U.S. patent application number 10/120042 was filed with the patent office on 2002-08-29 for treating hair by targeting enzymes.
This patent application is currently assigned to UNILEVER HOME & PERSONAL CARE USA, DIVISION OF CONOPCO, INC.. Invention is credited to Blount, Margaret Ann, Davis, Michael Arthur, Jackson, Kelly, Noel, Nathalie, Pearce, Matthew Leslie, Slusarewicz, Paul.
Application Number | 20020119112 10/120042 |
Document ID | / |
Family ID | 10852084 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119112 |
Kind Code |
A1 |
Blount, Margaret Ann ; et
al. |
August 29, 2002 |
Treating hair by targeting enzymes
Abstract
The invention provides the use of a hair benefit agent as an
ingredient in a topical hair treatment composition for the purpose
of targeting an active endogenous hair fibre enzyme when the
composition is applied to the hair, thereby delivering a hair
benefit via the interaction of the hair benefit agent with the
enzyme.
Inventors: |
Blount, Margaret Ann;
(Bedford, GB) ; Davis, Michael Arthur; (Bedford,
GB) ; Jackson, Kelly; (Bebington, GB) ; Noel,
Nathalie; (Bebington, GB) ; Pearce, Matthew
Leslie; (Bebington, GB) ; Slusarewicz, Paul;
(Bedford, GB) |
Correspondence
Address: |
UNILEVER
PATENT DEPARTMENT
45 RIVER ROAD
EDGEWATER
NJ
07020
US
|
Assignee: |
UNILEVER HOME & PERSONAL CARE
USA, DIVISION OF CONOPCO, INC.
|
Family ID: |
10852084 |
Appl. No.: |
10/120042 |
Filed: |
April 10, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10120042 |
Apr 10, 2002 |
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09878652 |
Jun 11, 2001 |
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6399052 |
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09878652 |
Jun 11, 2001 |
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09553634 |
Apr 20, 2000 |
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Current U.S.
Class: |
424/70.14 |
Current CPC
Class: |
A61Q 5/12 20130101; A61Q
5/065 20130101; A61K 8/64 20130101; A61K 8/88 20130101; A61Q 5/02
20130101 |
Class at
Publication: |
424/70.14 |
International
Class: |
A61K 007/06; A61K
007/11 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 22, 1999 |
GB |
9909294.2 |
Claims
1. The use of a hair benefit agent as an ingredient in a topical
hair treatment composition for the purpose of targeting an active
endogenous hair fibre enzyme when the composition is applied to the
hair, thereby delivering a hair benefit via the interaction of the
hair benefit agent with the enzyme.
2. The use according to claim 1, in which the hair benefit agent is
a catalytic substrate for endogenous hair fibre
transgltaminase.
3. The use according to claim 2, in which the catalytic substrate
for endogenous hair fibre transgltaminase is selected from the
group consisting of lysine, polylysine having a molecular weight
ranging from 100 to 2,000, lauroyllysine, soya amine, hydrogenated
tallow amine, stearyl amine, tallow amine, oleyl amine,
hexadecylamine, octadecylamine, and combinations thereof.
4. The use according to claim 1, in which the hair benefit agent is
an inhibitor of endogenous hair fibre protease.
5. The use according to claim 4, in which the inhibitor of
endogenous hair fibre protease is a cystatin or mixture of
cystatzins.
6. The use according to claim 1, in which the hair benefit agent is
a catalytic substrate for endogenous hair fibre protease.
7. The use according to claim 6, in which the catalytic substrate
for endogenous hair fibre protease contains at least 12% by weight
based on total weight of the amino acids lysine, histidine and/or
arginine, either singly or in admixture.
8. The use according to claim 1, in which the hair benefit agent is
a catalytic substrate for endogenous hair fibre lipase.
9. The use according to claim 8, in which the catalytic substrate
for endogenous hair fibre lipase is selected from the group
consisting of triglycerides derived from fatty acids having 8 to 22
carbon atoms.
10. The use according to claim 9, in which the fatty acids are
selected from the group consisting of pelargonic, lauric, myristic,
palmitic, stearic, isostearic, 12-hydroxystearic, oleic, linoleic,
ricinoleic, 18-methyleicosanoic, arachidonic, behenic and erucic
acids and mixtures thereof.
11. The use according to claim 1, in which the hair benefit agent
is an activator for endogenous hair fibre steroid sulphatase.
12. The use according to claim 1, in which the hair benefit agent
is an activator for endogenous hair fibre catalase.
13. The use according to claim 1, in which the hair benefit agent
is an inhibitor for endogenous hair fibre catalase.
14. The use according to claim 1, in which the hair benefit agent
is a catalytic substrate for endogenous hair esterase.
15. The use according to claim 14, in which the catalytic substrate
for endogenous hair esterase is selected from the group consisting
of perfumes, colourants, antioxidants, lipids and amino acids and
mixtures thereof.
16. The use according to claim 1, in which the topical hair
treatment composition is in the form of a shampoo composition
comprising one or more cleansing surfactants, or a conditioner
comprising one or more conditioning surfactants.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the treatment of hair with
activators, inhibitors, co-factors or catalytic substrates for
active endogenous hair fibre enzymes.
BACKGROUND AND PRIOR ART
[0002] The vast number of biochemical reactions that occur in every
human cell are nearly all mediated by enzymes. Enzymes are
biological catalysts, facilitating reactions under mild and
physiological conditions. All enzymes have a defined function and
are highly specific for the reactions they catalyse. A particular
enzyme could therefore be targeted by a specific reagent, whilst
other enzymes remain unaffected. In addition, the types of
reactions catalysed by different enzymes is extremely variable, and
each enzyme is capable of catalysing up to a million reaction
events per second.
[0003] Enzymes are of particular interest for the development of
new modes benefit delivey in hair care. For example, U.S. Pat. No.
5,490,980 describes a composition for topical application to skin,
hair or nails containing a beneficial active agent which has been
functionalised with an a alkylamine moiety, together with the
enzyme transgltaminase (a calcium and thiol dependent enzyme
responsible for the crosslinking of proteins by the formation of
covalent bonds between lysine and glutamine residues). The
transgltaminase in the composition is said to act as a catalyst to
crosslink the active ingredient with glutamine residues in skin,
hair or nails. The transgltaminase in the composition of U.S. Pat.
No. 5,490,980 is sourced from guinea pig liver, slime mould,
alfalfa or preferably bacterial fermentation.
[0004] Problems with the above approach include size exclusion of
the exogenous enzyme, the expense of obtaining the exogenous
enzyme, the possibility of immunological sensitisation from
enzyme-containing formulations, and instability of the enzyme when
stored in the formulation, particularly where high surfactant
levels are present as in cleansing compositions. Furthermore,
Gardner et al. (1995) J.Soc.Cosmet.Chem.,46, 11-28 provides
evidence that glutamine residues on the surface of human hair are
not, contrary to previous thought, recognised as a substrate for
guinea pig liver transgltaminase. The authors of that paper found
no conclusive evidence that virgin hair was modified by exogenously
applied transgltaminase, and considered that the outer fatty acid
layer of the hair probably restricted access to candidate glutamine
sites, despite the large abundance of glutamine residues in hair,
through hydrophobic repulsion or steric interactions. It was
suggested that future work should be directed to cross-linking
soluble protein or modified protein films co-deposited on hair
together with the exogenous enzyme, so that the hair was not
required to donate endogenous residues to the reaction. Tsushima et
al., Arch. Dermatol. Res. 284: 380-335 (1992) describes the
purification and characterisation of a cystatin-type cysteine
proteinase inhibitor (CPI) in the ruman hair shaft;. In that paper,
it is speculated that there are cysteine proteinases in hair, since
there is CPI. However, the presence of any such enzymes within the
hair fibre has, to date, not been reported in the literature.
