WO2000032220A1 - Anti-dandruff composition comprising an antifungal polypeptide - Google Patents

Abstract

The present invention relates to an anti-dandruff composition comprising an antifungal polypeptide, to the use of an antifungal polypeptide for the treatment or prophylaxis of dandruff, to a method for the treatment or prophylaxis of dandruff, and to the use of an antifungal polypeptide for the preparation of a composition for the treatment or prophylaxis of dandruff. It has been found that the antifungal polypeptide with the amino acid sequence shown in Fig. 1 efficiently impedes the growth of the yeast P. ovale, even in low concentrations.

Description

ANTI -DANDRUFF COMPOSITION COMPRISING AN ANTIFUNGAL POLYPEPTIDE

FIELD OF THE INVENTION

The present invention relates to an anti -dandruff composition comprising an antifungal polypeptide, to the use of an antifungal polypeptide for the treatment or prophylaxis of dandruff, to a method for the treatment or prophylaxis of dandruff, and to the use of an antifungal polypeptide for the preparation of a composition for the treatment or prophylaxis of dandruff.

BACKGROUND OF THE INVENTION

Dandruff results from a scalp malfunction but is not considered as being a skin disease and, accordingly, dandruff should be distinguished from the diseases that may mimic it in their early stages. Thus, although dandruff is only of cosmetic concern, a large research effort has been devoted to the field of treating this condition.

It is widely accepted that dandruff is mainly caused by the yeast Pi tyrosporum ovale and, consequently, substances which inhibit the growth of P. ovale might prove useful in the treatment or prophylaxis of dandruff.

Active substances which traditionally have been used for treating dandruff include for example various coal tar preparations, salicylic acid, selenium sulphide and zinc pyrithione.

More recently a novel anti-dandruff shampoo comprising ketoconazole or elubiol has been described in WO 95/27471. However, some of the prior art substances have a very unpleasant smell, while others are very potent substances that may only be applied to the scalp under medical guidance and control. Therefore, there is a need for the development of substances which are readily produced (e.g. by a microorganism) , which do not cause obnoxious smells and which possess no hazardous properties .

SUMMARY OF THE INVENTION

It has now surprisingly been found that the antifungal polypeptide with the amino acid sequence shown in Fig. 1 efficiently impedes the growth of the yeast P. ovale, even in low concentrations.

Accordingly, in a first aspect the present invention relates to an anti-dandruff composition suitable for application to the hair or scalp of a human, comprising an effective amount of an antifungal polypeptide comprising the amino acid sequence shown in Fig. 1, or a variant thereof,

said variant being a polypeptide which

I) reacts with an antibody reactive with at least one epitope of the polypeptide having the amino acid sequence shown in Fig . 1 , or

II) has an amino acid sequence which is substantially homologous with the amino acid sequence of the polypeptide shown in Fig. 1. In a second aspect, the present invention relates to the use of an antifungal polypeptide as defined above for the treatment or prophylaxis of dandruff.

In a third aspect, the present invention relates to a method for the treatment or prophylaxis of dandruff, said method comprising contacting hair with an effective amount of an antifungal polypeptide as defined above.

Finally, the present invention also relates to the use of an antifungal polypeptide as defined above for the preparation of a composition for the treatment or prophylaxis of dandruff.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the full amino acid sequence of the antifungal polypeptide.

Figure 2 is a schematic representation of the microtiter plates in which the yeast P. ovale was grown, cf. Example 1.

DETAILED DESCRIPTION OF THE INVENTION

The antifungal polypeptide and variants thereof In the present context, the three-letter code of the amino acids has been used in its conventional meaning. Unless indicated explicitly, the amino acids mentioned herein are L- amino acids.

The term "antifungal polypeptide" as used herein is intended to comprise the linear as well as the active folded structures of the polypeptide, and may, where appropriate, be used interchangeably with the term "antifungal protein" .

The term "variant" as used in connection with the antifungal polypeptide is intended to indicate a polypeptide which is derived from the polypeptide having the amino acid sequence shown in Fig. 1, or a naturally occurring variant. Typically, the variant differs from the native antifungal polypeptide by one or more amino acid residues, which may have been added or deleted from either or both of the N-terminal or C-terminal end of the polypeptide, inserted or deleted at one or more sites within the amino acid sequence of the polypeptide, or substituted with one or more amino acid residues within, or at either or both ends of the amino acid sequence of the polypeptide.

Furthermore, the variant of the invention has one or both of the characteristic properties I) and II) mentioned above.

Property I), i.e. the reactivity of the variant of the invention with an antibody raised against or reactive with at least one epitope of the polypeptide having the amino acid sequence shown in Fig. 1 below, may be determined using an antibody preparation produced as described under the heading " Production of antibodies against the AFPP" in the Materials and Methods section below. The antibody reactivity may be determined using assays known in the art, examples of which are Western Blotting or radial immunodiffusion assay, e.g. as described by Hudson et al . , 1989.

The homology referred to above of the AFPP is determined as the degree of identity between two protein sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package

(Program Manual for the Wisconsin Package, Version 8, August

5 1994, Genetics Computer Group, 575 Science Drive, Madison,

Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD.,

(1970), Journal of Molecular Biology, 48, p. 443-453). Using

Gap with the following settings for polypeptide sequence comparison: Gap creation penalty of 3.0 and Gap extension o penalty of 0.1, the mature part of a polypeptide encoded by an analogous DNA sequence of the invention exhibits a degree of identity preferably of at least about 60%, such as at least about 70%, at least about 80%, at least about 90%, at least about 95%, more preferably at least about 97%, and most s preferably at least about 99% with the mature part of the amino acid sequence shown in figure 1.

Preferably, the AFPP comprises the amino acid sequences of figure 1; or allelic variants thereof; or fragments thereof 0 that has AFPP activity.

A fragment of AFPP is a polypeptide which have one or more amino acids seleted from the amino and/or carboxyl terminus of this amino acid sequence. An allelic variant denotes any of two 5 or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino 0 acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene. The amino acid sequences of homologous AFPP may differ from the amino acid sequence of figure 1 by an insertion or deletion of one or more amino acid residues and/or the substitution of one or more amino acid residues by different amino acid residues. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain .

The term "effective amount" when used herein is intended to mean an amount of the antifungal polypeptide comprising the amino acid sequence shown in Fig. 1, or a variant thereof, which is sufficient to combat (thereby removing or at least arresting) scaling due to dandruff.

Typically, the polypeptide to be used according to the invention is derived from a microorganism. While it is contemplated that the antifungal polypeptide or a variant thereof may be produced by other microorganisms such as bacteria, protozoae and algae, the polypeptide is normally derived from a fungus, in particular a fungus of the genus Aspergillus .

As explained in detail in WO 94/01459, the antifungal polypeptide having the amino acid sequence shown in Fig. 1 has been found in several strains of the fungal species A . - giganteus, an example of which is the A . giganteus strain deposited with the Centraallbureau voor Schimmelcultures (CBS) under the deposition number CBS 526.65.

However, the antifungal polypeptide, or variants thereof, suitable for the use according to the invention are expected to be derivable from other fungal species, especially other Aspergillus species such as A . pallidus , A . clavatus, A . longivesica, A . rhizopodus and A . clavatonanicus, because of the close relationship which exists between these species and A . giganteus .

