl remains unclear, but appear to be acatalytic. The isozymes are distributed throughout the human body and have been identified, amongst other tissues, in the brain, kidney, eye, lung, epithelium, oesophagus, colon, pancreas and heart.

Several crystal structures of human CAs have been solved (CA I and II), both alone and in complex with ligands. These structures have contributed to the detailed understanding of the enzymatic mechanism of these proteins. All catalytically active human CAs contain a Zn ion bound at the active site by three histidine residues. A water molecule completes the tetrahedral co-ordination of the metal ion, which is ultimately responsible for nucleophilic attack at substrate. The Zn-bound water molecule interacts, through hydrogen bonding, with the active site residue Thr199 (based on CA Il numbering) and, via a cascade of histidine residues leading to the outer surface of the protein, a proton is abstracted to generate hydroxide. Attack at a suitably orientated carbon dioxide molecule, by this Zn bound hydroxide, leads to the formation of bicarbonate, which is subsequently released.

In addition to human isoforms, the CAs from specific organisms have been targeted for therapeutic gain, most notably to fight infectious disease. Thus the CAs from Plasmodium falciparum (malaria), Helicobacter pylori (stomach ulcers/gastric cancer), C. albicans (fungal infections), C. neoformans (meningitis) and Mycobacterium tuberculosis (tuberculosis) have all been proposed as potential targets.

All of the human CAs belong to the α-class of CAs, whilst most prokaryotes belong to the β-class. These gene families are evolutionarily unrelated. One of the significant differences between these classes is in the amino acid residues used to bind the catalytic Zn ion at the active site. The α-class of CAs utilise three histidine residues, whilst the β- class use an aspartate, histidine and two conserved cysteine residues to co-ordinate the Zn ion.

Example 2 M. globosa CA

The M. globosa protein database was searched using the E. coli CA Il sequence (SEQ ID NO. 2). BLAST analyses (Figure 3) identified two identical sequences, XP_001730815.1 and EDP43601.1 (SEQ ID NO. 1). Further sequence alignments (Figure 4), with CAN 2 from C. neoformans (SEQ ID NO. 3), showed the Zn binding residues to be conserved amongst these organisms (Cys47, Asp49, His103 and Cys106, based on the M. globosa numbering). Additional sequence comparisons of XP_001730815.1 /EDP43601.1 with E. coli CA I (SEQ ID NO. 4), C. neoformans CAN 1 (SEQ ID NO. 5) and the CA from Candida albicans (SEQ ID NO. 6), also showed the Zn binding residues to be conserved and appreciable identity throughout the sequences. In contrast, however, no significant sequence identity was found between human CA Il and XP_001730815.1/EDP43601.1. Thus, the proteins sequences XP_001730815.1 and EDP43601.1 have found to be identical and likely represent a single β-class CA from M. globosa. The corresponding DNA sequence for XPJ)OI 730815.1/EDP43601.1 is given as SEQ ID NO. 8.

BLAST searches of the available M. furfur and M. restricta databases were also performed using both the E.coli CA Il and XP_001730815.1/EDP43601.1 sequences. No protein sequences of similarity could be identified. This likely reflects the lack of protein sequences currently available for these particular Malassezia species rather than the absence of CA isoforms.

Additionally, using the E.coli CA Il crystal structure, a homology model of XP_001730815.1/EDP43601.1 was constructed (Figure 5). Consideration of the proposed Zn binding residues shows that they are ideally positioned to chelate a Zn ion at the supposed M. globosa CA active site.