[0005] The inventors have now found that the mature human hair
fibre contains endogenous enzymes, which furthermore have been
shown to be active and therefore capable of interaction with
exogenously supplied substrates. The precise origin of these active
endogenous hair fibre enzymes remains unclear. It is particularly
surprising that these endogenous hair fibre enzymes have not only
been shown by the inventors to be present, but also to be
biologically active. Maturation of hair fibre results in the death
of its constituent cells (Tamada et al. (1994) Br. J. Dermatol.
131: 521-524) and this coupled with the increased levels of
intracellular cross-linking results in a mature fibre which is
metabolically dead Unexpectedly, the inventors have found that
enzyme activity is in fact preserved, rather than denatured, during
the processes of cellular keratinisation and death that occur
during fibre growth.
SUMMARY OF THE INVENTION
[0006] The present invention provides the use of a hair benefit
agent as an ingredient in a topical hair treatment composition for
the purpose of targeting an active endogenous hair fibre enzyme
when the composition is applied to the hair, thereby delivering a
hair benefit via the interaction of the hair benefit agent with the
enzyme.
[0007] Advantageously, the above approach solves the problems
associated with the systems described previously involving
application of exogenous enzyme. For example, enzyme substrates can
be selected which are cheaper, safer and stabler to formulate into
hair treatment compositions than exogenous enzyme, and which,
unlike exogenous enzyme, are able to penetrate the hair and
actually deliver enzyme-linked benefits to the underlying fibre
matrix.
Detailed Description and Preferred Embodiments
[0008] Examples of active endogenous enzymes identified by the
inventors to date within the mature human hair fibre fall in
various and diverse classes and include:
[0009] Transgltaminase
[0010] Protease
[0011] Lipase
[0012] Steroid sulphatase
[0013] Catalase
[0014] Esterase
[0015] Hair Benefit Agents
[0016] Ingredients suitable for use as hair benefit agents
according to the invention for targeting endogenous hair fibre
enzymes may be any material which is capable of specifically
interacting with the selected enzyme, or is chemically modified in
order to do so. The material may serve as an enzyme activator,
enzyme inhibitor, enzyme co-factor or catalytic substrate for the
enzyme, in order to derive benefit.
[0017] Examples of the type of such ingredients include hair
conditioning agents, (such as humectants, softeners and cuticle
lubricants), hair colouring agents, antimicrobial compounds,
UV-absorbing compounds, fluorescers, hair strengthening agents
(such as fibre repair agents or fibre rebuilding agents),
antioxidants, perfumes, and mixtures thereof.
[0018] Preferred ingredients for use according to the invention are
selected to target the endogenous hair fibre enzymes which are
classified below:
[0019] (i) Transgltaminase
[0020] Transgltaminase has been found to be active in the hair
fibre, localised predominantly in the cuticle. It is a calcium and
thiol dependent enzyme which is responsible for the cross-linking
of proteins by the formation of covalent bonds between lysine and
glutamine residues.
[0021] Ingredients suitable for use as hair benefit agents
according to the invention for targeting endogenous hair fibre
transgltaminase include catalytic substrates for this class of
enzyme.
[0022] Catalytic substrates for transgltaminase are required to
include (or be modified to include) at least one--R'NH.sub.2 group
in which R' is a hydrocarbon or functionalised hydrocarbon chain.
Preferably R' is a straight aliphatic hydrocarbon chain containing
from 1 to 8 carbon atoms. Transgltaminase crosslinks the
substrate-through the alkyl amine (R'NH.sub.2) group to the
glutamine residues in hair.
[0023] Accordingly, hair benefit agents suitable for use according
to the invention as catalytic substrates for endogenous hair
transgltaminase may be any compound which has a beneficial effect
when delivered to human hair, as long as the compound contains, or
is modified to contain, an alkyl amine group.
[0024] Use of such hair benefit agents as catalytic substrates for
endogenous hair fibre transgltaminase enables a direct, in situ
interaction between substrate and enzyme for delivering a localised
and sustained hair fibre benefit derived from that interaction. In
this way, permanent or internal hair benefits may be obtained from
the transgltaminase-catalysed cross-linking reaction between a
benefit agent and the substance of the hair fibre itself. Preferred
benefit agents in this context are hair conditioning agents and
hair colouring agents. Advantageously, such enzyme-catalysed
interactions occur under physiological and naturally mild reaction
conditions, so harsh chemical treatments are not necessary.
[0025] Preferably, in order to optimise the cross-linking reaction
between transgltaminase and substrate, the R'NH.sub.2 group
contains at least 4 unbranched carbon atoms adjacent to the
NH.sub.2 group. Optimal transgltaminase activity is generally
achieved when the substrate has (a) alkylamine side-chain lengths
equivalent to 5 methylene groups (or 7.2-7.6 A long), (b) no
branching nor groups bulkier than methylene along the alkyl amine
chain, (c) hydrophobic moieties attached to the alkyl chain, and
(d) more than one alkyd amine group. However, this is not
particularly critical and other hydrocarbon chains can be used,
(including those incorporating functional groups such as ester,
ether or amide linkages or similar), provided the terminal amine
group is not sterically hindered from interacting with the active
site.
[0026] Suitable ingredients which inherently contain an alkyl amine
group include intact proteins, protein hydrolysates, chemically
modified (e.g. quaternized or acylated) proteins or protein
hydrolysates, peptides, non-proteinaceous amino acid polymers such
as amino acid polymers produced by chemical synthesis), amino acids
(or derivatives thereof), and primary amine compounds.
[0027] Preferred examples include the amino acid lysine, polymers
thereof (polylysine) having a molecular weight ranging from 100 to
2,000, and acylated derivatives thereof such as lauroyllysine.
[0028] Also preferred are primary amines having a carbon chain of
at least C.sub.10 preferably C.sub.12-22, such as soya amine,
hydrogenated tallow amine, stearyl amine, tallow amine, oleyl
amine, hexadecylamine, octadecylamine, and combinations thereof.
These materials are preferred since they are capable of imparting
smoothness and improved ease of combing to treated hair fibres.
[0029] Ingredients may also be used which have a beneficial effect
on human hair and which have been modified to contain at least one
alkyl amine moiety. U.S. Pat. No. 5,490,980 describes and
exemplifies a range of such ingredients and the corresponding
process by which they are appropriately modified. Preferred
examples are alkyl amine modified silicones which are capable of
imparting conditioning benefits to hair.
