Note: Descriptions are shown in the official language in which they were submitted.
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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 antifun-
gal 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
io a composition for the treatment or prophylaxis of dandruff.
BACKGROUND OF THE INVENTION
Dandruff results from a scalp malfunction but is not considered
i5 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 Pityrosporum ovate and, consequently, substances which
inhibit the growth of P. ovate 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 prepa-
rations, salicylic acid, selenium sulphide and zinc pyrithione.
3o More recently a novel anti-dandruff shampoo comprising
ketoconazole or elubiol has been described in WO 95/27471.
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2
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
s substances which are readily produced (e. g. by a micro-
organism), which do not cause obnoxious smells and which
possess no hazardous properties.
SUMMARY OF THE INVENTION
to
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
zo in Fig. 1, or a variant thereof,
said variant being a polypeptide which
I) reacts with an antibody reactive with at least one
z5 epitope of the polypeptide having the amino acid sequence
shown in Fig. l, or
II) has an amino acid sequence which is substantially
homologous with the amino acid sequence of the polypeptide
ao shown in Fig. 1.
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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.
s 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.
to 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
ao 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 aminc
acids has been used in its conventional meaning. Unless
indicated explicitly, the amino acids mentioned herein are L-
amino acids.
3o The term "antifungal polypeptide" as used herein is intended to
comprise the linear as well as the active folded structures of
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4
the polypeptide, and may, where appropriate, be used
interchangeably with the term "antifungal protein".
The term "variant" as used in connection with the antifungal
s 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
io 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
i5 polypeptide.
Furthermore, the variant of the invention has one or both of
the characteristic properties I) and II) mentioned above.
2o 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 "
25 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
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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
s 1994, Genetics Computer Group, 575 Science Drive, Madison,
Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, C.D.,
(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
io 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
i5 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 l; or allelic variants thereof; or fragments thereof
2o 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
2s 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
ao acid sequences. An allelic variant of a polypeptide is a
polypeptide encoded by an allelic variant of a gene.
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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-
to 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
zo 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
z5 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.
3o 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. -
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giganteus, an example of which is the A. giganteus strain
deposited with the Centraallbureau voor Schimmelcultures (CBS)
under the deposition number CBS 526.65.
s 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
io 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
i5 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.
ao 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
z5 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
ao York (1987), X-ray diffraction, computer modelling etc. When
one or more interesting modifications of the amino acid sequ-
ence of the antifungal polypeptide have been identified, the
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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
io 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
is 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
2o 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
as 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
ao the amino acid sequence shown in Fig. 1 in the following way:
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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.
s 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
io 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.
is 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
zo 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
zs 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
3o 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
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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
s 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
io 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
i5 and may be prepared in a manner well-known to the person
skilled in the art, e.g. such as described in " Woodruff'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
2o 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
2s 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
3o variant thereof, is employed in the shampoo composition in an
amount which is effective for treatment or prophylaxis of
dandruff.
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The shampoo composition of the present invention will typically
contain the antifungal polypeptide in an amount from about 10-'
to about 10 percent by weight calculated on the total weight of
s 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 5x10-6 to about 0 . 5%;
about 5x10-6 to about 0.1%; about 5x10-6 to about 10-5%; about
10-5 to about 10-'%; about 10-4 to about 10-3%; about 10-3 to about
io 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
is 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,
ao alkyl benzene sulphonates, alkyl sulphones, a-alkenyl
sulphonates, polyoxyethylenealkylphenyl sulphonates and
polyoxyethylenealkylphenyl sulphates. Mixtures of surfactants
may also be used.
as 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 (CHz) loCHz (OCHzCHZ) 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
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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.
s
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
io diethanolamine (lauramido DEA), cocamidopropyl betaine,
disodium oleamido MIPA sulfosuccinate, disodium cocamido MIPA
sulfosuccinate, disodium laureth sulfosuccinate,
cocoaamphocarboxy-glycinate and disodium MEA sulfosuccinate.
is 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):
R'-O- [G] p ( I )
in which R1 - a CQ_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
ao formula:
RICO-N(R3) - [Z) (II)
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in which RzCO - a C6_zz aliphatic acyl residue, R' - 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-O-[G]P and b) alkali and/or alkali metal salts of
Clz-zz secondary 2, 3-alkyl sulphates, where R1 - CQ_zz alkyl and/or
io 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
i5 and/or polysorbates.
