Topical cosmetic and/or pharmaceutical compositions

Topical cosmetic and pharmaceutical compositions are provided for the external protection of human or animal tissues from contact with heavy metals, said compositions comprising a metal sequestering component capable of binding metal ions and a physiologically inert carrier suitable for topical administration. The said metal sequestering component comprises one or more metal binding peptide having a high proportion of cysteine residues, for example a metallothionein.

DESCRIPTION OF THE INVENTION 
This invention relates to cosmetic and/or pharmaceutical compositions for 
use in protecting human or animal tissues, especially epithelial tissues, 
from the damaging effects of contact with heavy metals. 
Many metal ions are required by organisms in trace amounts as essential 
nutrients. For example, many functions carried out by polypeptides, e.g., 
enzymatic, structural and immunological functions, require metallic 
cofactors. However other metallic ions, in particular ions of the heavy 
metals, can adversely affect these functions, especially if the ions are 
present in non-physiological quantities. Thus, overexposure to metals in 
the environment can lead to toxic effects. 
Ecological studies conducted in the industrialized countries of the world 
have shown that the amounts of these metals present in the environment are 
increasing. This has led to an increase in levels of heavy metals in the 
tissues of organisms, including man, through ingestion of contaminated 
foodstuffs and exposure to metals in, for example, the atmosphere. 
The effects of an accumulation of heavy metal ions can be extremely 
dangerous and their toxicity is thought to be due in part to disruption of 
the tertiary and quaternary structure of proteins, causing reduction in 
catalytic, (i.e., enzymatic) activity. 
The thus disrupted proteins may be antigenic and cause an immune response. 
In this regard they may be recognized by the body as "foreign polypeptide 
biotic agents" which cause an auto-immune response (many common allergies 
are caused by heavy metals, in association with use of detergents or other 
environmental factors). 
A further mechanism that has been shown to be responsible for the toxic 
effects of metals is the competitive substitution of natural physiological 
enzymic cofactors by heavy metals when at non-physiological 
concentrations. 
Thus, the control of heavy metal pollutants in the atmosphere is essential 
if metal-related diseases are to be prevented. It is therefore expedient 
to seek products that efficiently and selectively block heavy metal ions, 
rendering them incapable of entering the body and thereby preventing them 
exerting their toxic effects. The extensive contamination of the 
environment by heavy metals and their ubiquitous presence in the 
ecosystem, mean that the skin and the accessible mucous membranes form the 
largest surface area available on which heavy metals accumulate and 
subsequently are absorbed into the body. It is also known that many of the 
cutaneous allergic manifestations that have until now been attributed to 
detergents or other causes have now been shown to involve heavy metals in 
their aetiopathogenesis. 
Of special note are copper, cobalt, zinc, manganese, mercury and nickel 
which present significant health hazards in the mining industry, and lead, 
from exhaust fumes. It has been postulated that the accumulation of lead 
in developing children may exert a deleterious effect on memory and powers 
of concentration. 
It is an object of the invention to provide a topical cosmetic and/or 
pharmaceutical composition for protecting the skin of a human or animal 
from exposure to heavy metals. U.S. Pat. Nos. 4,725,670 (Grill) and 
4,883,861 (Grill) disclose the extraction from plant material of 
metallothioneins and their use in the form of pharmaceutical preparations 
for treating acute and chronic heavy metal poisoning, and metal deficiency 
phenomena. The Grill compositions are suitable for internal consumption 
and use only. The Grill patents make no mention that metallothioneins may 
be used as protective agents against the effects of heavy metal ion 
contact. 
It has now been surprisingly found that certain polypeptides having a 
capacity to sequester heavy metals can be used to manufacture cosmetic 
and/or pharmaceutical compositions which enable the aforementioned 
problems to be overcome. 
Thus according to the present invention there is provided a topical 
cosmetic and/or pharmaceutical composition for the external protection of 
human or animal tissues from the toxic effect of contact with heavy 
metals, said composition comprising a metal sequestering component capable 
of binding metal ions and a physiologically inert carrier suitable for 
topical administration, characterised in that said metal sequestering 
component comprises one or more metal binding peptide having a high 
proportion of cysteine residues. 