[0030] Activators of endogenous hair fibre transgltaminase may also
be used as ingredients according to the invention. Such activators
may be used alone, in order to promote fibre strengthening due to
enhanced in situ fibre transgltaminase activity, or alternatively
in conjunction with one or more of the catalytic substrates
described above.
[0031] (ii) Protease
[0032] Various protease activities have been identified in the hair
fibre. These include cysteine and serine proteases, with the major
protease observed to date appearing to have similar properties to
the lysosomal cysteine proteases, cathepsins L and B. Catheosins L
and B are known to hydrolyse many cytosolic and structural
proteins, including collagen and elastin.
[0033] Ingredients suitable for use as hair benefit agents
according to the invention for targeting endogenous hair fibre
protease include inhibitors of this class of enzyme. Examples of
such protease inhibitors include cystatins, which are small
proteins found widely in nature, for example in eggs and avocado
mesocarp. Use of such inhibitors enables reduction or prevention of
autohydrolysis of the hair fibre by the active hair fibre protease,
thereby protecting against hair fibre damage or deterioration.
[0034] Further suitable ingredients for use as hair benefit agents
according to the invention for targeting endogenous hair fibre
protease are catalytic substrates for protease, i.e. protein,
proteinaceous or peptide containing substrates capable of being
hydrolysed by the protease into smaller peptides and/or free amino
acids.
[0035] Examples include collagen, keratin, fibroin, elastin,
ovalbumin, casein, gluten, ferritin, gliadin, zein, soy protein,
silk protein and hydrolysates or derivatives thereof.
[0036] Preferably the substrate is rich in the amino acids lysine,
histidine and/or arginine. In this way, hydrolysis of substrate by
the enzyme can deliver benefits such as moisturisation to the hair
via the release of the free amino acids directly into the hair
fibre. For example, preferred protein or proteinaceous substrates
contain at least 12%, most preferably at least 15% to about 70% of
these amino acids, either singly or in admixture, by weight based
on total weight.
[0037] Ingredients may also be used which have a beneficial effect
on human hair and which have been covalently modified to contain an
amino acid or amino acid sequence capable of interacting with
endogenous hair fibre protease. In this way, protease-mediated
catalysis facilitates the gradual release of benefit agent directly
into the hair fibre, where it may be allowed to penetrate further
into the fibre, without the need for harsh chemical modification
processes.
[0038] (iii) Lipase
[0039] Lipase activity has been found in the hair fibre. Lipases
hydrolyse ester linkages in lipids, and in particular those within
triacylglycerol. This reaction yields free fatty acids and
glycerol. Glycerolipids are the most common substrates that are
hydrolysed by the enzyme, although they are relatively non-specific
and will also hydrolyse other acyl esters. Lipases are believed to
have an important role in hair development where they are involved
in the recycling of hair glycerolipids during the formation of cell
membrane complex lipids. During hair cell differentiation, the
cellular lipids (predominantly glycerolipids) are destroyed and new
lipids synthesised such as steroids, ceramides and fatty acids.
[0040] Ingredients suitable for us as hair benefit agents according
to the invention for targeting endogenous hair fibre lipase include
catalytic substrates for this class of enzyme.
[0041] Examples are triglyceride fats and oils derived from
vegetable, animal and marine sources such as coconut oil, castor
oil, safflower oil, cotton seed oil, corn oil, olive oil, cod liver
oil, almond oil, avocado oil, palm oil, sesame oil, and soybean
oil.
[0042] Preferred are triglycerides derived from fatty acids having
8 to 22 carbon atoms. Suitable examples of such fatty acids include
pelargonic, lauric, myristic, palmitic, stearic, isostearic,
12-hydroxystearic, oleic, linoleic, ricinoleic,
18-methyleicosanoic, arachidonic, behenic, and erucic acids. In
this way, lipase-mediated catalysis facilitates the release of free
fatty acids, which are important components of the cell membrane
complex, and glycerol, an effective humectant, directly into the
hair fibre. Advantageously, targeting of endogenous hair fibre
lipase with triglyceride substrates of selected fatty acid content
provides a route to influencing the lipid environment of the hair
fibre without the necessity for harsh chemical or physical
modification processes.
[0043] Ingredients may also be used which have a beneficial effect
on human hair and which have been covalently tagged with a lipid
capable of interacting with endogenous hair fibre lipase.
[0044] (iv) Steroid Sulphatase
[0045] Steroid sulphatase activity has been identified in the hair
fibre. This enzyme catalyses the hydrolysis of
cholesterol-3-sulphate into cholesterol (and sulphate). Steroid
sulphatase is believed to have an important role in the formation
of normal healthy hair, since dramatic hair effects are noticeable
in people who are deficient in this enzyme. For example, in
patients with X-linked ichthiesis, the enzyme activity is reduced
(or non-existent) and the hair feels dry and brittle.
[0046] Ingredients suitable for use as hair benefit agents
according to the invention for targeting endogenous hair steroid
sulphatase include activators of this class of enzyme. Use of such
activators enables increased delivery of cholesterol directly into
the hair fibre. Cholesterol has been shown to protect hair against
damage, enhance hair flexibility and reduce dry feel.
[0047] Further suitable ingredients for use as hair benefit agents
according to the invention for targeting endogenous hair fibre
steroid sulphatase are catalytic substrates for steroid sulphatase
such as cholesterol-3-sulphate, which is capable of being
hydrolysed by the steroid sulphatase into cholesterol (and
sulphate).
[0048] (v) Catalase
[0049] Catalase activity has been identified in the hair fibre.
This enzyme catalyses the conversion of hydrogen peroxide
(H.sub.2O.sub.2) into water and molecular oxygen.
[0050] Ingredients suitable for use as hair benefit agents
according to the invention for targeting endogenous hair fibre
catalase include activators of this class of enzyme.
[0051] Use of such activators provides a route to additional
radical damage protection for the hair fibre through increased
removal of peroxide. This is particularly advantageous in the case
of chemically treated hair (e.g. bleached, coloured or permed)
which is especially vulnerable to oxidative damage, and also in
climates in which high levels of oxidative stress are
encountered.
[0052] Use of such activators additionally provides a route to
removal of surplus peroxide which is deliberately added to the hair
during treatment with products such as bleaches, colorants, and
perms in which peroxide is used for the oxidation step.
Alternatively, inhibitors of endogenous hair fibre catalase may be
used as hair benefit agents according to the invention, as
ingredients for increasing the efficacy of peroxide-containing
products which are applied to the hair fibre such as colourants,
bleaches or perms.
[0053] (vi) Esterase
[0054] Esterase activity has been identified in the hair fibre.
Esterases are a broad class of enzymes that catalyse the hydrolysis
of ester linkages.
[0055] Ingredients suitable for use as hair benefit agents
according to the invention for targeting endogenous hair fibre
esterase include catalytic substrates for this class of enzyme.
[0056] Catalytic substrates for esterase are required to include
(or be modified to include) at least one ester linkage.
[0057] Accordingly, hair benefit agents suitable for use according
to the invention as catalytic substrates for endogenous hair
esterase may be any compound which has a beneficial effect when
delivered to human hair, as long as the compound contains, or is
modified to contain, an ester linkage.