A sugar surfactant may also comprise 10-40% by weight of alkyl
and/or alkenyl-oligoglucoside of the formula R1-O-[G]P, 10-40%
by weight of an alkyl- and/or alkenyl-oligoglucoside of the
zo formula Rz-O- (G) p and 80-20% by weight of an alkyl ether
sulphate of the formula R3- (OCH2CHz) n O-S03M, in which Rl - CB_,1
alkyl or alkenyl; (G) - a glucose group; p - 1-10, preferably
1-3; Rz - Clz-zz alkyl or alkenyl; R3 - C6_zz alkyl or alkenyl; M
- an alkali or alkaline earth metal, or an ammonium or
z5 alkanolammonium ion, preferably Na or Mg; and n - 1-20,
preferably 2-7. Preferably, Rz and R3 are independently Clz-14
alkyl.
Another example of a sugar surfactant is a polyglycerin fatty
3o acid ester polyoxyalkylene ether Rl Rz R3 Ni-CH (Y) -CHz-O-CHz-
C (CH3) z-C (OH) (H) -C (=O) -NH-CHz-CHz-OH X- ( I ) where Rl, Rz - C1_z9
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alkyl or C8_z4 alkenyl; R3 - C1_le 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
s 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(Rz)-Z,
io where R1 CO - C6_~2 aliphatic acyl; Rz - H, C1_4 alkyl or Cl_4
hydroxyalkyl; Z - C3_lz polyhydroxyalkyl containing 3-10 hydroxy
groups.
Sugar surfactants can also be selected from alkyl
is oligoglycosides of formula (I) and carboxylic acid N-
polyhydroxyalkylamides of formula (II):
R1-O (G) (I)
Rz CO-NR3-Z (II)
where R1 - optionally hydroxylated C1_8 alkyl; G - a sugar
residue with 5 or 6 carbon atoms; p - 1-10; Rz CO - C1-a
aliphatic acyl; R' - H, C1_e alkyl or C1_8 hydroxyalkyl; Z - C3_lz
polyhydroxyalkyl containing 3-10 OH groups.
is
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
3o coconut acid diethanolamides), oleamide DEA and oleamide DEA.
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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
s derivatives such as hydroxypropyl methylcellulose, hydroxyethyl
cellulose, carboxymethyl cellulose, and the like. Addition of
small amounts (usually from 0.2 to 5.0°s by weight) of NaCl may
also be used in order to control the viscosity of the final
shampoo composition.
io
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-1-azonia-
i5 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
ao 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
2s 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
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16
the antifungal polypeptide. Examples of such therapeutically
active components are piroctone (an antiseborrheic agent whose
chemical name is 1-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,
io 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,
i5 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
2o 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%
3o AFPP 5x10-6 to 1.0%
B. Citric acid 0.07%
C. Sodium chloride 2.50%
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D. _Fraqrance (perfume) , colour Qs
100.00%
Shampoo Composition II (" Amino Acid Shampoo ")
A. Water (aqua) 30.00%
B. Hydroxypropyl guar hydroxy-
propyiltrimonium chloride 0.30%
C. Citric acid 0.05%
io Tetrasodium EDTA 0.10%
D.
Methyl paraben 0.15%
Sodium PCA 1.00%
Sodium laureth sulphate 30.00%
TEA-cocoyl glutamate 15.00%
i5 Cocamidopropyl betaine 9.50%
PEG-150 distearate 1.00%
E. Lauramide DEA 2.00%
F. Methylchloroisothiazolinone
and) methylisothiazolinone 0.05%
ao Lysine hydrochloride 0.05%
Proline 0.05%
AFPP 5x10-6 - 1.0%
Fragrance (perfume) 0.20%
Sodium chloride 0.80%
2s Water cts
G.