It is preferred that said metal-binding peptide comprises ligands capable 
of forming metal-thiolate clusters with heavy metal ions. 
Thus, it is preferred that the metal-binding peptide has between about 10 
and 50% cysteine residues, more preferably between about 25 and 45%, based 
on the total number of residues. 
Metal binding peptides having a high proportion of cysteine exist in 
nature. Examples of such naturally-occurring peptides include the 
metallothioneins. A sub-class of metallothioneins is the so-called 
phytochelatins. However many authorities now use the term 
"metallothionein" to cover both. 
In more detail, metallothioneins (MTs) are a group of proteins, first 
discovered in equine kidney in studies on cadmium accumulation by 
Margoshes and Vallee in 1957. The apoprotein (devoid of bound metal ions) 
may be capable of binding more than one type of metal. Since then, MTs 
have been found to bind cadmium, zinc, copper, mercury, silver, gold, lead 
and bismuth, amongst others. 
MTs have been isolated from the tissues of animals, plants and 
microorganisms. They are non-globular proteins, located intracellularly 
mainly in the cytoplasm and have been isolated in mammals in the greatest 
abundance from parenchymatous tissues of liver, kidney and intestines. 
The classification and nomenclature of MTs referred to herein is that 
recommended by "The Committee on the Nomenclature of Metallothionein" from 
the First International Meeting on Metallothioneins and other Low 
Molecular Weight Metal-Binding Proteins in Zurich, July 1978. 
Three classes of MT are now recognised, as follows: 
Class I 
Included in this class are all polypeptides related in primary structure to 
equine renal MT. This material is characterised by the following features: 
(i) Molecular weight 6000-7000; 
(ii) High metal content; 
(iii) Characteristic amino acid composition (high cysteine content, no 
aromatic amino acids); 
(iv) Unique distribution of cysteine residues in the amino acid sequence; 
(v) Spectroscopic features characteristic of 
metal-thiolate complexes and metal-thiolate clusters. 
Class II 
This class comprises forms displaying only slight or no evolutionary 
correspondence in terms of primary structure to mammalian forms. Examples 
are MTs from sea urchin and N. crassa. They share however the same ability 
to form metal-thiolate complexes and metal-thiolate clusters as Class I 
MTs. 
Class III 
This class comprises homologous, atypical oligo--and polypeptides of the 
general structure (Glu-Cys).sub.n X, where n=2-8 and X is an amino acid 
such as glycine of .beta.-alanine. They are often preferred to as 
phytochelatins and have been isolated from plant and fungal tissue. 
These MTs may occur as oligomeric structures composed of two or more 
chains, differing in weight from 500 to 2000 daltons, linked via 
metal-thiolate bridges and/of via disulphide bonds. 
Thus, it can be seen that the term metallothionein encompasses an extremely 
wide variety of proteins varying in structure and size, but shaping a 
common capacity to bind metal ions in complexes with cysteine side chains 
to form discrete metal-thiolate clusters. The amino acid sequences of 
typical Class I, II and III metallothioneins ape shown below in Table I: 
TABLE I 
______________________________________ 
Amino acid sequences of a Class I, II and II metallothionein. 
Organism Amino Acid Sequence 
______________________________________ 
Human (Class I) 
Ac-MDPNCSAA GDSCTCAGS 
MT-2 CKCKE CKCTSCKKS 
CCSCCPVGCA KCAQGCICKGASDK 
CSCCA 
Saccharomyces 
QNEGHECQC QCGSCKNNEQ 
cerevisiae CQKSCSCPTG 
(Class II) CNSDDKCPCG NKS 
EETKKSCCSGK 
Rauvolfia serpentia 
(Glu--Cys).sub.n Gly, n = 4-8 
______________________________________ 
Polypeptides having the functional characteristics of metallothioneins as 
described above may be used in accordance with the invention. These may be 
characterised by having specific sequences within the peptide chain such 
as Cys-Cys, Cys-X-Cys, and Cys-X-Y-Cys wherein X and Y are residues other 
than Cys. 
Thus the metal-binding peptide used in accordance with the invention may be 
a metallothionein of Class I, and/or II, and/or III as defined in 
accordance with the International Convention on nomenclature, although 
other metal-binding peptides having a high proportion of cysteine residues 
may be used. 