Example 3 - cloning, expression and purification of the M. globosa carbonic anhvdrase

M. globosa strain ATCC 96807 / CBS 7966 was used for the cloning experiments. Addition of the EcoRI recognition sequence (underlined in the following sequence) resulted in the following forward primer

3'. The 3'-primer sequence, including the Sail recognition site (underlined in the following sequence), was The PCR reaction was hot-started

with incubation for 5 min at 94°C and consisted of 40 cycles of 30 sec at 94°C, 30 sec at 57°C and 90 sec at 72°C, and terminated with incubation for 10 min at 72ºC. The PCR products were cleaved with EcoRI and Sa/I, and then ligated in-frame into the pGEX-4T2 vector (Amersham, UK). The DNA sequence of the MG-CA insert, subcloned into the vector, was confirmed by DNA sequencing. The constructs were then transformed into E. coll strain BL21 for production of the GST-MG-CA fusion protein. Following induction of protein expression, by addition of 1 mM isopropyl-γ-D-thiogalactopyranoside, the bacteria were harvested and sonicated in PBS. The sonicated cell extracts were further homogenized twice with a polytron (Brinkmann) for 30 sec each at 4ºC. Centrifugation at 30Kg for 30 min afforded the supernatant containing the soluble proteins. The obtained supernatants were then applied to prepacked Glutathione Sepharose 4B columns (Amersham). The columns were extensively washed with buffer and then the GST- MG- CA fusion protein (Figure 6) was eluted with a buffer consisting of 5mM reduced glutathione in 5OmM Tris-HCI pH 8.0. Finally, the GST part of the fusion protein was cleaved with thrombin. The obtained MG-CA recombinant protein was further purified by sulfonamide affinity chromatography and the amount of enzyme was determined spectrophotometricaliy as reported for similar β-CAs.

Example 4 - carbonic anhydrase assay, kinetic property determinations

An Applied Photophysics stopped flow instrument was used for measuring the carbonic anhydrase CO₂ hydration reaction for both the determination of kinetic properties and compound screening. Phenol red (0.2 mM), with an absorbance maximum of 557nm, was used as an indicator to monitor the progression of the reaction, which causes a change in pH of the solution. Activity was measured at 2OºC and pH 7.5 in 10 mM Hepes buffer for the human CA Il isozyme (α-CA). The activity of the β-CAs (from M. glohosa, C. neoformans and C. albicans) was measured at 20ºC, pH 8.3 in 20 mM Tris-HCI buffer and 20 mM NaCI.

For the measurement of kinetic parameters, CO₂ concentrations ranged from 1.7 to 17 mM with enzyme concentrations at 15nM. The reaction was followed for 10 - 50 seconds. Uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Catalytic activity was calculated from Lineweaver-Burk plots and represents the mean of triplicate experiments.

Table 1 below compares the kinetic properties of the MG-CA with those CAs from C. neoformans and C. albicans as well as the human CA I and Il isozymes.

From the above, it can clearly be seen that the MG-CA has kinetic properties consistent with those CAs from other organisms.

Example 5 - carbonic anhvdrase assay, compound screening

For inhibition measurements, using the above described spectrophometric assay, the enzyme and inhibitor were preincubated together at room temperature for 15 minutes to allow the formation of the enzyme-inhibitor complex. Inhibitor stock solutions (1 mM) were prepared in 10% DMSO, with subsequent dilutions to 0.01 nM in distilled water. Each inhibitor was typically tested in the concentration range from 0.01 nM to 100μM. Inhibition constants were obtained by nonlinear least-squares methods using PRISM 3.

K| values were determined, using the assay described above, for a series of compounds against the MG-CA. Results are shown in the Table 2 provided hereinafter.

The results clearly demonstrate that previously identified CA inhibitors also reduce the activity of the MG-CA, with a range of potencies. In particular, the para-amino substituted benzene sulphonamides (cmpds 11 - 13) appear to be particularly potent with Ki values from 79 - 245 nM. The ortho-amino equivalent is, however, of lower potency at 9.8 μM. The corresponding hydroxyl (cmpds 14 - 15) and carboxylic acid derivatives (cmpds 16 - 17) were of equivalent potency to the amino substituted benzene sulphonamides, as were the halogenated sulphanilamide analogues (cmpds 19 - 22). However, 4-methyl benzene sulphonamide (cmpd 18) was significantly less active (~ 30 fold) than 4-amino benezene sulphonamide/sulphanilamide (cmpd 11). Compounds 30 - 32 were also highly potent with double digit nanomolar K| values. Sulphamate and sulphamide derivatives of benzene sulphonamide (cmpds 36, 45, 47) were all of equivalent potency to one another. This was also observed for the sulphamate and sulphamide derivatives of para-bromo benzene sulphonamide (cmpds 39, 46, 48), suggesting that all three functional groups can be accommodated at the MG-CA active site.