100.00%
Shampoo Composition III (" Full-Family Ultra mild Shampoo" )
3o A. Water (aqua) 50.00%
Dye ( s ) qs
PEG-80 sorbitan laurate (and)
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cocamidopropyl betaine (and)
sodium trideceth sulfate (and)
sodium lauroamphoacetate
(and) PEG-150 distearate
s (and) sodium laureth-13
carboxylate (and) citric acid
(and) quaternium-15 (and)
tetrasodium EDTA (Miracare
BC-10, Rhone-Poulenc) 38.50%
io B. Butylene glycol (and) water
(aqua) (and) chamomile
extract (and) Aloe barba-
densis extract (and) balm
of gilead (Commiphora
i5 gileadensis) extract (and)
Althea officinalis extract
(Actiplex, Active Organics) 1.00%
AFPP 5x10-6 - 1.0%
Quaternium-15 0.20%
2o Fragrance (perfume) qs
C. Citric acid 0.10%
D. Sodium chloride 1.50%
Water (aqua) qs
100.00%
Shampoo Composition IV (" Marine Poultice Shampoo ")
A. Water (aqua) 30.00%
B. Methylparaben 0.05%
3o Propylparaben 0.15%
C. Laminaria Micronized 15.00%
Lithothamne Lithothamne T-450 9.00%
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Marine silts (Int'1 Sourcing) 5.00%
D. Ammonium laureth sulphate 32.50%
PEG-7 glyceryl cocoate 1.50%
E. Fragrance (perfume) qs
AFPP 5x10-6 - 1.0%
F. Water (aaua) Qs
100.00%
Shampoo Composition V (" Mild Shampoo ")
io
A. Water (aqua) 50.00%
B. Polyquaternium-1 0 0.20%
C. Sodium laureth sulphate,28% active 25.00%
Caprylyl/capryl glucoside 8.00%
is Cocamidopropyl betaine,30% active 9.00%
AFPP 5x10-6 - 1.0%
D. PEG-120 methyl glucose dioleate 1.50%
E. Sodium chloride 0.30%
F. Citric acid qs
Zo Preservative qs
Fragrance (perfume) qs
Water (actua) qs
100.00%
z5 Shampoo Composition VI (" Nutritive Shampoo ")
A. Sodium laureth sulphate 25.00%
Preservative qs
PEG-120 methyl glucose dioleate 1.00%
ao Water (aqua) qs
B. Polyquaternium-11 2.00%
hydrolysed collagen 2.00%
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Cocamidopropyl betaine 8.00%
Oleamide DEA 1.30%
Soluble collagen 3.00%
Hydrolysed milk protein 2.00%
s AFPP 5x10-6 - 1.0%
100.00%
Shampoo Composition VII (" Oatmeal Shampoo with Ceramides ")
io Water (aqua) 15.00%
A.
Xanthan gum, 0.25% soln 30.00%
B. Water (aqua) (and) glycerin
(and) hydroxypropyl methylcellulose
(and) glucuronic acid (and) glucose
i5 (and) glyceryl polyacrylate 25.0 0%
AFPP 5x10-6 - 1.0%
C. Colloidal oatmeal 2.00%
D. Cocamide DEA 3.00%
Sodium laureth sulphate 20.00%
20 lsocetyl alcohol (and) butylene
glycol (and) ceramide 3 0.50%
E. Methylchloroisothiazolinone
(and) methylisothiazolinone 0.05%
F. Water (aqua) cts
100 . 00%
Shampoo composition VIII ("Protein Conditioning Shampoo")
A. Water (aqua) 35.00%
3o Sodium laureth sulfate 50.00%
DL-Panthenol 2.00%
Cocamide DEA 4.00%
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21
Sodium isostearoyl lactylate 3.00%
B. Hydroxypropyltrimonium
hydrolysed keratin 2.00%
AFPP 5x10-6 - 1.0%
s Fragrance (perfume) 0.20%
DMDM hydantoin 0.20%
C Water (aqua) qs
100.00%
io Shampoo Composition IX ("Pearlescent Treatment Shampoo")
Decyl glucoside 5.00%
Lauryldimonium hydroxypropyl
hydrolysed wheat protein 4.00%
is Sodium myreth sulphate 27.00%
Glycol distearate (and) laureth-4
(and) cocamidopropyl betaine
(Euperian PK 3000-AM 3.00%
Propylene glycol (and) water (aqua)
ao (and) white nettle (Lamium album)
extract (and) ivy (Hedera helix) extract
(and) pine cone (Pinus) extract (and)
chamomile (Anthemis nobilis)
extract (and) watercress
25 (Nasturtium officinale) extract
(and) Arnica montana extract
(and) burdock (Arctium lappa)
extract (and) rosemary
(Rosemarinus officinalis)
3o extract (Prodhyactif Hair
Treatment, Prod'Hyg Labs) 5.00%
AFPP 5x10-6 - 1.0%
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Sodium chloride 2.10%
Water (agua) and preservative Qs
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
io (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
i5 (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%
2o 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 - 5x10-6 -
25 100 . 00%
Shampoo Composition XI
Ingredient Level % Function of
Ingredient
Triethanolamine lauryl 10.00 Foaming/cleansing
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sulphate agent
Sodium lauroyl sulphate 2.80 do
AFPP 5x10-6-1.0 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.