The compositions of the invention are all anhydrous, water-repellant oily 
preparations or water-repellant water-oil emulsions considered as barrier 
creams. The compositions of the invention are not dispersible in water and 
therefore if administered via an oral route would not have any activity. 
Preferably, the composition according to the invention is substantially 
free of heavy metal ions, in order that the metal sequestering facility is 
not impaired. Thus, preferably the metallothionein(s) present is/are 
apometallothionein(s). 
It is preferred that the compositions of the invention are formulated in 
the form of gels, creams, ointments, or body lotions which are not 
suitable for oral consumption or use. Additionally, it is envisaged that 
the compositions of the invention are formulated to be applied to exposed 
parts of the body in the form of a barrier cream or cosmetic make-up 
foundation which may preferably comprise a dermatologically inert 
colouring agent and/or perfume. 
It is most preferred that the compositions are formulated in the form of a 
film-forming, water-resistant mixture which may comprise for example oils, 
waxes, emulsions of an oil and emulsions of a wax silicone oils or other 
similar inert hydrophobic carrier materials. It is of course desirable 
that such materials do not interact with sulfhydryl groups of the 
metal-binding peptide. The compositions ape preferably water resistant and 
preferably are capable of remaining on the skin throughout the normal 
activities of the day, whilst being capable of being removed simply by 
washing with a detergent such as soap. 
The compositions of the invention preferably contain between about 0.01 and 
percent by weight of said metal-binding peptide, and most preferably 
between about 0.1 and 5 percent by weight of said metal-binding peptide. 
Examples of excipients for compositions of the invention are excipients 
selected from: 
Cyclomethicone 
Stearal alkonium hectorite (Bentone 27, NL Chemical) 
Hydrogenated castor oil 
Hydroxyoctacosanyl hydroxystearate (Elfacos C.sub.26, Atzo Chemical) 
Isostearylstearate 
Cetyl palmitate 
Peg 45, dodecylglycol copolymer (Elfacos TS9, Atzo Chemie) 
Isopropylmyristate 
Glyceryl mono-dipalmitostearate (Geleol, Gattefosse) 
Cetyl dimethicone copolyol (Abil EM 90, Th. Goldschmidt) 
Stearyl dimethicone (Abil Wax 9800) 
Caprylic/Capric triglycerides (Migliol 810, Dynamit-Nobel) 
Polyglyceryl-4-stearate (Witconiol 18F, Witco Organic) 
As a rule, all these excipients are not used for formulations intended for 
oral use. 
In a further embodiment of the present invention, there is provided a mask 
for the protection of human tissues from the hazardous effects of 
environmental, particularly airborne heavy metal ion contamination, said 
mask comprising a filter medium carrying an adsorbent material comprising 
a metal sequestering component capable of binding metal ions, 
characterised in that said metal sequestering agent comprises one or more 
metal-binding peptide having a high proportion of cysteine residues. 
Metal-binding peptides can, according to the invention, also be employed 
in disposable cartridges fop masks for the protection of human epithelial 
tissues from the hazardous effects of airborne heavy metal ion contact. 
The metal-binding peptide used according to the invention may be capable of 
binding many of the heavy metal ions which may be present in the 
environment. However it is envisaged that it will be most important to 
protect against lead, cadmium, chromium, mercury, copper and nickel. 
Metal-binding peptides useful in producing the compositions, methods, masks 
and disposable cartridges of the invention are of widespread occurrence 
and can be obtained from plants, and/or animals and/or microorganisms. 
Although metal-binding peptides may be obtained from any of these sources 
for use in the invention, those obtained from plants or microorganisms are 
preferred. 
For example EP-A 0,242,799 discloses a method for extracting 
metallothioneins from plant material and metallothioneins produced 
according to this method may advantageously be used in producing 
compositions according to the invention. 
It will be appreciated that the use of a product of vegetable or microbial 
origin belonging to the metallothionein group therefore provides an 
effective "molecular" method of intercepting heavy metals. Using the 
method of the invention it is possible to bind the metals with a thiolic 
bond to a polypeptide structure and the bound metals are then prevented 
from diffusing into the body. This prevents the metals from exerting their 
toxic effects (e.g. enzymatic inhibition and immunological reactions). 