[0058] In this way, benefit agents may be used in esterified form,
(for example, conjugated via an ester linkage to an inert carrier
molecule), and used to target endogenous hair fibre esterase, so
that upon hydrolysis by endogenous hair fibre esterase, the benefit
agent is released and thus activated, allowing a controlled release
of the benefit agent directly into the hair fibre. This is
particularly advantageous in the case of benefit agents which in
free (unesterified) form are sensitive to factors such as heat,
oxidation, light, moisture, pH, or microbial attack, or which tend
to react with other ingredients in a formulation. Examples of such
benefit agents include perfumes, colourants, antioxidants such as
vitamins A, C and E, and natural hair nutrients such as lipids and
amino acids.
[0059] Product Form
[0060] The final product form of hair treatment compositions
according to the invention may suitably be, for example, shampoos,
conditioners, sprays, mousses or lotions. Particularly preferred
product forms are shampoos, post-wash conditioners (leave-in and
rinse-off) and hair treatment products such as hair essences and
hair oils.
[0061] Shampoo Compositions
[0062] A particularly preferred hair treatment composition in
accordance with the invention is a shampoo composition.
[0063] Cleansing Surfactant
[0064] Such a shampoo composition will comprise one or more
cleansing surfactants which are cosmetically acceptable and
suitable for topical application to the hair. Further surfactants
may be present as an additional ingredient if sufficient for
cleansing purposes is not provided as emulsifying agent for oily or
hydrophobic components (such as silicones) which may typically be
present in the shampoo.
[0065] It is preferred that shampoo compositions of the invention
comprise at least one further surfactant (in addition to that used
as emulsifying agent) to provide a cleansing benefit.
[0066] Suitable cleansing surfactants, which may be used singularly
or in combination, are selected from anionic, amphoteric and
zwitterionic surfactants, and mixtures thereof. The cleansing
surfactant may be the same surfactant as the emulsifier, or may be
different.
[0067] Examples of anionic surfactants are the alkyl sulphates,
alkyl ether sulphates, alkaryl sulphonates, alkanoyl isethionates,
alkyl succinates, alkyl sulphosuccinates, N-alkyl sarcosinates,
alkyl phosphates, alkyl ether phosphates, alkyl ether carboxylates,
and alpha-olefin sulphonates, especially their sodium, magnesium,
ammonium and mono-, di-and triethanolamine salts. The alkyl and
acyl groups generally contain from 8 to 18 carbon atoms and may be
unsaturated. The alkyl ether sulphates, alkyl ether phosphates and
alkyl ether carboxylates may contain from 1 to 10 ethylene oxide or
propylene oxide units per molecule.
[0068] Typical anionic surfactants for us in shampoos of the
invention include sodium oleyl succinate, ammonium-lauryl
sulphosuccinate, ammonium lauryl sulphate, sodium dodecylbenzene
sulphonate, triethanolamine dodecylbenzene sulphonate, sodium
cocoyl isethionate, sodium lauryl isethionate and sodium N-lauryl
sarcosinate. The most preferred anionic surfactants are sodium
lauryl sulphate, triethanolamine monolauryl phosphate, sodium
lauryl ether sulphate 1EO, 2EO and 3EO, ammonium lauryl sulphate
and ammonium lauryl ether sulphate 1EO, 2EO and 3EO.
[0069] Examples of amphoteric and zwitterionic surfactants include
alkyl amine oxides, alkyl betaines, alkyl amidopropyl betaines,
alkyl sulphobetaines (sultaines), alkyl glycinates, alkyl
carboxyglycinates, alkyl amphopropionates, alkylamphoglycinates,
alkyl amidopropyl hydroxysultaines, acyl taurates and acyl
glutamates, wherein the alkyl and acyl groups have from 8 to 19
carbon atoms. Typical amphoteric and zwitterionic surfactants for
use in shampoos of the invention include lauryl amine oxide,
cocodimethyl sulphopropyl betaine and preferably lauryl betaine,
cocamidopropyl betaine and sodium cocamphopropionate.
[0070] The shampoo composition can also include co-surfactants, to
help impart aesthetic, physical or cleansing properties to the
composition. A preferred example is a nonionic surfactant, which
can be included in an amount ranging from 0% to about 5% by weight
based on total weight. For example, representative nonionic
surfactants that can be included in shampoo compositions of the
invention include condensation products
of-aliphatic-(C.sub.8-C.sub.18) primary or secondary linear or
branched chain alcohols or phenols with alkylene oxides, usually
ethylene oxide and generally having from 6 to 30 ethylene oxide
groups.
[0071] Other representative nonionics include mono- or di-alkyl
alkanolamides. Examples include coco mono- or di-othanolamide and
coco mono-isopropanolamide.
[0072] Further nonionic surfactants which can be included in
shampoo compositions of the invention are the alkyl polyglycosides
(APGs). Typically, the APG is one which comprises an alkyl group
connected (optionally via a bridging group) to a block of one or
more glycosyl groups. Preferred APGs are defined by the following
formula:
RO-(G).sub.n
[0073] wherein R is a branched or straight chain alkyl group which
may be saturated or unsaturated and G is a saccharide group.
[0074] R may represent a mean alkyl chain length of from about
C.sub.5 to about C.sub.20. Preferably R represents a mean alkyl
chain length of from about C.sub.8 to about C.sub.12. Most
preferably the value of R lies between about 9.5 and about 10.5. G
may be selected from C.sub.5 or C.sub.6 monosaccharide residues,
and is preferably a glucoside. G may be selected from the group
comprising glucose, xylose, lactose, fructose, mannose and
derivatives thereof. Preferably G is glucose.
[0075] The degree of polymerisation, n, may have a value of from
about 1 to about 10 or more. Preferably, the value of n lies in the
range of from about 1.1 to about 2. Most preferably the value of n
lies in the range of from about 1.3 to about 1.5.
[0076] Suitable alkyl polyglycosides for use in the invention are
commercially available and include for example those materials
identified as: Oramix NS10 ex Seppic; Plantaren 1200 and Plantaren
2000 ex Henkel.
[0077] The total amount of surfactant (including any co-surfactant,
and/or any emulsifying agent) in shampoo compositions of the
invention is generally from 0.1 to 50% by weight, preferably from 5
to 30%, more preferably from 10% to 25% by weight of the total
shampoo composition.
[0078] Cationic Polymer
[0079] A cationic polymer is a preferred ingredient in shampoo
compositions of the invention, for enhancing conditioning
performance of the shampoo. Typically such a polymer enhances
deposition of conditioning components such as silicone from the
shampoo composition onto the intended site during use, i.e. the
hair and/or the scalp.
[0080] The cationic polymer may be a homopolymer or be formed from
two or more types of monomers. The molecular weight of the polymer
will generally be between 5 000 and 100 00 000, typically at least
10 000 and preferably in the range 100 000 to about 2 000 000. The
polymers will have cationic nitrogen containing groups such as
quaternary ammonium or protonated amino groups, or a mixture
thereof.
[0081] The cationic nitrogen-containing group will generally be
present as a substituent on a fraction of the total monomer units
of the cationic polymer. Thus when the polymer is not a homopolymer
it can contain spacer non-cationic monomer units. Such polymers are
described in the CTFA Cosmetic Ingredient Directory, 3rd edition.