As explained above the variant of the antifungal polypeptide may be a naturally derived variant, i.e. a polypeptide isolated from any of the fungal species listed above, or may be a synthetically produced variant constructed on the basis of the knowledge of the amino acid sequence of the antifungal polypeptide .

As indicated previously, other variants of the antifungal polypeptide may also be useful for the purposes of the present invention. Therefore, it is contemplated that interesting variants of the polypeptide shown in Fig. 1, which will also be useful in the treatment or prophylaxis of dandruff, may be identified on the basis of an analysis of the conformational structure and the active site of the antifungal polypeptide shown in Fig. 1 using techniques known in the art, examples of which are NMR (e.g. as described in Protein Engineering, Editors Oxender, D.L. and Fox, C.F., Alan R. Liss, Inc. New York (1987), X-ray diffraction, computer modelling etc. When one or more interesting modifications of the amino acid sequence of the antifungal polypeptide have been identified, the corresponding variants may be prepared by suitably modifying a nucleotide sequence encoding the antifungal polypeptide.

Previously, Nakaya et al . (1990) disclosed the amino acid sequence of an A. giganteus antifungal polypeptide. Wnendt et . al . (1990) described the corresponding cDNA sequence, and in WO 91/19738 the use of this cDNA sequence in the construction of trangenic plants capable of producing the polypeptide was described. The antifungal polypeptide used in the examples given herein (i.e. the antifungal polypeptide having the amino acid sequence shown in Fig. 1) differs from the antifungal polypeptide disclosed in WO 91/19738 as follows:

Asn-4 has been replaced with Pro, Ala-24 has been replaced with Gly, Lys-32 has been replaced with Arg, Phe-42 has been replaced with Leu and Tyr-50 has been replaced with His.

Thus, a specific example of an interesting variant of the polypeptide shown in Fig. 1 for use in the treatment or prophylaxis of dandruff is an antifungal polypeptide which comprises, and preferably consists of, the amino acid sequence shown in Fig. 1, in which Pro in position 4 has been substituted with Asn, Gly in position 24 has been substituted with Ala, Arg in position 32 has been substituted with Lys, Leu in position 42 has been substituted with Phe, and/or His in position 50 has been substituted with Tyr.

Another interesting variant has been disclosed in WO 94/01459. The amino acid sequence disclosed in WO 94/01459 differs from the amino acid sequence shown in Fig. 1 in the following way: Pro-4 has been replaced with Asp, Gly-24 has been replaced with Ala, Arg-32 has been replaced with Val , Leu-42 has been replaced with Phe and His-50 has been replaced with Tyr.

Therefore, another specific example of an interesting variant of the polypeptide shown in Fig. 1 for use in the treatment or prophylaxis of dandruff is an antifungal polypeptide which comprises, and preferably consists of, the amino acid sequence shown in Fig. 1, in which Pro in position 4 has been substituted with Asp, Gly in position 24 has been substituted with Ala, Arg in position 32 has been substituted with Val, Leu in position 42 has been substituted with Phe, and/or His in position 50 has been substituted with Tyr.

It is contemplated, however, that other variants than those mentioned above, may prove useful in the treatment or prophylaxis of dandruff. Thus, the use according to the invention of such variants, wherein one or more of the amino acid residues in position 4, 24, 32, 42 and 50 of the antifungal polypeptide shown in Fig. 1 have been substituted with another naturally occurring amino acid residue is also within the scope of the present invention.

The antifungal polypeptide shown in Fig. 1, as well as variants thereof, may be prepared by methods known in the art. A detailed description of the preparation of the polypeptide shown in Fig. 1 is given in WO 94/01459

Anti-dandruff compositions Although the compositions described herein will, for the sake of simplicity, generally be referred to as "shampoo compositions" it will be understood by persons skilled in the art that the antifungal polypeptide may also be incorporated into any other composition suitable for application to the scalp or hair. Therefore, such non-shampoo compositions comprising the antifungal polypeptide are also within the scope of the present invention. Examples of such other compositions are solutions, suspensions, emulsions, gels, foams, creams, etc. Thus, while a shampoo is the presently preferred composition for applying the antifungal polypeptide of the invention, it is also contemplated that the antifungal polypeptide may be applied by other means, e.g. formulated into a hair gel or mousse, a hair tonic, a hair spray, a hair conditioner, a hair dressing, etc.

Shampoo compositions may be adapted to oily, dry or normal hair and may be prepared in a manner well-known to the person skilled in the art, e.g. such as described in ^,xWoodruff's ingredients & formulary handbook", 1997, Miller Freeman.

Similarly, a hair gel or mousse, a hair tonic, a hair spray, a hair conditioner, etc. may also be prepared using ingredients and methods known in the art for the preparation of such compositions adapted to be applied to the hair or scalp. It will be clear to persons skilled in the art, however, that anti-dandruff compositions according to the invention should not be produced using ingredients or methods (e.g. excessively high temperatures) that would tend to inactivate the antifungal polypeptide .

The active ingredient, i.e. the antifungal polylpeptide comprising the amino acid sequence shown in Fig. 1, or a variant thereof, is employed in the shampoo composition in an amount which is effective for treatment or prophylaxis of dandruff . The shampoo composition of the present invention will typically contain the antifungal polypeptide in an amount from about 10^"7 to about 10 percent by weight calculated on the total weight of the shampoo composition. Preferably the amount of antifungal polypeptide is in the range from about 5x10^"6 to about 5% such as in any of the below sub-ranges: about 5xl0^"6 to about 0.5%; about 5xl0^"6 to about 0.1%; about 5xl0^"6 to about 10^"5%; about 10^"5 to about 10^"%; about 10^"4 to about 10^~3%; about 10^"3 to about 0.01%; about 0.01 to about 0.1%; about 0.1% to about 1.0%; and about 1.0% to about 5.0%.

Shampoo compositions according to the present invention may contain one or more surfactants, which are employed principally as cleansing agent. Selection of suitable surfactants is a matter of common general knowledge for the person skilled in the art. Usually anionic surfactants are employed in shampoo compositions. Examples of such anionic surfactants are alkali metal, ammonium or hydroxyalkylamine salts of alkyl sulphates, alkyl benzene sulphonates, alkyl sulphones, α-alkenyl sulphonates, polyoxyethylenealkylphenyl sulphonates and polyoxyethylenealkylphenyl sulphates . Mixtures of surfactants may also be used.

A specific example of a suitable surfactant which may be included in the shampoo composition according to the invention is sodium laureth sulphate, which has the formula CH₃ (CH₂) ₁₀CH₂ (OCH₂CH₂) _nOS0₃Na, wherein n represents a number having an average value of from 1 to 4.

Another specific example of a suitable surfactant which may be incorporated in the shampoo composition of the present invention is sodium cocoyl sarcosinate. Cocoyl sarcosine is the coconut oil fatty acid amide of sarcosine (N-methylglycine) and coconut fatty acid is a mixture of myristic acid, lauric acid, palmitic acid and stearic acid.