The plants most suitable for use in producing metallothioneins are those, 
for example, belonging to the Cruciferae and Caryophyllaceae families. 
Metallothionein can have the ability specifically to sequester selected 
metals and it is especially preferred, in accordance with the invention, 
to utilise metallothioneins which are adapted to sequester toxic metals, 
in preference to metals which serve a useful purpose in metabolism, such 
as copper or iron. 
Thus it is especially preferred to use a metallothionein which has a 
relatively high capability to sequester chromium, nickel, lead and/or 
cadmium, but a relatively low ability to sequester copper or iron. It is 
possible to prepare metallothioneins that are adapted to sequester 
predetermined cation species by cultivating plants such as Eruca sativa, 
Brassica napus, etc. under glass or in open fields in areas of low 
contamination by the metals. Growth may then continue in the presence of 
appropriate salts of the selected heavy metal acting as inducer. Once the 
metallothioneins formed in response to the applied metal have been 
isolated, they may be treated to remove the heavy metal, thereby freeing 
the active centres to resequester the same metal when they come into 
contact with it again. 
When incorporated in appropriate pharmaceutical or cosmetic products, these 
metallothioneins will block the specific metal, for example one involved 
in the aetiopathogenesis of a specific metal-associated toxic 
manifestation. 
The extraction of metallothioneins from vegetable material is described in 
EP-A-0 242 799 and U.S. Pat. No. 5,116,749 and these metallothioneins can 
be used according to the invention. Useful metallothioneins can be 
obtained from vegetable matter, yeasts or microorganisms that contain them 
by means of extraction of vegetable or microbial material homogenates with 
water. 
The homogenates may then be centrifuged to separate the cell residues, and 
the supernatant, after partial dilution with C.sub.1-3 aliphatic alcohols 
or with C.sub.3-6 aliphatic ketones, may be filtered to eliminate 
undesired glycoprotein products of high molecular weight and then 
concentrated to a reduced volume. 
The crude metallothioneins (often still containing bound metal) portion may 
be separated from the aqueous extract after being rendered insoluble by 
adding C.sub.1-3 aliphatic alcohols or C.sub.3-6 aliphatic ketones. The 
precipitate can be resolubilized in water and reprecipitated in the same 
solvents at a predetermined ratio to water of between 40 and 90% so as to 
enable products with a low molecular weight such as simple sugars, amino 
acids and inorganic salts to be eliminated completely. The 
metallothioneins, whose molecular weight can vary between 500 and 8,000 
depending on their origin, can then be purified from the precipitate by 
conventional methods known in the literature For example using Sephadex 
G-50 columns or by means of ultrafiltration with a suitable cut-off 
membrane. 
After being isolated, the metallothioneins prior to their intended cosmetic 
or therapeutic use, may be treated with acid resins in an atmosphere of 
inert gases and in the presence of antioxidants to remove the cation. 
In this form they may then be incorporated into preparations for 
application to the skin in formulations such as aqueous gels, cleansing 
milks, or simple emulsions. It will be understood that it is preferred 
that only excipients and surfactants that do not interfere with the 
sulfhydryl groups of the polypeptide may be used. 
As an example of cosmetic treatment in accordance with the invention, the 
formulations can be applied to the exposed parts of the body such as the 
face, neck, legs, etc. This is preferably carried out in the mornings when 
one is going into areas with a high urban traffic density or staying in 
areas of high pollution. 
At the end of the day, normal washing will remove from the skin the residue 
of the formulation that has retained the heavy metals over the course of 
the day, preventing them from being absorbed through the skin. 
Suitable formulations can be applied to the hands or other parts of the 
body after prolonged use of detergents in order to cleanse the lipid layer 
and at the same time pick up any traces of contaminating metals.

The following examples illustrate suitable formulations for the 
compositions of the invention: 
EXAMPLE I 
Formulation of a gel containing Pb, Cr, Cd and Ni sequestering 
metallothioneins. 