The ratio of the cationic to non-cationic monomer units is selected
to give a polymer having a cationic charge density in the required
range.
[0082] Suitable cationic polymers include, for example, copolymers
of vinyl monomers having cationic amine or quaternary ammonium
functionalities with water soluble spacer monomers such as
(meth)acrylamide, alkyl and dialkyl (meth)acrylamides, alkyl
(meth)acrylate, vinyl caprolactone and vinyl pyrrolidine. The alkyl
and dialkyl substituted monomers preferably have C1-C7 alkyl
groups, more preferably C1-3 alkyl groups. Other suitable spacers
include vinyl esters, vinyl alcohol, malelc anhydride, propylene
glycol and ethylene glycol.
[0083] The cationic amines can be primary, secondary or tertiary
amines, depending upon the particular species and the pH of the
composition. In general secondary and tertiary amines, especially
tertiary, are preferred.
[0084] Amine substituted vinvl monomers and amines can be
polymerised in the amine form and then converted to ammonium by
quaternization.
[0085] The cationic polymers can comprise mixtures of monomer units
derived from amine- and/or quaternary ammonium-substituted monomer
and/or compatible spacer monomers. Suitable cationic polymers
include, for example:
[0086] copolymers of 1-vinyl-2-pyrrolidine and
1-vinyl-3-methyl-imidazoliu- m salt (e.g. chloride salt), referred
to in the industry by the Cosmetic, Toiletry, and Fragrance
Association, (CTFA) as Polyquaternium-16. This material is
commercially available from BASF Wyandotte Corp. (Parsippany, N.J.,
USA) under the LUVIQUAT tradename (e.g. LUVIQUAT FC 370);
[0087] copolymers of 1-vinyl-2-pyrrolidine and dimethylaminoethyl
methacrylate, referred to in the industry (CTFA) as
Polyquaternium-11. This material is available commercially from Gaf
Corporation (Wayne, N.J., USA) under the GAFQUAT tradename (e.g.,
GAFQUAT 755N);
[0088] cationic diallyl quaternary ammonium-containing polymers
including, for example, dimethyldiallyammonium chloride homopolymer
and copolymers of acrylamide and dimethyldiallylammonium chloride,
referred to in the industry (CTFA) as Polyquaternium 6 and
Polyquaternium 7, respectively;
[0089] mineral acid salts of amino-alkyl esers of homo-and
co-polymers of unsaturated carboxylic acids having from 3 to 5
carbon atoms, (as described in U.S. Pat. No. 4,009,256);
[0090] cationic polyacrylamides(as described in WO95/22311).
[0091] Other cationic polymers that can be used include cationic
polysaccharide polymers, such as cationic cellulose derivatives,
cationic starch derivatives, and cationic guar gum derivatives.
[0092] Cationic polysaccharide polymers suitable for use in
compositions of the invention include those of the formula:
A--O--[R--N.sup.+(R.sup.1) (R.sup.2) (R.sup.3)X.sup.-],
[0093] wherein: A is an anhydroglucose residual group, such as a
starch or cellulose anhydroglucose residual. R is an alkyl-ene,
oxyalkylene, polyoxyalkylene, or hydroxyalkylene group, or
combination thereof. R.sup.1, R.sup.2 and R.sup.3 independently
represent alkyl, aryl, alkylaryl, arylalkyl, alkoxyalkyl, or
alkoxyaryl groups, each grouo containing up to about 18 carbon
atoms. The total number of carbon atoms for each cationic moiety
(i.e., the sum or carbon atoms in R.sup.1, R.sup.2 and R.sup.3) is
preferably about 20 or less, and X is an anionic counterion.
[0094] Cationic cellulose is available from Amerchol Corp. (Edison,
N.J., USA) in their Polymer JR (trade mark) and LR (trade mark)
series of polymers, as salts of hydroxyethyl cellulose reacted with
trlmethyl ammonium substituted epoxide referred to in the industry
(CTFA) as Polyquaternium 10. Another type of cationic cellulose
includes the polymeric quaternary ammonium salts of hydroxyethyl
cellulose reacted with lauryl dimethyl ammonium-substituted
epoxide, referred to in the industry (CTFA) as Polyquaternium 24.
These materials are available from Amerchol Corp. (Edison, N.J.,
USA) under the tradename Polymer LM-200.
[0095] Other suitable cationic polysaccharide polymers include
quaternary nitrogen-containing cellulose ethers (e.g. as described
in U.S. Pat. No. 3,962,418), and copolymers of etherified cellulose
and starch (e.g. as described in U.S. Pat. No. 3,958,581).
[0096] A particularly suitable type of cationic polysaccharide
polymer that can be used is a cationic guar gum derivative, such as
guar hydroxypropyltrimonium chloride (Commercially available from
Rhone-Poulenc in their JAGUAR trademark series).
[0097] Examples are JAGUAR C13S, which has a low degree of
substitution of the cationic groups and high viscosity. JAGUAR C15,
having a moderate degree of substitution and a low viscosity,
JAGUAR C17 (high degree of substitution, high viscosity), JAGUAR
C16, which is a hydroxypropylated cationic guar derivative
containing a low level of substituent groups as well as cationic
quaternary ammonium groups, and JAGUAR 162 which is a high
transparency, medium viscosity guar having a low degree of
substitution. Preferably the cationic polymer is selected from
cationic cellulose and cationic guar derivatives. Particularly
preferred cationic polymers are JAGUAR C13S, JAGUAR C15, JAGUAR C17
and JAGUAR C16 and JAGUAR C162.
[0098] Conditioners
[0099] Conditioning Surfactant
[0100] Compositions in accordance with the invention may also be
formulated as conditioners for the treatment of hair (typically
after shampooing) and subsequent rinsing.
[0101] Such a conditioner will comprise one or more conditioning
surfactants which are cosmetically acceptable and suitable for
topical application to the hair.
[0102] Suitable conditioning surfactants are selected from cationic
surfactants, used singly or in admixture.
[0103] Examples of suitable cationic conditioning surfactants
include quaternary ammonium cationic surfactants.
[0104] Suitable quaternary ammonium cationic surfactants for use in
hair conditioners of the invention include cetyltrimethylammonium
chloride, behenyltrimethylammonium chloride, cetylpyridinium
chloride, tetramethylammonium chloride, tetraethylammonium
chloride, octyltrimethylammonium chloride, dodecyltrimethylammonium
chloride, hexadecyltrimethylammonium chloride,
octyldimethylbenzylammonium chloride, decyldimethylbenzylammonium
chloride, stearyldimethylbenzylammo- nium chloride,
didodecyldimethylammonium chloride, dioctadecyldimethylammo- nium
chloride, tallowtrimethylammonium chloride, cocotrimethylammonium
chloride, and the corresponding hydroxides thereof. Further
suitable cationic surfactants include those materials having the
CTFA designations Quaternium-5, Quaternium-31 and Quaternium-18.
Mixtures of any of the foregoing materials may also be suitable. A
particularly useful cationic surfactant for use in hair
conditioners of the invention is cetyltrimethylammonium chloride,
available commercially, for example as GENAMIN CTAC, ex Hoechst
Celanese.