s 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 5x10-6-1.0 Antifungal polypepide
Water/perfume/etc. to 100
The pH of the composition is adjusted with citric acid.
io
Shampoo Composition XIII (Conditioning Antidandruff Shampoo)
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Ingredient Levels 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 5x10-6-1.0 Antifungal polypeptide
Water/perfume/etc. to 100
Shampoo Composition XIV
s
Ingredient Levels Function of
Ingredient
Sodium lauryl ether 10.50 Foaming/cleansing
sulphate agent
Coconut 2.50 Foam
diethanolamide stabiliser/shampoo
thickener
AFPP 5x10-6-1 . 0 Antifungal
polypeptide
Water/perfume/etc. to 100
The pH of the composition is adjusted to 6.0-6.5 with citric
acid/NaOH.
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Shampoo Composition XV
Ingredient Levels Function of
Ingredient
Triethanolamine 14.00 Foaming/cleansing
lauryl sulphate agent
Briphos 03D* 1.50 Conditioning
ingredient
AFPP 5x10-6-1 . 0 Ant i f ungal
polypeptide
Empicol WP40** 10.00 Opacifying/pearling
agent
Water/perfume/etc. to 100
5
* Oleyl-3E0 phosphate
** Proprietary mixture lauryl ether sulphate,
of sodium
ethyleneglycol mono and distearates supplied by Albright and
Wilson.
io
Shampoo Composition XVI
Ingredient Levels 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
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AFPP 5x10-6-1. 0 Ant i fungal
polypeptide
Water/perfume/etc. to 100
~ Coco amido propyl betaine.
s Shampoo Composition XVII
Ingredient Levels 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 5x10-6-1. 0 Ant i fungal
polypeptide
Water/perfume/etc. to 100
~ Proprietary mixture of propylene glcol / PEG-55 propylene
glycol oleate supplied by Yh. Goldschmidt AG.
io
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
i5 following
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WO 00/32220 PCT/DK99/00659
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WO 99/13830 disclosing shampoo compositions comprising: a) an
effective amount of a triazole, b) a detersive surfactant, and
c) a carrier;
s 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
io 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-
is 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
ao 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
2s 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
3o 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
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28
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%
s 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;
io 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
i5 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
zo 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
z5 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;
ao 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
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surfactant system, anionic surfactants which are alkyl
ethoxylated sulfates and alkyl sulfates in a ratio between
about 1 :1 and 1: 0 , and ( i i ) f rom about 1 % to 2 0 % by we fight of
the surfactant system, polyhydroxy fatty acid amide
s 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
io surfactant, platelet zinc pyridinethione active of specific
particle size, a suspending agent, water, and a synergizer for
the active.