Other specific examples of suitable surfactants which may be incorporated in the shampoo composition of the present invention include sodium C₁₄, C₁₅ and C₁₆ olefin sulphonates, sodium lauryl sulphate, cocamidopropylamine oxide, lauramido diethanolamine (lauramido DEA) , cocamidopropyl betaine, disodium oleamido MIPA sulfosuccinate, disodium cocamido MIPA sulfosuccinate, disodium laureth sulfosuccinate, cocoaamphocarboxy-glycinate and disodium MEA sulfosuccinate.

Another interesting class of surfactants which may be incorporated in the composition of the present invention are sugar surfactants. Examples of surfactants in the form of sugar surfactants include (a) alkyl- and/or alkenyloligoglycosides and/or (b) fatty acid-N-alkylpolyhydroxyalkylamides .

The alkyl- and/or alkenyloligoglycoside (a) may have the formula (I) :

R¹-0-[G]_p (I)

in which R¹ = a C₄_₂₂ alkyl and/or alkenyl group, G = a sugar residue with 5 or 6 carbon atoms, and p = 1-10.

The fatty acid-N-alkylpolyhydroxyalkylamide (b) may have the formula:

R²CO-N(R³) - [Z] (II) in which R²CO = a C₆_₂₂ aliphatic acyl residue, R³ = H, alkyl or hydroxyalkyl with 1-4 carbon atoms, and [Z] = a linear or branched polyhydroxyalkyl residue with 3-12 carbon atoms and 3- 10 OH groups.

It also includes a) alkyl and alkenyl oligoglycosides of the formula R¹-0-[G]_p and b) alkali and/or alkali metal salts of ^ci₂-₂₂ secondary 2, 3 -alkyl sulphates, where R¹ = C₄_₂₂ alkyl and/or alkenyl; G = a sugar residue with 5 or 6 carbon atoms, and p = 1-10. The weight ratio (I): (II) is preferably 1:99-99:1.

Also included are fatty acid-N-alkyl polyhydroxyalkyl amides; and sugar surfactants of: saccharose esters, sorbitan esters and/or polysorbates .

A sugar surfactant may also comprise 10-40% by weight of alkyl and/or alkenyl -oligoglucoside of the formula R¹-0-[G]_p, 10-40% by weight of an alkyl- and/or alkenyl -oligoglucoside of the formula R²-0-(G)_p and 80-20% by weight of an alkyl ether sulphate of the formula R³- (OCH₂CH₂) _n 0-S0₃M, in which R¹

alkyl or alkenyl; (G) = a glucose group; p = 1-10, preferably 1-3; R² = C₁₂_₂₂ alkyl or alkenyl; R³ = C₆_₂₂ alkyl or alkenyl; M = an alkali or alkaline earth metal, or an ammonium or alkanolammonium ion, preferably Na or Mg; and n = 1-20, preferably 2-7. Preferably, R² and R³ are independently C₁₂_₁₄ alkyl .

Another example of a sugar surfactant is a polyglycerin fatty acid ester polyoxyalkylene ether R¹ R² R³ N⁺-CH (Y) -CH₂-0-CH₂-

C(CH,),-C(0H) (H) -C(=0) -NH-CH₂-CH,-OH X^" (I) where R¹ , R² = C^,. alkyl or C₈_₂₄ alkenyl; R³ =

alkylene; X^" = a monovalent organic or inorganic anion; and Y = OH or H.

A sugar surfactant may also include (A) 1-5 % by weight of a fatty alcohol polyglycol ether, 1-5% by weight of a Guerbet alcohol, and 1-5% by weight of a polyol partial ester, (B) 1-5% by weight of a anionic polymer, (C) 15-30% by weight of a fatty alcohol polyglycol ether sulphate, (D) 15-30% by weight of an alkyloligoglycoside; and a sulphated product of fatty acid-N- alkylpolyhydroxyalkyl amides of the formula R¹ CO- N(R)-Z, where R¹ CO = C₆_₂₂ aliphatic acyl; R² = H, C_x_₄ alkyl or C_x_₄ hydroxyalkyl ; Z = C₃_₁₂ polyhydroxyalkyl containing 3-10 hydroxy groups .

Sugar surfactants can also be selected from alkyl oligoglycosides of formula (I) and carboxylic acid N- polyhydroxyalkylamides of formula (II) :

R^x-0(G) (I)

R² C0-NR³-Z (II)

where R¹ = optionally hydroxylated C₁_₈ alkyl; G = a sugar residue with 5 or 6 carbon atoms; p = 1-10; R² CO =

aliphatic acyl; R" = H,

alkyl or C_x_₈ hydroxyalkyl; Z = C_{3 12} polyhydroxyalkyl containing 3-10 OH groups.

The shampoo composition of the present invention may also contain foaming agents such as fatty acid mono- and di- alkanolamides such as, for example, cocamide MEA (a mixture of coconut acid monoethanolamides) , cocamide DEA (a mixture of coconut acid diethanolamides) , oleamide DEA and oleamide DEA. The shampoo composition of the present invention may also contain one or more viscosity enhancing agents in order to confer the shampoo a suitable "thickness" . Examples of such viscosity enhancing agents are Carbopol 1342 and cellulose derivatives such as hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxyτnethyl cellulose, and the like. Addition of small amounts (usually from 0.2 to 5.0% by weight) of NaCl may also be used in order to control the viscosity of the final shampoo composition.

In addition, the compositions of the present invention may also include one or more preservatives such as tetrasodium EDTA (the tetrasodium salt of ethylenediamine tetraacetic acid) , quaternium-15 (1- (3 -chloroallyl) -3,5, 7-triaza-l-azonia- adamantane chloride) , Kathon CG biocide (a mixture of 5-chloro- 2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3- one) , paraben, methylparaben, benzyl alcohol, and the like.

As will be understood by the person skilled in the art the compositions of the present invention may also contain other accessory agents such as perfume, colouring agents, opacifiers, conditioning agents such as polyquaternium-7 (which is the polymeric quaternary ammonium salt of acrylamide and dimethyl diallyl ammonium chloride) , pearlizing agents such as ethylene glycol distearate and ethylene glycol monostearate, antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) , and/or pH adjusting agents such as, for example, citric acid, phosphoric acid, sodium hydroxide, and the like.

Furthermore, the compositions of the present may also contain one or more therapeutically active components in addition to the antifungal polypeptide. Examples of such therapeutically active components are piroctone (an antiseborrheic agent whose chemical name is l-hydroxy-4 -methyl -6- (2 , 4 , 4-trimethylpentyl) - 2 (1H) -pyridinone) , ketoconazole and elubiol .