A number of carbonic anhydrase inhibitors that have been used clinically were also tested. Of these, the most potent compounds were dichlorophenamide (cmpd 4, 346 nM), sulpiride (cmpd 33, 32OnM) and indisulam (cmpd 7, 113nM). Acetazolamide (cmpd 1), methazolamide (cmpd 3) and their derivatives (cmpds 25 - 27), were of relatively lower activity with K, values in the micromolar range. The exception to this was benzolamide (cmpd 28, 482 nM) and its para-amino substituent (cmpd 29, 236 nM). Ethoxzolamide (cmpd 2), brinzolamide (cmpd 5), dorzofamide (cmpd 6), topiramate (cmpd 8), zonisamide (cmpd 9), valdecoxib (cmpd 34) and celecoxib (cmpd 35) were all relatively weaker inhibitors of the MG-CA.

Example 6 - MIC determinations against Malassezia sp

In order to evaluate the effect of the identified MG-CA inhibitors on Malassezia growth, susceptibility tests were performed on Malassezia dermatis CBS 9145, Malassezia furfur CBS 9569, Malassezia pachydermatis CBS 6536, and Malassezia globosa CBS 7966. Malassezia strains were cultured in ambient air at 30°C on modified Leeming Notman agar for up to 120 hours before testing, to ensure their purity The inoculum for each strain was prepared by picking distinct colonies from the culture flasks and suspending them in 3 ml of sterile water. The turbidity was then adjusted to McFarland standard 5.0. The inoculum was completely resuspended by vigorous shaking on a vortex mixer for 15s. Any remaining particulates were then disrupted by passing the inoculum through a fine 27G sterile needle. The inocula were then adjusted by diluting 1 :10 in sterile water. All assays were performed in either Christensen's urea broth (supplemented with 0.2% tween 80 and 1.0% tween 60), or Christensen's urea agar (supplemented with 0.2% tween 80 and 1.0% tween 60 and solidified using 1% agar). Stock solutions of each of the compounds were prepared at a concentration of 2.5g/L in either DMSO or sterile water. All stock samples were then either further diluted in modified double strength Christensen's urea broth (broth assay) or in water (agar assay) to give a starting concentration of 640 mg/L In a 96 well plate, column 1 was filled with 200 μl of the appropriate test compound, 100μl amounts were taken from wells in column 1 and diluted two-fold by transferring them to column 2 with a multichannel pipette (± 2% coefficient of variation). Samples (100μl) were then removed from column 2 and transferred to column 3, and so on through to column 10. For the broth assay, Malassezia inoculum suspension (100μl) in sterile water was then added to the appropriate well, producing a well containing 200μl final volume (made up of 100μl diluted compound and 100 μl of inoculum). For the agar assay, 100μl of double strength molten Christensen's urea agar was then added to each well in a 5OºC water bath. The plates were then removed from the 5OºC water bath and the drug/agar media allowed to solidify. Malassezia inoculum suspension (10μl) in sterile water was then added to the appropriate well on top of the solidified agar/drug mixture. All plates were incubated at 3OºC in air within a darkened incubator for up to 7 days. Plates were read visually with the endpoint taken as the lowest concentration of drug that inhibited growth by more than 50% compared to the drug free control.

MIC values were determined for a number of the MG-CA inhibitors used in the enzymatic studies (against purified MG-CA) together with additional compounds. The results are shown in Table 3 hereinafter.