ns 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
2o 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
as 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
ao 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
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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
s 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
to 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
i5 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
ao 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
25 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
3o 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%
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WO 00/32220 PCT/DK99/00659
31
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 polyalkyleneamine and a detersive
surfactant, in further embodiment, a conditioning shampoo
composition comprising from about by weight 0.01% to about 10%
io of a quaternary polyalkoxylated polyalkyleneamine, 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;
i5 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
20 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;
25 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
3o 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
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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
s 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
io 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
is 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
2o 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
is 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;
3o 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
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WO 00/32220 PCT/DK99/00659
33
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
s 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
io 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
is 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
ao 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
zs 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
3o weight water;
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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
s 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;
io
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
i5 of from about 1 to about 300 centipoise, and which conforms to
either of formulaes (I) and (II) wherein R1 is an alkyl,
alkenyl, hydroxyalkyl or hydroxyalkenyl 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
2o 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
2s consisting of hydrocarbon oils, fatty esters other than the
above synthetic esters, and combinations thereof. The organic
conditioning oil is preferably a polyolefin
[R1-CO-O]n-Y (I) [R2-O-CO)n-Y (II);
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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
s 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
io 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%
is 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
2o 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-
z5 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)
3o a non-ionic detersive surfactant, and e) a carrier, wherein the
composition has a pH of no higher than about 7;
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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;
s
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
to 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
2o insoluble, dispersed conditioning agent, a low viscosity
organic conditioning oil and a select soluble cellulosic
cationic organic polymer hair conditioning agent.
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EXAMPLES
MATERIALS AND METHODS
The production and subsequent purification of the antifungal
s 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. qiqanteus strains
io 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. Qiqanteus strain producing the AFPP
i5 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.
ao Submerged cultivation
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
z5 culture of A. g.iganteus obtained as described above containing
10E 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 seguence determination
30 of the AFPP
The fermentation broth obtained as described above was sub-
jected to centrifugation, the mycelium was suspended in Tris-
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38
buffered saline at pH 7 and then subjected to a second cen-
trifugation. The supernatants from the two centrifugations were
combined and subjected to sterile filtration. The pH of the
resulting supernatant preparation was adjusted to between 6.5
s 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
io 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 6Ø Elution from this second
step was, if necessary, carried out as a gradient elution. The
is purity of the active fractions was assessed by HPLC.
The combined active fractions were subjected to sterile
filtration on a 0.22 ~m 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
3o amino acid sequence shown in Fig. 1 using the formula
s°'1~ (280 nm) =5690 ~ No Trp) +1280 ~ (No . TYr) +120 ~ (No. Cys)
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39
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
s acid sequence.
Based on this formula the extinction coefficient at 280 nm has
been calculated to 1.3. All polypeptide determinations are
based on ODZeo measurements using this calculated extinction co
io efficient.
Production of antibodies against the AFPP
The AFPP produced and purified as described above was diluted
to a concentration of 25-250 ~g/ml in phosphate-buffered
i5 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
2o 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
z5 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
30 of the invention
Reactivity of a variant of the AFPP with an antibody or an
antibody-containing serum obtained as described above may be
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determined in single radial immunodiffusion or western blotting
performed essentially as described by Hudson et al., 1989.
EXAMPLE 1
5
Inhibition of the yeast P. ovate
P. ovate 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
io 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.
is 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
zo 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.
ovate) .
zs - One well identical to the above-mentioned well except
being inoculated with P. ovate.
- One well inoculated with P. ovate and containing AFPP in a
concentration of 0.005 ODZBO (about 5 mg AFPP/litre).
- One well inoculated with P. ovate and containing AFPP in a
concentration of 0 . 027 ODzao
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- One well inoculated with P. ovale and containing AFPP in a
concentration of 0 . 068 ODzeo -
s - One well inoculated with P. ovale and containing AFPP in a
concentration of 0.136 ODzeo.
The growth of P. ovale was followed as a function of time. The
yeast did not grow uniformly, but rather in large
io 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 ~l was plated on
the same medium containing agar. Determination of the
approximate growth in microtiter plates and on solid medium
is 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
ao 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
2s was shortened.
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Table 1
Concentration day 3 day 6 day 7 day day
8 10
day
13
of AFPP (ODZ$o)
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 centreof the well
xx: Some growth observed in entre of the well and minor
the c
growth along the edge
xxx: Vigorous growth on all the med ia
surface
Conclusion:
The present example clearly shows that the AFPP impedes the
to growth of the yeast
Pityrosporum ovate. The fact that even a very low concentration
of AFPP (for example about 5 mg/litre) inhibits the growth of
P. ovate renders the AFPP suitable for use in the treatment or
prophylaxis of dandruff.
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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);
io 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:
is 3987, 1990, "Cloning and nucleotide sequence of a cDNA encoding
the antifungal-protein of Aspergillus giganteus and preliminary
characterization of the native gene".