In general, the components of the shampoo composition are employed in conventional amounts, for example:

i) 10-65% surfactants, ii) 1-10% foaming agents, iii) 0.1-2% viscosity enhancing agents, iv) preservatives and/or antioxidants sufficient to retard degradation of the shampoo composition, thereby providing an adequate shell life, v) acids, bases or buffers to yield a pH in the desired range (typically from 4 to 10) vi) water qs ad 100%

Below, a number of specific examples of various shampoo compositions comprising the antifungal polypeptide defined above is given (in the examples the antifungal polypeptide as defined above is abbreviated AFPP) :

Shampoo Composition I (* 'All -Purpose Shampoo for Normal Hair"

A. Water (aqua) 50.00%

Sodium laureth sulphate 40.00%

Cocamidopropyl betaine 5.00%

Lauramide DEA 1.50%

AAFFPPPP 55xxll00^""δb to 1.0%

B. Citric acid 0.07%

C. Sodium chloride 2.50% D. Fragrance (perfume) , colour gs

100.00%

Shampoo Composition II (^,vAmino Acid Shampoo")

A. Water (aqua) 30.00%

B. Hydroxypropyl guar hydroxy- propyiltrimonium chloride 0.30%

C. Citric acid 0.05% D. Tetrasodium EDTA 0.10%

Methyl paraben 0.15%

Sodium PCA 1.00%

Sodium laureth sulphate 30.00% TEA-cocoyl glutamate 15.00% Cocamidopropyl betaine 9.50%

PEG-150 distearate 1.00%

E. Lauramide DEA 2.00%

F. Methylchloroisothiazolinone and) methylisothiazolinone 0. 05% Lysine hydrochloride 0. 05%

Proline 0. 05%

AFPP 5 5xxll00^""b6 - 1.0%

Fragrance (perfume) 0. 20%

Sodium chloride 0. 80% G. Water as

10C ).00%

Shampoo Composition III ( ^{x λ} Full -Family Ultra mild Shampoo"

A. Water (aqua) 50.00%

Dye (s) qs

PEG-80 sorbitan laurate (and) cocamidopropyl betaine (and) sodium trideceth sulfate (and) sodium lauroamphoacetate

(and) PEG-150 distearate (and) sodium laureth-13 carboxylate (and) citric acid

(and) quaternium-15 (and) tetrasodium EDTA (Miracare

BC-10, Rhone-Poulenc) 38.50% B. Butylene glycol (and) water

(aqua) (and) chamomile extract (and) Aloe barba- densis extract (and) balm of gilead (Commiphora gileadensis) extract (and)

Althea officinalis extract

(Actiplex, Active Organics) 1.00%

AFPP 5xl0^"6 - 1.0%

Quaternium-15 0.20% Fragrance (perfume) qs

C. Citric acid 0.10%

D. Sodium chloride 1.50% Water (aqua) gs

100.00%

Shampoo Composition IV (^κxMarine Poultice Shampoo")

A. Water (aqua) 30.00%

B. Methylparaben 0.05% Propylparaben 0.15%

C. Laminaria Micronized 15.00% Lithothamne Lithothamne T-450 9.00% Marine silts (Int'l Sourcing) 5.00%

D. Ammonium laureth sulphate 32.50% PEG-7 glyceryl cocoate 1.50%

E. Fragrance (perfume) qs

AFPP 5xl0^"6 - 1.0%

Water (aqua) qs

100.00%

Shampoo Composition V ("Mild Shampoo")

A. Water (aqua) 50.00%

B. Polyquaternium-1 0 0.20%

C. Sodium laureth sulphate, 28% active 25.00% Caprylyl/capryl glucoside 8.00% Cocamidopropyl betaine,30% active 9.00%

AFPP 5xl0^"6 - 1.0*

D. PEG-120 methyl glucose dioleate 1.50%

E. Sodium chloride 0.30%

F. Citric acid qs Preservative qs

Fragrance (perfume) qs

Water (aqua) qs

100.00%

Shampoo Composition VI ("Nutritive Shampoo")

A. Sodium laureth sulphate 25.00% Preservative qs PEG-120 methyl glucose dioleate 1.00% Water (aqua) qs

B. Polyquaternium-11 2.00% hydrolysed collagen 2.00% Cocamidopropyl betaine 8.00%

Oleamide DEA 1.30%

Soluble collagen 3.00%

Hydrolysed milk protein 2.00% AFPP 5xl0^"6 - 1.0%

100.00%

Shampoo Composition VII ("Oatmeal Shampoo with Ceramides"

A. Water (aqua) 15.00%

Xanthan gum, 0.25% soln 30.00%

B. Water (aqua) (and) glycerin

(and) hydroxypropyl methylcellulose (and) glucuronic acid (and) glucose (and) glyceryl polyacrylate 25.00%

AFPP 5xl0^"6 - 1.0%

C. Colloidal oatmeal 2.00%

D. Cocamide DEA 3.00% Sodium laureth sulphate 20.00% lsocetyl alcohol (and) butylene glycol (and) ceramide 3 0.50%

E. Methylchloroisothiazolinone

(and) methylisothiazolinone 0.05%

F. Water (aqua) qs 100.00%

Shampoo composition VIII ("Protein Conditioning Shampoo")

A. Water (aqua) 35.00% Sodium laureth sulfate 50.00%

DL-Panthenol 2.00%

Cocamide DEA 4.00% Sodium isostearoyl lactylate 3.00%

B. Hydroxypropyltrimonium hydrolysed keratin 2.00%

AFPP 5xl0^~6 - 1.0% Fragrance (perfume) 0.20%

DMDM hydantoin 0.20%

C. Water (aqua) qs

100.00%

Shampoo Composition IX ("Pearlescent Treatment Shampoo")

Decyl glucoside 5.00%

Lauryldimonium hydroxypropyl hydrolysed wheat protein 4.00% Sodium myreth sulphate 27.00%

Glycol distearate (and) laureth-4

(and) cocamidopropyl betaine

(Euperian PK 3000-AM 3.00%

Propylene glycol (and) water (aqua) (and) white nettle (Lamium album) extract (and) ivy (Hedera helix) extract

(and) pine cone (Pinus) extract (and) chamomile (Anthemis nobilis) extract (and) watercress (Nasturtium officinale) extract

(and) Arnica montana extract

(and) burdock (Arctium lappa) extract (and) rosemary

(Rosemarinus officinalis) extract (Prodhyactif Hair

Treatment, Prod'Hyg Labs) 5.00%

AFPP 5xl0^~6 - 1.0% Sodium chloride 2.10%

Water (aqua) and preservative

100.00%

Shampoo Composition X ("Shampoo for Dry Scalp and Dandruff"

A. Water (aqua) qs

B. Magnesium aluminum silicate 1.00%

C. Oat (Avena sativa) protein (Microat E, Tri-K) 1.00%

D. Tetrasodium EDTA

(Kelate 220, Tri-K) 0. .20% Methylparaben (Trisept M, Tri-K) 0. ,30%

E. Hydroxypropyl methylcellulose

(Methocel J 1 2MS, Dow Chem) 1. .00%

F. Cocamidopropyl betaine 5. ,00%

Glycerin 2. ,00%

Sodium lauroyl sarcosinate 20. ,00%

Lauramide DEA 2. ,00% Sodium laureth sulphate 20. ,00%

G. Phosphoric acid, to pH 6.0-6.5 qs J. Panthenol (DL-Panthenol , Tri-K) 0.50% imidazolidinyl urea (Tristat IU, Tri-K) 0.50%

AFPP 5xl0 1.0%

100.00%

Shampoo Composition XI

Ingredient Level % Function of Ingredient

Triethanola ine lauryl 10.00 Foaming/cleansing sulphate agent

Sodium lauroyl sulphate 2.80 do

AFPP 5x10 -1.0 Antifungal polypepide

Hydroxypropyl methyl 1.00 Thickener cellulose

Water/preservative/perfume/ to 100 etc .

The pH of the composition is adjusted with NaOH/citric acid.