[0105] A further preferred class of cationic conditioning
surfactants are acid-neutralised amidoamine compounds of the
general structural formula (I):
R1--C(O)--NH--R2--N(R3) (R4) (I)
[0106] wherein R1 is a fatty acid chain containing from 12 to 22
carbon atoms, R2 is an alkylene group containing from one to four
carbon atoms, and R3 and R4 are, independently, an alkyl group
having from one to four carzon atoms.
[0107] Examples of suitable amidoamine compounds of general
structural formula (I) include stearamidopropyl dimethylamine,
stearamidopropyl diethylamine, stearamidoethyl dimethylamine,
stearamidoethyl diethylamine, palmitamidopropyl dimethylamine,
behenamidopropyl dimethylamine, myristamidopropyl dimethylamine,
oleamidopropyl dimethylamine, ricinoleamidopropyl dlmethylamine,
and combinations thereof.
[0108] The acid used to neutralise the amidoamine compound can be
essentially any organic acid or mineral acid of sufficient acid
strength to neutralise a free amine nitrogen. Such acids include
hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid,
lactic acid, citric acid, tartaric acid, acetic acid, gluconic
acid, glycolic acid and propionic acid, or combinations thereof. A
preferred acid is lactic acid, since neutralisation of the
amidoamine compound with this acid yields an exceptionally stable
composition.
[0109] In general, a sufficient amount of acid is added to
neutralise the amidoamine compound and to adjust the final pH of
the composition to within a range of from about 2.5 to about 6,
preferably in a pH range of from about 3 to about 5.
[0110] In conditioners of the invention, the level of cationic
surfactant is preferably from 0.01 to 10%, more preferably 0.05 to
5%, most preferably 0.1 to 2% by total weight of cationic
ssurfactant based on the total weight of the composition.
[0111] Fatty Alcohol
[0112] Conditioners of the invention advantageously incorporate a
fatty alcohol. The combined use of fatty alcohols and cationic
surfactants in conditioning compositions is believed to be
especially advantageous, because this leads co the formation of a
lamellar phase, in which the cationic surfactant is dispersed.
[0113] Representative fatty alcohols comprise from 8 to 22 carbon
atoms, more preferably 16 to 20. Examples of suitable fatty
alcohols include cetyl alcohol, stearyl alcohol and mixtures
thereof. The use of these materials is also advantageous in that
they contribute to the overall conditioning properties of
compositions of the invention.
[0114] The level of fatty alcohol in conditioners of the invention
is conveniently from 0.01 to 10%, preferably from 0.1 to 5% by
weight of the composition. The weight ratio of cationic surfactant
to fatty alcohol is suitably from 10:1 to 1:10, preferably from 4:1
to 1:8, optimally from 1:1 to 1:4.
[0115] Silicone
[0116] Silicone is a particularly preferred ingredient in hair
treatment compositions of the invention. In particular, hair
shampoos and conditioners of the invention will preferably also
comprise emulsified particles of silicone, for enhancing
conditioning performance. The silicone is insoluble in the aqueous
matrix of the composition and so is present in an emulsified form,
with the silicone present as dispersed particles.
[0117] Suitable silicones include polydiorganosiloxanes, in
particular polydimethylsiloxanes which have the CTFA designation
dimethicone. Also suitable for use compositions or the invention
(particularly shampoos and conditioners) are polydimethyl siloxanes
having hydroxyl end groups, which have the CTFA designation
dimethiconol, and polydimethylsiloxanes having containing at least
one amino functional group, which have the CTFA designation
amodiethicone or trimethylsilylamodimethicone.
[0118] Examples of suitable materials include:
[0119] the DC200 series of silicone fluids, available from Dow
Corning (e.g. DC200, viscosity 350 cs), or SF96 or the VISCASIL
series of silicones, available from General Electric Silicones;
[0120] silicone gums such as SE30, SE54 and SE76, available from
General Electric Silicones;
[0121] silicone gum/fluid blends such as Q2-1403 available from Dow
Corning, or CF 1251, available from General Electric Silicones;
[0122] pre-formed emulsions of dimethlconol such as emulsions
DC2-1766, DC2-1784, DC2-1787 and microemulsions DC2-1391, DC2-1865
and DC2-1870, all available from Dow Corning;
[0123] amino functional silicones such as Q2-8220 and Q2-8466
fluids, available from Dow Corning, and also SF-1708-D1, available
from General Electric Silicones; pre-formed emulsions of amino
functional silicones such as DC929 Cationic Emulsion, DC939
Cationlc Emulsion, and the non-ionic emulsions DC2-7224, DC2-8467,
DC2-8177 and DC2-8154 (al available from Dow Corning)
[0124] silicone gum/silicone fluid/amino functional silicone
blends.
[0125] Most preferably, the silicone for inclusion in the hair
treatment composition of the invention, is sourced as a preformed
aqueous emulsion, for example a mechanically-formed aqueous
emulsion. In such emulsions, it is highly preferable that the
emulsion additionally includes at least one emulsifier in order to
stabilise the silicone emulsion. Suitable emulsifiers are well
known in the art and include anionic and nonionic surfactants.
[0126] Preferably, the average particle size of the silicone
droplets in the emulsion and also in the final hair treatment
composition is less than 20 microns, more preferably less than 10
microns. A smaller silicone particle size enables a more uniform
distribution of silicone on the hair for the same amount of
silicone in the composition.
[0127] Silicone particle size in the emulsion may be measured by
means of a laser light scattering technique, for example using a
2600D Particle Sizer from Malvern Instruments.
[0128] Viscosity (of the silicone itself and not the emulsion or
the final hair treatment composition) can be measured by means of a
glass capillary viscometer as set out further in Dow Corning
Corporate Test Method CTM004, Jul. 20 1970.
[0129] The tocal amount of silicone incorporated into hair
treatment compositions of the invention depends on the level of
conditioning desired and the material used. A preferred amount is
from 0.01 to about 10% by weight of the total composition although
these limits are not absolute. The lower limit is determined by the
minimum level to achieve conditioning and the upper limit by the
maximum level to avoid making the hair and/or skin unacceptably
greasy.
[0130] We have found that a total amount of silicone of from 0.3 to
5%, preferably 0.5 to 3%, by weight of the total composition is a
suitable level.
[0131] Solvents and Carriers
[0132] Compositions of the invention are preferably aqueous based,
but non-aqueous solvents also can be used in order to help
solubilise ingredients that are not sufficiently soluble in water.
Suitable non-aqueous solvents include the lower alcohols like ethyl
alcohol and propyl alcohol; polyols like glycerol; glycols or
glycol ethers, like 2-butoxyethanol, ethylene glycol, ethylene
glycol monoethyl ether, propylene glycol and diethylene glycol
monoethyl ether or monomethyl ether and mixtures thereof.
[0133] Optional Ingredients
[0134] Compositions of this invention may contain any other
ingredient normally used in hair treatment formulations. These
other ingredients may include viscosity modifiers, preservatives,
colouring agenzs, polyols such as glycerine and polypropylene
glycol, chelating agents, antioxidants, fragrances, antimicrobials
and sunscreens. Each of these ingredients will be present in an
amount effective to accomplish its purpose. Generally these
optional ingredients are included individually at a level of up to
about 5% by weight of the total composition.