Following compound testing, MIC values as low as 10 μg/ml were observed, with growth inhibition apparent against all the Malassezia strains screened. Although the MIC values for a number of the compounds was high (640 μg/ml), this may be a reflection of low cell permeability, a frequent problem with cell based assays

Example 7 - murine model of M. pachvdermatis skin infection

The in vitro data indicated that inhibitors of the MG-CA also limited the growth of Malassezia sp. A murine model of M. pachydermatis skin infection was used to assess the in vivo efficacy of the compounds.

Mice used in this study, male CD1 mice (an outbred strain that is very similar to Swiss mice), were supplied by Charles River (Margate UK) and were specific pathogen free (16- 18g at delivery). All mice weighed 22-25g at the beginning of the experiment.

Mice were housed in individual ventilated cages supplied with HEPA filtered air. Sterile aspen chip bedding was supplied in pre-autoclaved boxes. Sterile water was provided ad libitum using disposable pouches. Standard mouse chow was provided ad libitum. Mice experienced a 12hour light dark cycle at 22±1°C, 55-60% relative humidity and background noise of <60db.

For the study, animals were treated in groups of 6 mice per treatment group. Cortisone acetate was prepared as a 12.5mg/ml suspension in PBS containing 0.05% tween 80. All animals were immunosuppressed with a dose of 125mg/kg cortisone acetate subcutaneously 5 days before infection and re-immunosuppressed at the same dose 24 hours before infection.

On the day of infection, the mice were lightly anaesthetized with 2.5% isofluorane. Both flanks were then shaved (2cm x 2cm) and just prior to infection the shaved flanks were lightly scarified with a scrubbing brush. Following surface abrasion, flanks were coated with a suspension of M. pachydermatis containing 2x10⁷ blastoconidia/ml. Following infection all mice were observed as regularly as their clinical conditioned required. Animals exceeding the severity band of the experiment (mild) would have been humanely euthanized.

Mice were treated 48hrs post-infection (just before the appearance skin lesions), once daily for 6 days by liberally spreading the relevant cream or vehicle control on to the infected flank using cotton tipped swabs. The skin of the shaved flanks was examined daily by investigators that were blinded to the treatment mice received or the infecting organism.

Post infection (8 days) all mice were humanely terminated by cervical dislocation. Following euthanasia, approximately 10 hairs were removed from each flank of the mouse, were cultured onto Leeming Notman agar and incubated at 3OºC for up to 20 days. Skin biopsies were also removed from the flanks of the mice and cultured as indicated.

The compound chosen for the in vivo studies was 4-(2-Aminoethyl) benzenesulfonamide (cmpd 13), which was one of the most potent molecules from the enzymatic studies with a Ki value of 79nM. Large scale quantities of this material could also be readily purchased from a commercial supplier. A 5% cream was prepared by dissolving 100mg of 4-(2- Aminoethyl) benzenesulfonamide powder in 200 μl of DMSO and then mixing this solution with 2g of E45 cream and vortexing for 2 minutes to disperse. A 2% Nizoral (Ketoconazole) cream was used as a positive control. The vehicle control was 5% E45 cream alone.

Results for both skin appearance and the culture experiments for all three treatment options are given in Table 4 below:

For mice treated with 5% 4-(2-Aminoethyl) benzenesulfonamide, 67% (4/6) demonstrated clinical improvement following infection with Malassezia and subsequent compound application.

No treatment, including ketoconazole, was effective at clearing the skin or hair samples of Malassezia as determined by culture, which may reflect the need for a prolonged treatment period. However, it should be noted that immunosupression of the mice for such a duration would not be possible and hence experimental results would then be conflicted by the host's natural immune response.

Treatment with 4-(2-Aminoethyl) benzenesulfonamide caused damage to the Malassezia resulting in hypha fragmentation, which is indicative of compound effect on fungal growth.

SEQ ID NO. 2 is the amino acid sequence of Escherichia coli CA II (P61517), which shares 35% sequence identity with the proposed Malassezia globosa carbonic anhydrase (XP_001730815.1/EDP43601.1).