Shampoo Composition XII (Conditioning Antidandruff Shampoo)

Ingredient Level % Function of Ingredient

Triethanolamine 8.00 Foaming/cleansing lauryl sulphate agent

Coconut 4.00 Foam booster/foam monoethanolamide stabiliser

Ethylene glycol 4.00 Opacifying/pearling mono-stearate agent

NaCl 0.50 Thickening agent

Polyethylene Glycol 0.50 Adjust viscosity

AFPP 5xl0^"6-1.0 Antifungal polypepide

Water/perfume/etc. to 100

The pH of the composition is adjusted with citric acid.

Shampoo Composition XIII (Conditioning Antidandruff Shampoo) Ingredient Level% Function of Ingredient

Sodium lauryl ether 9.25 Foaming/cleansing sulphate agent

Sodium lauroyl 2.45 do sarcosinate

Coco-amido propyl 0.60 Mildness additive/foam amine oxide stabiliser

PEG-7 Glycerol 2.00 Emollient/mildnessaddi cocoate -tive

Crotein Q 0.25 Conditioning (quaternised ingredient collagen AFPP 5x10 -1.0 Antifungal polypeptide

Water/perfume/etc. to 100

Shampoo Composition XIV

Ingredient Level% Function of

Ingredient

Sodium lauryl ether 10.50 Foaming/cleansing sulphate agent

Coconut 2.50 Foam diethanolamide stabiliser/shampoo thickener

AFPP 5xl0^"b-1.0 Antifungal polypeptide

Water/perfume/etc. to 100

The pH of the composition is adjusted to 6.0-6.5 with citric acid/NaOH. Shampoo Composition XV

Ingredient Level% Function of

Ingredient

Triethanolamine 14.00 Foaming/cleansing lauryl sulphate agent Briphos 03D* 1.50 Conditioning ingredient

AFPP 5xl0^~b-1.0 Antifungal polypeptide

Empicol WP40** 10.00 Opacifying/pearling agent

Water/perfume/etc to 100

* Oleyl-3EO phosphate

** Proprietary mixture of sodium lauryl ether sulphate, ethyleneglycol mono and distearates supplied by Albright and

Wilson.

Shampoo Composition XVI

Ingredient Level* Function of

Ingredient

Sodium lauryl ether 7.00 Foaming/cleansing sulphate agent

Triethanolamine 5.64 do lauryl sulphate

Tegobetane L7* 4.80 Foam stabiliser

PEG-2 Starate 3.00 Viscosity modifier AFPP 5x10 - 1 . 0 Antifungal polypeptide

Water/perfume/etc. to 100

Coco amido propyl betaine .

Shampoo Composition XVII

Ingredient Level% Function of

Ingredient

Sodium lauryl ether 9.80 Foaming/cleansing sulphate agent

Cocoamido propyl 4.80 Foam betaine stabiliser/mildness additive

Antil 141 liquid 0.50 Viscosity modifier AFPP 5x10 -1.0 Antifungal polypeptide

Water/perfume/etc. to 100

Proprietary mixture of propylene glcol / PEG-55 propylene glycol oleate supplied by Yh. Goldschmidt AG.

Examples of other antidandruff shampoo composition wherein the antifungal polypeptide of the present invention may be incorporated and optionally substituted with the antidandruff substance present in the composition are disclosed in the following: WO 99/13830 disclosing shampoo compositions comprising: a) an effective amount of a triazole, b) a detersive surfactant, and c) a carrier;

WO 99/39683 disclosing shampoo compositions comprising: a) from about 7% to about 30% by weight of a detersive surfactant selected from the group consisting of anionic surfactant, amphoteric surfactant, and a mixture thereof, b) from about 0.1 % to about 10 % by weight of an antimicrobial agent, c) from about 0.5 % to about 10% by weight of a suspending agent, d) from about 0.01 % to about 1.0 % by weight of a cationic guar polymer having a charge density of from about 0.1 to about 3 meq/mg, e) a cool-feeling agent selected from the group consisting of borneol , camphor, cineol, methane, glycosil-mono- mentyl-oacetate, menthol, 3-1-menthoxypropane, peppermint, speamint, and mixtures thereof, and f) from about 40% to about 92% by weight of water, wherein at least about 50% by weight of the cationic guar polymer is in coacervate form, said coacervate comprising detersive surfactant and cationic guar polymer;

WO 97/39096 disclosing a shampoo composition comprising an antimicrobial compound selected from certain 1-hydroxy- 6- substituted pyridine-2 -one and 2-thione compounds, and a shampoo surfactant, in which the pH of the hair treatment composition is adjusted to at least 7 ;

WO 97/26854 disclosing a shampoo composition comprising : a) from about 7% to about 30% by weight of a detersive surfactant selected from the group consisting of anionic surfactant, amphoteric surfactant, zwitterionic surfactant, and compositions thereof, b) from about 0.1% to about 10% by weight of an antimicrobial agent, c) from about 0.5% to about 10% by weight of a suspending agent, d) from about 0.01% to about 1.0% by weight of a cationic guar polymer having a charge density of from about 0.01 meq/mg to about 3 meq/mg, and e) from about 40% to about 92% be weight of water, wherein at least 50% be weight of the cationic guar polymer is in coacervate form, said coacervate comprising detersive surfactant and cationic uar polymer;

WO 97/47275 disclosing an anti-dandruff agent containing clotrimazol as the active agent, especially rinse-off products like shampoos, medicated shampoos or rinses;

WO 95/02389 disclosing an anti-dandruff shampoo composition comprising: a) from about 5% to about 29.5% by weight of anionic surfactant selected from the group consisting of alkyl sulfates and alkyl ehtoxylated sulfates, acids thereof, and mixtures thereof, b) from about 0.5% to about 5% by weight of N-acylamino acid anionic surfactant and salts thereof, c) from about 0.1% to about 10% by weight of particulate anti-dandruff agent, d) from about 0.5% to about 10% by weight of crystalline suspending agent for said anti -dandruff active, e) from about 40% to about 89% by weight of water, wherein the total detersive surfactant concentration in said composition is from about 10% to about 30%, the weight ratio of components b) : component a) is at least about 1:20, and said composition is substantially free of betaine surfactants and alkanol amide foam boosters;

WO 95/17880 disclosing a shampoo composition comprising: a) from about 5% to about 40% by weight surfactant system comprising: (i) from 80% to about 99% by weight of the surfactant system, anionic surfactants which are alkyl ethoxylated sulfates and alkyl sulfates in a ratio between about 1:1 and 1:0, and (ii) from about 1% to 20% by weight of the surfactant system, polyhydroxy fatty acid amide surfactants, b) from about 0.05% to about 25% by weight functional materials, and c) from about 35% to about 95% by weight water; and

WO 92/14440 disclosing anti-dandruff shampoos comprising a surfactant, platelet zinc pyridinethione active of specific particle size, a suspending agent, water, and a synergizer for the active.