The formulation has the following percentage composition: 
______________________________________ 
% 
______________________________________ 
Metallothioneins 1 
Beeswax 10 
Cyclosilicone pentamer 
53 
Vaseline 30 
Stearal alkonium hectorite 
2 
Hydrogenated castor oil 
2 
Pyrogenic silica 1 
Perfume 1 
______________________________________ 
EXAMPLE II 
body milk A/0 
The formulation has the following percentage composition: 
______________________________________ 
% 
______________________________________ 
Metallothioneins 1 
Cetyl dimethicone copolyol 
5 
Tetraglyceryl stearate hexyl laurate 
3 
Stearyl dimethicone 6 
Isopropyl myristate 6 
Mineral oil 4 
Triglycerides C8-10 3 
Glycerine 5 
Vaseline 3 
NaCl 2 
Perfume 0.5 
Water 61.5 
______________________________________ 
EXAMPLE III 
______________________________________ 
% 
______________________________________ 
Metallothioneins 1 
Cetyl palmitate 10 
Cyclomethicone 53 
Isostearylstearate 30 
Stearal alkonium hectorite 
2 
Hydrogenated castor oil 
2 
Pyrogenic silica 1 
Perfume 1 
______________________________________ 
EXAMPLE IV 
______________________________________ 
% 
______________________________________ 
Metallothioneins 0.5 
Hydroxyoctacosanyl hydroxystearate 
10 
Peg 45, dodecylglycol copolimer 
10 
Cyclomethicone 30 
Isopropylmyristate 40 
Glyceryl mono-,dipalmitostearate 
7 
Perfume 0.5 
Colloidal silicon dioxide 
2 
______________________________________ 
EXAMPLE V 
______________________________________ 
% 
______________________________________ 
Metallothioneins 1 
Cetyl dimethicone copolyol 
5 
Polyglyceryl-4-stearate 
3 
Stearyl dimethicone 6 
Isopropyl myristate 6 
Mineral oil 4 
Caprilic/Capric triglycerides 
3 
Glycerine 5 
Isostearylstearate 3 
Sodium chloride 2 
Perfume 0.5 
Water 61.5 
______________________________________ 
EXAMPLE VI 
Materials and Methods: Ten healthy female volunteers of ages ranging 
between 23 and 31 (average 25.+-.3.7) were selected for the investigation. 
In the initial conditions an examination was made of the microcirculation 
of the skin of the cheeks, the biomicroscopoic observation being repeated 
30 days after daily application (cf. annexed Report). A placebo (product 
A) was applied to the left cheek, a metallothionein-containing composition 
according to the invention (product B) being applied to the right cheek. 
The products applied had the following percentual composition and were 
prepared at the Indena Laboratories: 
______________________________________ 
Placebo (Product A) g 
______________________________________ 
Polytrimethylsiloxysilicate/dimethicone 
3 
Mineral oil 0.5 
Isopropyl lanolate 3 
Stearic acid 1 
Cetyl alcohol 1 
Tea extract 1.2 
Perfume 0.2 
Water 90.1 
Total 100 
______________________________________ 
______________________________________ 
Genuine (Product B) g 
______________________________________ 
Metallothioneins (Pb - Cd specific) 
0.5 
Polytrimethylsiloxysilicate/dimethicone 
3 
Mineral oil 0.5 
Isopropyl lanolate 3 
Stearic acid 1 
Tea extract 1.2 
Perfume 0.2 
Water 89.6 
Total 100 
______________________________________ 
A quantity of approximately 1.5 g of these two formulations was separately 
applied, with a gentle massage, to half the face of the experimental 
subjects; application was performed in the morning. The treatment period 
selected was the winter period, since at that time there is very heavy 
pollution in city centres. The determinations of the heavy metals were 
carried out on the second day from the start of the experiment, using the 
following procedure. 
The treated part was washed with a cotton wad impregnated with a 0.5% 
aqueous solution of citric acid, using approximately 5ml of solution to 
wash half the face. The acid solution was concentrated to 1 ml, the heavy 
metals being determined from said solution by atomic absorption. 
Instrument: Automatic sequential spectrometer ARL 3410 ICP (inductively 
coupled plasma). 