Ciyptococcus neoformans CAN 2 (Q314V7)

>tr| Q3I4V7 J Q3I4V7_CRYNV Carbonic anhydrase 2 OS=Cryptococcus neoformans var. grubii GN=CAN2 PE=2 SV=I

MPPHAEPLKPSDEIDMDLGHSVAAQKFKEIREVLEGNRYWARKVTSEEPEFMAEQVKGQA PNFLWIGCADSRVPEVTIMARKPGDVFVQRNVANQFKPEDDSSQΆLLNYΆIMNVGVTHVM WGHTGCGGCIAAFDQPLPTEENPGGTPLVRYLEPIIRLKHSLPEGSDVNDLIKENVKMA VKNVVNSPTIQGAWEQARKGEFREVFVHGWLYDLSTGNIVDLNVTQGPHPFVDDRVPRA

SEQ ID NO. 3 is the amino acid sequence of Cryptococcus neoformans CAN 2 (Q314V7), which shares 33% sequence identity with the proposed Malassezia globosa carbonic anhydrase (XP_001730815.1/EDP43601.1). Escherichia coli CA I (P0ABE9)

SEQ ID NO. 4 is the amino acid sequence of Escherichia coli CA I (P0ABE9), which shares 22% sequence identity with the proposed Malassezia globosa carbonic anhydrase (XP_001730815.1/EDP43601.1).

SEQ ID NO. 5 is the amino acid sequence of Cryptococcus neoformans CAN 1 (Q30E70), which shares 34% sequence identity with the proposed Malassezia globosa carbonic anhydrase (XP_001730815.1/EDP43601.1).

SEQ ID NO. 6 is the amino acid sequence of Candida albicans carbonic anhydrase (Q5AJ71), which shares 34% sequence identity with the proposed Malassezia globosa carbonic anhydrase (XP_001730815.1/EDP43601.1).

Human carbonic anhydrase II (P00918)

>sp|P00918|CAH2_HUMAN Carbonic anhydrase 2 OS=Homo sapiens GN=CA2 PE=I SV=2

MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYDQATSLRILNNGHA

FNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHTVDKKKYAAELHLVHWNTKYGD

FGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSADFTNFDPRGLLPESLDYWTYPG

SLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK

SEQ ID NO. 7 is the amino acid sequence of human carbonic anhydrase II (P00918), which shares no significant sequence identity with the proposed Malassezia globosa carbonic anhydrase (XP_001730815.1/EDP43601.1).

XM_001730763

atgctgccca tttctcgtga ggtggtggac tcgttgctcg atcgcaacgc gtcctggtcc aaagacttta tcacgcatca gccggatcta gcttgtgcgc tccgagaggg tcagcaccca aaggtatttt ggattggatg cagtgactct cgtgtccctg aaagtgtggt ttgcaatgca cgtcctggtg agctgttcgt cttgcgcaac gtagcgaatc aattccattt gcacgacgac agcgcggtaa gtgcgttgac gtfctgctgtg caagcacttg gcgtcgagca cgtcattgtc gfccggacaca cgagctgcgg tggtgtagcg gcagcggtga aacaggcgct caaagagcag gaagatgact atgagccacc gccgtcgtca gcgctagcga ggcacttgtc gtcgctgact gagctcgcac gctatttccg agtgcgagtg cgcgaacgca atatcatgtc tggcaagtcg atgcaagagc ggctcgtccc tctgctgacc gaggcgagcg tgcgtcgaca aatccaaaac attgtagagc accctgtgat ccaggacaac tggaaccagc gtgtatcgcc cttgaacggc aaggtgaacc cgcgtgttac catccatggc tggattcata acctgcatga caatcgcttg tttgacctca atgtctcggt ccctccgccc ccgttgaacg aggagaagaa gcaaagcaca aactaa

SEQ ID NO. 8 is the DNA sequence encoding the proposed Malassezia globosa carbonic anhydrase (XP_001730815.1/EDP43601) of SEQ ID NO. 1. 

Claims

1 A method of treating and\or preventing dandruff, dermatitis or tinea versicolor in a subject which method comprises topically administering a composition comprising an inhibitor of a β-class carbonic anhydrase to the subject.