Examples of other shampoo composition wherein the antifungal polypeptide of the present invention may be incorporated are disclosed in the following:

WO 98/00498 disclosing a composition comprising a surfactant system containing a diaionic or alkoxylated diaionic surfactant having a structural skeleton of at least five carbon atoms to which two anionic substituent groups spaced at least three atoms apart are attached, wherein one anionic substituent group is a sulfate group or an alkoxy-linked sulfate group and the other anionic substituent is selected from sulfate and sulfonate optionally alkoxy-linked, in combination with one or more other co-surfactants;

WO 97/14405 disclosing a shampoo compositions comprising from about 5% to about 50% by weight of a detersive surfactant, from about 0.05% to about 10% by weight of a silicone hair conditioning agent, from about 0.1% to about 10% by weight of a suspending agent, from about 0.025% to about 1.5% by weight of selected polyalkylene glycols, preferably polyethylene glycols having from about 1,500 to about 25,000 degrees of ethoxylation, and water, and optionally one or more additional materials known for use in shampoo or conditioning compositions ;

WO 97/25965 disclosing a shampoo composition comprising, per 100 parts by weight, from about 5 to about 50 parts of a sensitive surfactant mixture comprising a) from about 10 to about 60 percent by weight of the mixture of at least ne strong anionic surfactant, b) from about 10 to about 40 percent by weight of at least one amphoteric surfactant and c) from about 10 to about 60 percent by weight of the mixture of at least one alkyl saccharide nonionic surfactant;

WO 97/14406 disclosing a shampoo composition comprising a detersive anionic surfactant, other than anionic amino acid derivative surfactants, a stabilizing surfactant selected from the group consisting on anionic amino acid derivative surfactants, amphoteric surfactants, cationic surfactants, and mixtures thereof, a fatty compound selected from the group consisting of fatty alcohols, fatty acids, fatty alcohol derivatives, fatty acid derivatives, and mixtures thereof, a hair conditioning agent selected from the group consisting of nonvolatile dispersed silicone conditioning gents, and mixtures thereof, and water;

WO 98/04233 disclosing shampoo compositions comprising an alkoxylated polyalkyleneamine and one or more detersive surfactant, in further embodiment, air conditioning shampoo composition comprising by weight from about 0.01% o about 10% of alkoxylated polyalkyeneamine, from about 0.01% to about 20% of a cationic surfactant conditioning agent, from about 5% to about 50% of a detersive surfactant and from about 20% to about 90% water;

WO 98/04238 disclosing a shampoo composition comprising a quaternary polyalkoxylated polyalkyleneamme and a detersive surfactant, in further embodiment, a conditioning shampoo composition comprising from about by weight 0.01% to about 10% of a quaternary polyalkoxylated polyalkyleneamme, from about 0.01% to about 20% of a cationic surfactant conditioning agent, from about 5% of a detersive surfactant, and from about 20% to about 90% of water;

WO 98/04239 disclosing a shampoo composition comprising: a) from about 0.1% to about 10% by weight of short chain alkyl sulfate surfactant having a carbon chain length of 10 carbon atoms or less, b) from about 0.1% to about 10% be weight of ethoxylated alcohol and c) from about 1% to about 60% by weight of auxiliary surfactant selected from anionic surfactant other than he short chain alkyl sulfate of a) , nonionic surfactant other than the ethoxylated alcohol of b) , amphoteric and cationic surfactants and mixtures thereof, d) water;

WO 98/18433 disclosing shampoo compositions comprising from about 5% to about 50% by weight of a surfactant selected from the group consisting of anionic surfactants, zwitterionic or amphoteric surfactants having an attached group that is anionic at the pH of the composition, and combinations thereof, from about 0.025% to about 3% by weight of an organic cationic polymer having a cationic charge density of from about 0.2 meq/mg to about 7 meq/mg and a molecular weight of from about 5,000 to about 10 million, from about 0.1% to about 10% by weight of a water-insoluble hair styling polymer, from about 0.1% to about 10% by weight of a water- insoluble volatile solvent, and from about 0.05% to about 5% by weight of selected cationic materials for use as spreading agents for the styling polymer, and from about 22% to about 94.3% by weight water;

WO 98/04241 disclosing shampoo compositions comprising: a) from about 1% to about 60% by weight of surfactant selected from anionic surfactant, nonionic, amphoteric and cationic surfactants and mixtures thereof, b) from about 0.001% to about 10% by weight of liquid polyol carboxylic acid ester containing at least four acid ester groups wherein the polyol moiety is selected from sugars and sugar alcohols containing from about 4 to about 12 hydroxyl groups, and wherein each carboxylic acid moiety has from abut 8 to about 22 carbon groups, and wherein the liquid polyol carboxylic acid ester has a complete melting point of less than about 30 °C, c) from about 0.001% to about 4% by weight of nonionic polyethylene glycol polymers having the general formula: -H (OCH2CH2) nOH wherein n is an average value of ethoxylation in the range of from about 1,000 to about 25,000, and d) water;

WO 98/18435 disclosing shampoo compositions containing a surfactant component in a shampoo with a particulate insoluble, dispersed, non- ionic conditioning agent having a particle size range of less than about 0.15 microns, crystalline agent and a deposition polymer;

WO 98/50007 disclosing shampoo compositions comprising a) from about 5% to about 50% by weight of a surfactant component selected from the group consisting of a combination of an anionic surfactant and an amphoteric surfactant, and a combination of an anionic surfactant and a zwitterionic surfactant, b) from about 0.025% to about 3% by weight of a cationic deposition polymer having a cationic charge density of from bout 0.2 meq/g to about 2 meq/g and which is selected from the group consisting of cationic cellulose polymers, cationic guar gum derivatives, and mixtures thereof, c) from about 0.5% to about 10% by weight of an organic cationic hair styling polymer having a cationic charge density of greater than about 2 meq/g to less than about 4.75 meq/g, and d) from about 27% to about 94.5% by weight water. Especially effective are those styling shampoo compositions containing a combination of Polyquaternium-16 as the cationic styling polymer, Polyquaternium-10 as a cationic deposition polymer, and a surfactant matrix comprising a combination of ammonium laureth sulfate and cocoamidopropyl betaine;

WO 99/38475 disclosing shampoo compositions comprising from about 5% to about 50% by weight of a surfactant selected from the group consisting of anionic surfactants, zwitterionic or amphoteric surfactants having attached group that is anionic at the pH of the composition, and combinations thereof, from about 0.025% to about 3% by weight of an organic cationic polymer having a cationic charge density of from about 0.2 meq/mg to about 7 meq/mg and a molecular weight of from about 5,000 to about 10 million, from about 0.1% to about 10% by weight of a water-insoluble volatile solvent, and from about 0.005% to about 2.0% by weight a crystalline hydroxyl -containing stabilizing agent, and from about 26.5% to about 94.9% by weight water; WO 99/38476 disclosing shampoo compositions comprising from about 5% to about 50% by weight of a surfactant selected from the group consisting of anionic surfactants, zwitterionic or amphoteric surfactants, and combinations thereof, from about 0.1% to about 1.0% by weight of an organic or silicone-grafted hair styling polymer, and from about 40% to about 94.9% by weight of water, wherein the Hair Feel Index (HFI) is at least about 0.65 and the Curl Retention Value (CRV) is at least about 70;