[0135] The invention is further illustrated by way of the following
non-limitative examples. All percentages quoted are by weight based
on total weight unless otherwise stated.
EXAMPLES
Example 1
[0136] Determination of the Presence of Steroid Sulphatase in Hair
Fibres
[0137] Hair fibres taken from approx. 5 mm from the scalps of male
individuals, aged 25-35, were cleaned, dried and then ground in
liquid N.sub.2, and homogenised with an Ystral Homogeniser (high
shear) using a 5 mm probe in imidazole-HCl (pH 7), 1 M NaCl and
0.1% Triton X-100. Sucrose extracts were prepared in a similar way
to the above in 250 mM sucrose.
[0138] Both homogenates were centrifuged at 13 000 rpm for 5
minutes. Both supernatant and insoluble proteinaceous pellet were
retained. The pellets were resuspended in 20% glycerol in 100 mM
imidazole-HCl (pH 7). The clear supernatants (imidazole and sucrose
extracts) were dialysed against 20% glycerol in 100 mM
imidazole-HCl (pH 7). The extracts were frozen at -20.degree. C.
until required.
[0139] Cholesterol-sulphate sulphatase activity of the hair fibre
extracts was measured using the following assay:
[0140] The assay mixture (per assay) contained 20 .mu.M [.sup.14C]
cholesterol-3-sulphate (250 000 dpm total) in 50 mM imidazole-HCl
pH7, 0.05% Triton X-100 and hair fibre extract in a final assay
volume of 100 .mu.l. A typical assay was conducted by preparing a
5.times.substrate solution as follows: 22.72 .mu.l of [.sup.14C]
cholesterol-3-sulphate was placed in a clean glass vial and the
solvent removed under a steady stream of N.sub.2 gas. The lipid was
resuspended into 1 ml of 0.25% Triton X-100 in 250 mM imidazole-HCl
(pH 7) and mixture vigorously mixed. The K.sub.m of human steroid
sulphatase is 5 .mu.M, and thus using 20 .mu.M substrate in the
assay, the velocity of the reaction would be tending towards
maximum.
[0141] For each assay, 20 .mu.l of the 5.times.substrate solution
was added to 80 .mu.l of hair fibre extract, incubated at
37.degree. C. for 3 hours, followed by up to 21 hours at room
temperature. The reaction was stopped by the addition of
chloroform:methanol (2:1). This was then transferred to a clean
vial, and any residual reaction mixture was reclaimed with two 500
.mu.l washes with the solvent. Milli-Q water (0.5 ml) was added to
the pooled solvent and vigorously mixed for 5 seconds and allowed
to stand to enable the phases to separate. The lower (chloroform)
phase was placed in a fresh vial and the solvent removed under a
steady stream of N2 gas. The dried lipid was resuspended in 50
.mu.l of chloroform:methanol (2:1) and the whole sample loaded onto
a preparative silica TLC plate.
[0142] A line in the silica plate was scored at 13 cm from the
origin, ensuring that the solvent front migration was fixed. The
TLC plates were developed in chloroform:methanol:acetic acid
(100:2:1), alongside appropriate standards. Using this solvent
system cholesterol-3-sulphate and cholesterol had Rf values of 0.27
and 0.74, respectively. From each sample lane, the area
corresponding to cholesterol was scraped from the TLC plate and the
silica finings placed in a glass vial. To each vial 250 .mu.l
Chloroform:methanol (2:1) was added and the vial gently agitated.
Scintillation fluid (10 ml) was added, the samples mixed and the
radioactivity measured by scintillation counting.
[0143] The results showed that steroid sulphatase activity was
present in the hair fibre extracts, located primarily in the
insoluble protein fraction. A radioactive region corresponding to
cholesterol was not observed in control samples of pre-boiled hair
fibre extract or substrate only respectively. Cholesterol
production was linear for 24 hours, and a concomitant linear
decrease in cholesterol-3-sulphate was observed.
[0144] Using the same assay protocol as described above, steroid
sulphatase activity was also observed in samples of finely ground
hair which had not been subjected to an extraction procedure.
Example 2
[0145] Determination of the Presence of Lipase in Hair Fibres
[0146] A hair fibre extract was prepared from finely ground hair in
imidazole buffer using the extraction procedure described above in
Example 1. The extract was stored in aliquots in the presence of
20% glycerol at -20.degree. C. until required. All solutions were
syringe filtered through a nylon 0.2 .mu.m filter in order to
reduce micro-organism contamination.
[0147] Lipase activity of the hair fibre extract was measured using
the following assay:
[0148] Tri[1-.sup.14C]oleoylglycerol (triacylglycerol, 56 mCi/mmol,
100 .mu.Ci/ml) was from obtained from Amersham-Pharmacia Biotech.
Lipase activity was measured by following the formation of
[1-.sup.14C]oleic acid, mono[1-.sup.14C]oleoylglycerol and
di[1-.sup.14C]oleoylglycerol by TLC. A stock solution (5.times.) of
tri[1-.sup.14C]oleoylglycerol in 250 mM Imidazole-HCl (pH 7)
containing 0.25% Triton X-100 was prepared and 20 .mu.l of this
added to 80 .mu.l of hair enzyme extract. The reactions were
incubated at 37.degree. C. for 3.5 hours. The final assay mixture
(100 .mu.l) contained 25 .mu.M tri[1-.sup.14C ]oleoylglycerol (250
000DPM), 0.05% Triton X-100, and 50 mM imidazole-HCl (pH 7.0). The
reaction was stopped and the lipid phase extracted as in the
steroid sulphatase assay described above for Example 1. The lower
chloroform layer was dried and resuspended into 20 .mu.l
chloroform:methanol (2:1) and loaded onto a preparative silica TLC
plate. The lipids were developed with petroleum ether:diethyl
ether:acetic acid (90:10:1) to the end of the plate (17 cm). The
TLC plate was removed from the developing chamber and dried. The
plate was exposed to photographic film in a cassette for 3 days.
The film was removed from the cassette and the image developed.
[0149] The results showed that lipase activity was present in the
hair fibre extracts, with several new reaction products observed.
The reaction products were identified as dioleoylglycerol,
monooleoylglycerol and free oleic acid. Consequently the hair
enzyme had the ability to hydrolyse the triacylglycerol into
glycerol and free fatty acids. The lipid profile in control samples
containing pre-boiled extract was similar to that of control
samples containing substrate only. Therefore the possibility of
non-enzymatic artefact in the assays could be ruled out.
Example 3
[0150] Determination of the Presence of Protease in Hair Fibres
[0151] Hair fibres taken from approx. 5 mm from the scalps of male
and female individuals, aged 25-35, were cleaned, dried and then
ground in liquid N.sub.2, and homogenised with an Ystral
Homogeniser (high shear) using a 10 mm probe in extraction buffer
(50 mM Citrate-Phosphate pH6.0, 2 M NaCl).