2 A method as claimed in claim 1 for treating and\or preventing dandruff in the subject wherein the inhibitor is an inhibitor of the Malassezia globosa carbonic anhydrase.

3 A method as claimed in any one of claims 1 to 2 wherein the composition further comprises additional active ingredients having activity against Malassezia spp.

4 A method as claimed in any one of claims 1 to 3 wherein the composition further comprises an active ingredient selected from the list consisting of:

(i) a heavy metal salt of pyrithione; (ii) selenium sulfide; (iii) ketoconazole; (iv) particulate sulfur; (v) coal tar.

5 A method as claimed in claim 4 wherein the composition further comprises an active ingredient which is zinc pyrithione.

6 A method as claimed in any one of claims 1 to 5 wherein the composition is a shampoo, conditioner or skin lotion.

7 A method as claimed in any one of claims 1 to 6 wherein administering the composition comprises applying an effective amount of the composition to hair or skin that has optionally been wetted with water, and then rinsing the composition off.

8 A method as claimed in any one of claims 1 to 7 wherein the inhibitor of the carbonic anhydrase is selected from the list consisting of:

(i) sulphonamide derivatives; (ii) sulphamide derivatives; (iii) sulphamate derivatives. 9 A method as claimed in claim 8 wherein the inhibitor of the carbonic anhydrase is a compound of the following general formula (1), or a pharmaceutically acceptable salt, hydrate or solvate thereof:

in which:

-L₁- is independently a single covalent bond,

wherein:

-L_A- is independently C_1-3 alkylene; and

-L_B- is independently a single covalent bond, -

and wherein:

-A is independently C_6-10 carboaryl, C₅.₁₀ heteroaryl, non-aromatic C_3-7 carbocyclyl, or non-aromatic C_5--_I2 heterocyclyl, and is optionally substituted.

10 A method as claimed in claim 9 wherein the inhibitor of the carbonic anhydrase is a compound of the following general formula (3), or a pharmaceutically acceptable salt, hydrate or solvate thereof:

wherein:

-B- is independently phenylene, and is optionally substituted with n groups R¹; and wherein:

n is independently 0, 1 , 2, or 3;

and wherein:

each -R¹ is independently selected from -SO₂NH₂, -F, -Cl, -Br, -I₁ C_1-4 alkyl, haloalkyl, -R²-O-R³, and -NHR³;

wherein:

and wherein:

-L₁- is independently a single covalent bond,

-L₂- is independently a single covalent bond or C_1-4 alkylene;

and wherein:

11 A method as claimed in claim 9 wherein the inhibitor of the carbonic anhydrase is a compound of the following general formula (4), or a pharmaceutically acceptable salt, hydrate or solvate thereof:

wherein: -B- is independently 1 ,4-phenylene or [1 ,3,4j-thiadiazol-2,5-di-yl, and is optionally substituted with n groups R¹;

and wherein:

n is independently 0, 1 , 2, or 3;

and wherein:

each -R¹ is a group independently selected from -SO₂NH₂, -F, -Cl, -Br, -I, C_1-4 alky!, C_1-4 haloalkyl, -R²-O-R³, and -NHR³;

wherein:

and wherein:

wherein:

-Z- is independently selected from a single covalent bond and C_1-4 alkylene optionally substituted with =O;

and wherein:

-X is independently -D; wherein:

-D is independently C_6-10 carboaryl, C_5-10 heteroaryl or non-aromatic C_5-12 heterocyclyl, optionally substituted with one or more groups independently selected from C_1-4 alkyl, -NH₂, -F, -Cl, -Br, -I, C_1-4 haloalkyl, and -E;

wherein:

-E is independently phenyl, C₅ heteroaryl, or non-aromatic C₅ heterocyclyl, and is optionally substituted.

12 A method as claimed in any one of claims 8 to 11 wherein the inhibitor of the carbonic anhydrase is selected from those shown in Table 2.