WO 97/35546 disclosing shampoo compositions comprising an anionic detersive surfactant component, an anionic, cationic, hair conditioningpolymer, and from about 0.01% to about 1.0% by weight of a water insoluble, synthetic ester having a viscosity of from about 1 to about 300 centipoise, and which conforms to either of formulaes (I) and (II) wherein Rl is an alkyl, alkenyl, hydroxyalkyl or hydrox alkenyl group, having from 7 to 9 carbon atoms, n is a positive integer having a value of from 2 to 4, R2 is an alkyl, alkenyl, hydroxyalkyl or hydroxyalkenyl group, having from 8 to 10 carbon atoms, and y is an alkyl, alkenyl, hydroxy or carboxy substituted alkyl or alkenyl, having from about 2 to about 20 carbon atoms, and an organic, water insoluble conditioning oil having a viscosity of from about 1 to about 399 centipoise, selected from the group consisting of hydrocarbon oils, fatty esters other than the above synthetic esters, and combinations thereof. The organic conditioning oil is preferably a polyolefin

[Rl-CO-0]_n-Y (I) [R2-0-CO]_n-Y (II); WO 99/24004 disclosing a shampoo composition comprising by weight: a) from about 0.05% to about 50% of a polyhydrophilic anionic surfactant, b) from about 0.05% to about 20% of a cationic conditioning agent selected from the group consisting of cationic surfactants, cationic polymers, and mixtures thereof. C) from about 0.01% to about 20% of a silicone compound, and d) an aqueous carrier;

WO 98/50011 disclosing shampoo compositions comprising a) from about 5% to about 50% by weight of a surfactant component having an anionic moiety at the pH of the composition, b) from about 0.025% to about 3% by weight of cationic deposition polymer, c) from about 0.1% to about 10% by weight of a malodor-producing hair styling polymer, d) from about 0.0075% to about 2.5% by weight of an odor masking base, and e) from about 25% to about 94.5% by weight water. The odor masking base comprises (by weight of the base) from about 15% to about 75% by weight of an ionone perfume having a boiling point of more than about 250 °C, from about 5% to about 65% by weight of a musk having a boiling point of more than about 250 °C, and from about 20% to about 80% by weight of a highly volatile perfume having a boiling point of less than about 250 °C . Also disclosed are shampoo compositions further comprising a malodor producing liquid carrier to solubilize or disperse the malodor- producing polymer in the composition;

WO 99/13828 disclosing shampoo compositions comprising: a) an effective amount of an optical brightener, b) an anionic detersive surfactant, c) an amphoteric detersive surfactant, d) a non-ionic detersive surfactant, and e) a carrier, wherein the composition has a pH of no higher than about 7; WO 99/13829 disclosing shampoo compositions comprising: a) an effective amount of an optical brightener, b) a detersive surfactant, c) an effective amount of a polyvalent cation, and d) a carrier;

WO 93/18737 disclosing shampoo compositions comprising alkyl ethoxylated sulfate surfactant, a specific type of imidazolinium-type amphoteric surfactant, a crystalline suspending agent that is preferably solubilized and recrystallized in the premix composition, and a relatively low amount of water. The preferred composition comprises alkyl ehtoxylated sulfate surfactant, imidazoline-type amphoteric aurfactant, betaine surfactant, a recrystallized suspending agent, and water; and

WO 97/35549 disclosing shampoo compositions comprising a specific surfactant component comprising and ethoxylated alkyl sulfate surfactant having from about 1 to about 8 moles of ethoxylation and an amphoteric surfactant in a shampoo with insoluble, dispersed conditioning agent, a low viscosity organic conditioning oil and a select soluble cellulosic cationic organic polymer hair conditioning agent.

EXAMPLES

MATERIALS AND METHODS

The production and subsequent purification of the antifungal polypeptide (AFPP) shown in Fig. 1 was carried out as described in WO 94/01459. In brief, the antifungal polypeptide was prepared as follows:

A. giganteus strains The A . giganteus strain deposited with the Centraalbureau voor Schimmelculturen (CBS) under the accession number CBS 526.65 was used for the experiments.

Cultivation of an A. giganteus strain producing the AFPP Cultivation on agar slants was carried out using an agar medium prepared from 39 g of Potato Dextrose Agar (from Difco) and distilled water up to 1000 ml. The agar slants were inoculated with the A . giganteus strain and grown for one week at 26°C.

Submerged cul tivation

A 500 ml Erlenmeyer flask containing 100 ml of AMC substrate

(15 g meat extract, 20 g Peptone, 20 g corn starch, 5 g NaCl , 1 ml Pluronic, 1 litre H₂0) was inoculated with 5 ml of a spore suspension containing 10⁶ spores/ml prepared from an agar slant culture of A . giganteus obtained as described above containing 10⁶ spores per ml. The flask was shaken at 220 rpm at 30°C for 3 days, after which the AFPP could be recovered.

Isolation, purification and amino acid sequence determination of the AFPP

The fermentation broth obtained as described above was subjected to centrifugation, the mycelium was suspended in Tris- buffered saline at pH 7 and then subjected to a second centrifugation. The supematants from the two centrifugations were combined and subjected to sterile filtration. The pH of the resulting supernatant preparation was adjusted to between 6.5 and 9 and the supernatant preparation was applied to a cation exchange resin (S Sepharose Fast Flow) which prior to application was equilibrated with a phosphate buffer at pH 6.5. Elution of active fractions from this resin was accomplished by the application of a buffer with a high ionic strength such as a buffer comprising 20 mM phosphate, 1.5 mM NaCl, pH 6.5. If necessary, the procedure was repeated after dilution with or dialysis against a buffer with ionic strength below or near that of 10 mM phosphate at pH 6.0. Elution from this second step was, if necessary, carried out as a gradient elution. The purity of the active fractions was assessed by HPLC .

The combined active fractions were subjected to sterile filtration on a 0.22 μm filter (Millipore) prior to testing of the antifungal properties against P . ovale .

The purified AFPP was S-carboxymethylated using the method described by Nakaya et al . 1990 and de-salted using reverse phase HPLC. The S-carboxymethylated AFPP was subjected to N- terminal amino acid sequence determination on an Applied Bio- systems 473A sequencer operated in accordance with the manufacturer's instructions.

Extinction coefficient

The extinction coefficient of the AFPP was estimated from the amino acid sequence shown in Fig. 1 using the formula

ε°-^1%(280 nm) =5690- (No. Trp) 4-1280 • (No . Tyr) +120 ^• (No . Cys) molecular mass

(Gill & von Hippel, 1989), where (No. Trp) , (No. Tyr) and (No. Cys) are the number of Trp, Tyr and Cys residues in the amino acid sequence.

Based on this formula the extinction coefficient at 280 nm has been calculated to 1.3. All polypeptide determinations are based on OD₂₈₀ measurements using this calculated extinction co- efficient.

Production of antibodies against the AFPP

The AFPP produced and purified as described above was diluted to a concentration of 25-250 μg/ml in phosphate-buffered saline, pH 6.5-8. Rabbits were immunised with the purified polypeptide in Freund's complete adjuvant by use of an immunisation scheme essentially according to the standard procedures described by Hudson et al . , 1989. Serum may be collected and tested for polyspecific antibodies against the AFPP in a single radial immunodiffusion assay or a western blot assay using the purified AFPP obtained as described above as an antigen. The assays may be carried out in accordance with standard procedures, e.g. as described by Hudson et al . , 1989. The antibodies may be purified by standard methods such as standard immunoadsorbent techniques using the purified AFPP immobilised to a carrier, e.g. as described by Hudson et al . , 1989.