[0152] Triton X-100 (2% stock) was added to the homogenate to a
final concentration of 0.1%,.and intermittently mixed by gentle
agitation and incubated on ice for 1 hour. Large hair debris was
allowed to precipitate and the solution was centrifuged at 13 000
rpm for 5 minutes to remove insoluble protein. The resulting
supernatant, containing soluble protein was dialysed (MWCO ca 14
000 kDa) in 50 mM citrate-phosphate, pH 6.0. Glycerol was added to
the dialysed extract to 20% (v/v). The extracts were then
aliquotted and stored at -20.degree. C. until required.
[0153] The protease catalysed hydrolysis of casein was determined
using the EnzCheck Protease Kit (Molecular Probes) with BODIPY
FL-casein as substrate. In this reagent, the casein is heavily
labelled with the fluorescent dye BODIPY-FL, causing autoquenching
of the fluorescence. Upon hydrolysis into peptides, the quenching
is eliminated, causing a concomitant increase in fluorescence.
Casein serves as a relatively non specific substrate to different
classes of proteases and was used in order to observe the overall
protease activity in hair extracts.
[0154] In a typical assay, sample protein (approx. 10 g) was added
to a final reaction volume of 100 1, containing: 50 mM Tris-HCl pH
7.8 and 10-20 g BODIPY-casein. The reactions were initiated with
the addition of substrate and incubated at 30.degree. C. After 30
minutes, the reactions were terminated with the addition of 1 ml
7.5% acetic acid. The fluorescence of the terminated reaction was
measured using a Spex Fluoromax fluorimeter set to 503 nm
(excitation) and 510 nm (emission). The fluorescence of each sample
was measured at these wavelengths with 1 second increments over a
period of 10 seconds.
[0155] The results showed that caseinolytic activity was present in
the hair fibre extracts.
[0156] The effect of pH on caseinolysis was examined using the
following buffered assay systems: citrate-phosphate (pH4-7),
tris-HCl (pH 7-9) and glycine-NaOH (pH 9-11). Two peaks of protease
activity were observed at pH 5.5 and pH 8.0.
[0157] The pH profile allowed the identification of two main groups
of protease activity. The peaks of protease activity at pH 5.5 and
8.0 was indicative of cysteine and serine proteases,
respectively.
[0158] With the above information, it was decided to investigate
the protease profile of the hair extracts with the use of more
specific peptide substrates (peptidyl-7-amido-4-methylcoumarin
(peptidyl-AMC) substrates). Seven peptidyl-AMC substrates were used
to study the protease types within the hair extract. These were
selected on the basis of their specificity for individual protease
species.
[0159] Stock solutions of each peptidyl-AMC substrate (Bachem) were
prepared at 10 mM in DMSO and stored in aliquots at 4.degree. C.
Each aliquot was diluted to 1 mM with MQ water, prior to use. All
assays (100 .mu.l final volume) were routinely conducted in
triplicate. For the measurement of cysteine protease activity, the
standard assay contained 50 mM citrate-phosphate buffer pH5.0, 100
.mu.M peptidyl-AMC, 5 mM cysteine and approximately 1 .mu.g protein
extract. The assays were initiated by the addition of substrate and
incubated at 30.degree. C. for 2 hours. The reactions were
terminated with the addition of 1 ml `stop` solution (10 mM sodium
monochloroacetate, 30 mM sodium acetate, 70 mM acetic acid).
[0160] The hydrolysed AMC was measured with the use of a
fluorimeter set to 370 nm(excitation) and 460 nm (emission).
Protease activity was quantified using standard concentrations of
AIMC. The fluorescence of each substrate and the hair extract were
also measured and these values subtracted from the final
fluorescence of each reaction, to give the value of fluorescence
increase due to AMC liberation.
[0161] The results showed that of the various specific peptide
substrates tested, the substrate for cathepsin L, namely
z-Phe-Arg-AMC, was found to be the most hydrolysed by the hair
extracts. Further tests showed that the activity was stimulated by
cysteine, potently inhibited by E-6464
(L-trans-epoxysuccinyl-leucylamido(4-guanido)butane), and most
active under acidic conditions at 37.degree. C., thereby confirming
the activity to be that of a cathepsin-L type protease. Cathepsin-L
is a lysosomal enzyme that is considered to have a broad
specificity, and has been shown to hydrolyse many cytosolic and
structural proteins, including collagen and elastin. The apparent
V.sub.max of cathepsin L-type activity was calculated to be 1.66
nmoles AMC produced per minute per mg hair extract (1 mg of extract
is obtained from approx. 9 g hair).
[0162] No increase in fluorescence was observed in samples that had
been boiled for 20 minutes prior to assaying.
Example 4
[0163] Determination of the Presence of Transgltaminase (TGase) in
Hair Fibres
[0164] Hair fibre clippings of 1-2 mm in length were obtained from
within 5 mm of the scalp. Aliquots of 5 mg were placed into a
conical, screw-capped microfuge tube (Jencons) and 20 .mu.l of 20
mM cystamine (a TGase inhibitor) or 20 .mu.l of water added.
Samples were then made up to 50 .mu.l with water. To this 50 .mu.l
of a 2.times.solution of assay mixture containing 200 mM Tris/HCl
pH8.5, 10 mM CaCl2, 20 mM DTT, 1% (w/v) TX-100 and 2 .mu.Ci/ml
[14C]-putrescine (118 Ci/mol stock) was added.
[0165] Samples were incubated at 37.degree. C. for 1 hr and
reactions stopped by the addition of 10% (w/v) trichloroacetic acid
(TCA). Fibres were recovered by centrifugation at 13,000 rpm for 10
sec and washed three times with 1 ml of TCA at 20 min intervals
between incubations at 95.degree. C. with shaking. The fibres were
then solubilised by the addition of 200 .mu.l of 90% (w/v) toluene
(Packard) and incubated at 95.degree. C. with shaking until
complete dissolution had occurred (usually after about 10 min).
After addition of 1 ml of ReadySafe scintillation cocktail
(Beckman) the tubes were placed into small scintillation vials and
the incorporation of radioactive putrescine was measured in a
Beckman LS 6000IC scintillation counter.
[0166] Data were corrected by subtraction of non-specific
background represented by the samples containing cystamine. The
results showed that TGase activity was present in the hair fibre
extracts.
Example 5
[0167] Determination of the Presence of Esterase in Hair Fibres
[0168] Unfixed cryosections of hair fibres were prepared and used
to detect esterase activity. The assay mixture consisted of 50 mM
Tris/HCl, pH 7.5, 100 .mu.M fluorescein diacetate (FDA). FDA is a
recognised esterase substrate (Molecular Probes Handbook) and was
made up as a 10 mM stock in dimethylsulphoxide and diluted into the
assay mixture.
[0169] 100 .mu.l of assay mixture or 100 .mu.l of 50 mM Tris/HCl,
pH7.5 was placed onto the slides and overlaid with a glass
coverslip. The slides were incubated at room temperature for 30 min
before detection of activity by fluorescence microscopy.
Photographs were taken with a digital camera with the gain set to
give no signal in the sections treated with Tris buffer alone (i.e.
the negative control). The results showed that esterase activity
was present in the hair fibre extracts.
* * * * *