13 A method as claimed in claim 12 wherein the inhibitor of the carbonic anhydrase is selected from the list consisting of: 4-(2-Aminoethyl) benzenesulfonamide (compound 13); aminobenzolamide (compound 29); 4-(sulfanilyl-amidoethyl)-benzene sulphonamide (compound 30); 4-(sulfanilylaminomethyl)-benzene sulphonamide (compound 31); 4-(2- amino-pyrimidin-4-ylamino)-benzene sulphonamide (compound 32); 5- (trifluoromethyl)aniline - 2,4 - disulphonamide (compound 24); 3-chloroaniline-4,6- disulphonamide (compound 23).

14 A method as claimed in claim 12 wherein the inhibitor of the carbonic anhydrase is selected from the list consisting of:

(i) acetazolamide; (ii) ethoxzolamide; (iii) methazolamide; (iv) dichlorophenamide (v) brinzolamide; (vi) dorzolamide; (vii) indisulam; (viii) topiramate; (ix) zonisamide. (x) sulpiride (xi) benzolamide 15 A method as claimed in any one of claims 1 to 14 wherein the inhibitor of the carbonic anhydrase has a Ki of less than 1 μM.

16 A method for identifying a compound capable of treating and\or preventing dandruff, dermatitis or tinea versicolor in a subject, which method comprises:

(a) providing a test compound;

(b) comparing under comparable reaction conditions the activity of a polypeptide, which polypeptide is the Malassezia globosa carbonic anhydrase or a variant or active portion thereof, in the presence and absence of the test compound.

17 A method for identifying a compound capable of treating and\or preventing dandruff, dermatitis or tinea versicolor in a subject, which method comprises:

(a) providing a test compound;

(b) providing a polypeptide, which polypeptide is the Malassezia globosa carbonic anhydrase or a variant or active portion thereof, in isolated form;

(c) incubating the polypeptide in the presence of the test compound,

(d) measuring the activity of the polypeptide,

(e) optionally comparing the value obtained in step (d) with a reference value.

18 A method as claimed in claim 17 wherein the polypeptide is provided in isolated form from a host cell comprising a recombinant vector including nucleic acid encoding said polypeptide.

19 A method for identifying a compound capable of treating and\or preventing dandruff, dermatitis or tinea versicolor in a subject, which method comprises:

(a) providing a test compound which is believed to be a carbonic anhydrase inhibitor and which is selected from the list consisting of: zinc binding compounds comprising a sulphonamide, sulphamide or sulphamate moiety;

(b) testing its antifungal activity against M. globosa.

20 A method as claimed in claim any one of claims 16 to 19 further comprising formulating the identified compound as a therapeutic or cosmetic composition for treating and\or preventing dandruff, dermatitis or tinea versicolor. 21 A therapeutic or cosmetic composition adapted for topical application to a subject comprising as an active ingredient an inhibitor of the Malassezia globosa carbonic anhydrase

22 A composition as claimed in claim 21 wherein the composition is a shampoo, conditioner or skin lotion.

23 A composition as claimed in claim 22 wherein:

(i) said shampoo comprises one or more cleansing surfactants which are cosmetically acceptable and suitable for topical application to the hair, and

(ii) said conditioner comprises one or more cationic surfactants which are cosmetically acceptable and suitable for topical application to the hair.

24 A composition as claimed in claim 22 or claim 23 further comprising an additional active ingredient selected from the list consisting of:

25 A composition as claimed in claim 24 comprising a heavy metal salt of pyrithione which is zinc pyrithione.

26 An inhibitor of the Malassezia globosa carbonic anhydrase for use in a therapeutic or cosmetic method as claimed in any one of claims 1 to 15.

27 An inhibitor as claimed in claim 26 which is in the form of a composition of any one of claims 22 to 25.

28 Use of an inhibitor of the Malassezia globosa carbonic anhydrase in the preparation of a therapeutic or cosmetic composition for use in a method of treating and\or preventing dandruff and\or dermatitis and\or tinea versicolor in a subject.

29 Use as claimed in claim 28 wherein the method is a method as claimed in any one of claims 1 to 15.