Determination of antibody reactivity of a variant of the AFPP of the invention

Reactivity of a variant of the AFPP with an antibody or an antibody-containing serum obtained as described above may be determined in single radial immunodiffusion or western blotting performed essentially as described by Hudson et al . , 1989.

EXAMPLE 1

Inhibition of the yeast P. ovale

P. ovale was grown in microtiter plates in a medium recommended by American Type Culture Collection at 37°C. The medium contains 30 g Sabouraud's glucose broth, 5 g yeast extract and 2 ml olive oil pr. litre.

A standard solution comprising the AFPP was prepared by dissolving the AFPP in 10 mM phosphate buffer, pH 7.5.

As illustrated in Fig. 2, each microtiter plate contained 4 identical lanes, each lane having:

One well containing medium only (200 ml) and 50 ml 10 mM phosphate buffer, pH 7.5 (included as a control in order to test the sterility of the medium. The phosphate buffer was included in order to ensure identical experimental conditions compared to the wells inoculated with P. ovale) .

- One well identical to the above-men ioned well except being inoculated with P. ovale .

One well inoculated with P. ovale and containing AFPP in a concentration of 0.005 OD₂₈₀ (about 5 mg AFPP/litre) .

One well inoculated with P. ovale and containing AFPP in a concentration of 0.027 OD.,_Dn. One well inoculated with P. ovale and containing AFPP in a concentration of 0.068 OD₂₈₀.

- One well inoculated with P. ovale and containing AFPP in a concentration of 0.136 OD₂₈₀.

The growth of P. ovale was followed as a function of time. The yeast did not grow uniformly, but rather in large agglomerations. This fact, and the presence of olive oil, made it impossible to measure the growth by spectroscopic means. Therefore, the cells were re-suspended and 10 μl was plated on the same medium containing agar. Determination of the approximate growth in microtiter plates and on solid medium plates gave the same results (see Table 1) . The results shown in Table 1 are the average growth in the four lanes.

The growth was dependent on the size of the colony used for inoculation. The larger the colony, the faster the growth on the plates. In the present example the medium was inoculated with only a small colony. As it appears from Table 1 below, this gave rise to an extended period of growth. However, the results shown in Table 1 were the same as if the medium was inoculated with a larger colony, except that the growth period was shortened. Table 1

Concentration day 3 day 6 day 7 day 8 day 10 day 13 of AFPP (OD₂₈₀)

Medium only 0 0 0 0 0 0

0 0 X XX XX XXX XXX

0.005 0 0 X X XX XXX

0.027 0 0 0 X X XX

0.068 0 0 0 X X X

0.136 0 0 0 0 0 X

0: No growth observed in the well x: Little growth observed in the centre of the well xx: Some growth observed in the centre of the well and minor growth along the edge xxx: Vigorous growth on all the media surface

Conclusion:

The present example clearly shows that the AFPP impedes the growth of the yeast

Pi tyrosporum ovale . The fact that even a very low concentration of AFPP (for example about 5 mg/litre) inhibits the growth of P. ovale renders the AFPP suitable for use in the treatment or prophylaxis of dandruff.

REFERENCES CITED IN THE SPECIFICATION

Gill & von Hippel, Anal. Biochem. 182, 319-26, 1989;

Hudson, L., and Hay, F. in Practical Immunology, Third edition (1989) , Blackwell Scientific Publications;

Lipman and Pearson, Science 227, 1435 (1985);

Nakaya, K. et al . , Eur. J. Biochem. 193, 31-38, 1990, "Amino acid sequence and disulfide bridges of an antifungal protein isolated from Aspergillus giganteus";

Wnendt , S. et al . , Nucleic Acids Research, Vol. 18, No. 13: 3987, 1990, "Cloning and nucleotide sequence of a cDNA encoding the antifungal -protein of Aspergillus giganteus and preliminary characterization of the native gene" .

Claims

1. An anti-dandruff composition suitable for application to the hair or scalp of a human, comprising an effective amount of an antifungal polypeptide comprising the amino acid sequence shown in Fig. 1, or a variant thereof,

said variant being a polypeptide which

I) reacts with an antibody reactive with at least one epitope of the polypeptide having the amino acid sequence shown in Fig. 1, or

II) has an amino acid sequence which is substantially homologous with the amino acid sequence of the polypeptide shown in Fig. 1.

2. A composition according to claim 1, wherein the antifungal polypeptide is derived from a microorganism.

3. A composition according to claim 2, wherein the antifungal polypeptide is derived from a fungus, in particular a fungus of the genus Aspergillus .

4. A composition according to claim 3, wherein the antifungal polypeptide is derived from a fungus belonging to the species A . giganteus, A . pallidus, A . clavatus , A . longivesica , A . rhizopodus or A . clava tonanicus .

5. A composition according to claim 4, wherein the antifungal polypeptide is derived from a fungus belonging to the species A . giganteus .

6. A composition according to claim 5, wherein the antifungal polypeptide is derived from the A . giganteus strain CBS 526.65.

7. A composition according to any of the preceding claims, wherein the antifungal polypeptide comprises the amino acid sequence shown in Fig. 1.

8. A composition according to claim 7, wherein the antifungal polypeptide consists of the amino acid sequence shown in Fig.

1.

9. A composition according to any of claims 1-6, wherein one or more of the amino acid residues in position 4, 24, 32, 42 and 50 of the antifungal polypeptide shown in Fig. 1 have been substituted with another naturally occurring amino acid residue .

10. A composition according to claim 9, wherein the antifungal polypeptide comprises the amino acid sequence shown in Fig. 1, in which Pro in position 4 has been substituted with Asn or Asp, Gly in position 24 has been substituted with Ala, Arg in position 32 has been substituted with Lys or Val, Leu in position 42 has been substituted with Phe, and/or His in position 50 has been substituted with Tyr.

11. A composition according to claim 10, wherein the antifungal polypeptide consists of the amino acid sequence shown in Fig. 1, in which Pro in position 4 has been substituted with Asn or Asp, Gly in position 24 has been substituted with Ala, Arg in position 32 has been substituted with Lys or Val, Leu in position 42 has been substituted with Phe, and/or His in position 50 has been substituted with Tyr.

12. A composition according to any of the preceding claims, which is in the form of a shampoo, solution, suspension, emulsion, gel, foam or cream.

13. A composition according to claim 12, which is in the form of a shampoo.

14. A composition according to any of the preceding claims, wherein the active ingredient is present in an amount from about 5xl0^"6% to about 10% by weight, calculated on the weight of the composition.

15. Use of an antifungal polypeptide as defined in any of claims 1-11 for the treatment or prophylaxis of dandruff.

16. A method for the treatment or prophylaxis of dandruff, said method comprising contacting hair with an effective amount of an antifungal polypeptide as defined in any of claims 1-11 or with a composition as defined in any of claims 12-14.

17. Use of an antifungal polypeptide as defined in any of claims 1-11 for the preparation of a composition for the treatment or prophylaxis of dandruff.

18. Use according to claim 17, wherein the composition is as defined in any of claims 12-14.