Procedure: Reference solutions of the selected metals were prepared 
containing between 0.01 and 0.05 ppm, whereafter said solutions were 
aspirated to obtain the lead and cadmium calibration lines at the 
wavelengths specific to the two elements, i.e.: 
Pb=220,353 nm 
Cd=226,502 nm. 
Then the solution of the sample being examined was analyzed: the instrument 
automatically supplied the concentration of the two elements in ppm. No 
treatment of the sample was requested. 
Limits of detectability: lead 0.0025 ppm cadmium 0.003 ppm. 
Results: 
The following Table shows the results of the investigation of the 
volunteers: 
______________________________________ 
Lead content (ppm) in the washing liquids of the skin of 
the half face treated with placebo (A) or genuine (B) 
Case No. Placebo Genuine 
______________________________________ 
1 31.7 74.8 
2 34.1 90.6 
3 28.7 82.9 
4 31.6 53.2 
5 22.9 65.6 
6 32.4 32.8 
7 27.9 62.3 
8 21.4 75.6 
9 37.6 83.4 
10 26.4 55.9 
Mean 29.47 67.1 
______________________________________ 
______________________________________ 
Cadmium content (ppm) in the washing liquids of the 
skin of the half face treated with placebo (A) or genuine (B) 
Case No. Placebo Genuine 
______________________________________ 
1 11.7 44.8 
2 14.1 50.6 
3 18.7 22.9 
4 11.6 33.2 
5 22.9 25.6 
6 12.4 32.8 
7 17.9 42.3 
8 11.4 25.6 
9 17.6 33.4 
10 16.4 25.9 
Mean 15.47 33.62 
______________________________________ 
The contents of the two metals were higher in the solution obtained from 
the cheek treated with the product containing metallothionein, in 
comparison with the placebo, since the metallothionein retained the metal 
on the surface and inhibited its absorption. 
EXAMPLE VII 
We set up an experiment with the purpose of documenting the protective 
activities of the compositions of the invention on the skin 
microcirulation of women exposed to city traffic during the winter period. 
MATERIALS AND METHODS 
For the investigation 10 healthy female subjects were selected whose ages 
ranged between 23 and 31 (average age 25.+-.3.7). 
In the starting conditions an examination was made of the microcirculation 
in the skin of the cheeks, the biomicroscopic observation being repeated 
thirty days following daily application. A placebo (product A) was applied 
to the left cheek, a metallothionein-containing composition of the 
invention (product B) being applied to the right cheek. The instrument 
used was a hand-held video microscope imaging system (Scope Moritex MS509, 
Heisi, Japan), Formed by a telecamera, a computer, an optical probe and a 
monitor. Contact objectives D800.times.and D400.times.were selected. Prior 
to examination the skin was washed with luke warm water and dried with a 
cotton pad, whereafter a drop of microscopic immersion oil was deposited 
over an area and spread delicately over an area of approximately 2.times.2 
cm. The object of this procedure was to make the horny layer more 
transparent. 
OBSERVATIONS 
The following Table shows the data obtained. 
______________________________________ 
Capillary density of the skin of the cheeks in the initial conditions 
and after the administrations of products A and B for 30 days 
After (%) 
Case No. Before (%) A B 
______________________________________ 
1 30.7 29.6 34.8 
2 24.2 25.4 30.6 
3 28.6 30.2 32.9 
4 32.8 31.6 33.2 
5 19.3 21.2 25.6 
6 32.0 28.6 32.8 
7 28.9 28.4 32.3 
8 17.4 20.2 25.6 
9 33.6 31.6 33.4 
10 16.4 20.2 25.9 
Mean 26 .+-. 2.0 26.7 .+-. 1.4 
30.71 .+-. 1.1 
______________________________________ 
From an examination of the Table it can be deduced that no statistically 
significant differences in comparison with the initial conditions were 
found in the cheeks treated with the placebo (A) after 30 days of 
application. In contrast, a statistically significant difference (p&lt;0.001) 
was found in the cheeks treated with the composition of the invention (B). 
CONCLUSIONS 
The results clearly indicate that the protection by compositions of the 
invention of skin areas exposed to atmospheric pollution contaminated with 
heavy metals allows a statistically significant improvement in the blood 
irrigation, expressed in the increase in capillary density.