Patent Application
20100278882
The present invention relates to the use of protein microbeads in cosmetics, in particular in skin cosmetics.
EP 1110534 describes a cosmetic product of ultrafine crystalline silk powder which is produced by treating silk under alkaline conditions at a temperature above 100° C. and subsequent mechanical comminution.
The present invention relates, in a first embodiment, to the use of protein microbeads in cosmetics.
Protein microbeads consist of polypeptides which are constructed from amino acids, in particular from the 20 naturally occurring amino acids. The amino acids can also be modified, for example acetylated, glycosylated, farnesylated.
In addition, the protein microbeads have a globular structure with an average particle diameter of from 0.1 to 100 μm, in particular from 0.5 to 20 μm, preferably from 1 to 5 μm and particularly preferably from 2 to 4 μm.
Protein microbeads can preferably be produced by the method described below:
The protein is dissolved in a first solvent. Solvents which can be used are, for example, aqueous salt solutions. In particular, highly concentrated salt solutions with a concentration greater than 2 molar, in particular greater than 4 molar and particularly preferably greater than 5 molar, whose ions have more marked chaotropic properties than sodium ions and chloride ions are suitable. One example of such a salt solution is 6 M guanidinium thiocyanate or 9 M lithium bromide. Furthermore, organic solvents can be used for dissolving the proteins. In particular, fluorinated alcohols or cyclic hydrocarbons are suitable. Examples thereof are hexafluoroisopropanol and cyclohexane. The protein microbeads can be produced in the described solvents. Alternatively, this solvent can be replaced by a further solvent, e.g. low-concentration salt solutions (c<0.5 M) through dialysis or dilution. The end concentration of the dissolved protein should be between 0.1-100 mg/ml. The temperature at which the method is carried out is usually 0-80° C., preferably 5-50° C. and particularly preferably 10-40° C.
When using aqueous solutions, these may also be admixed with a buffer, preferably in the pH range 4-10, particularly preferably 5 to 9, very particularly preferably 6 to 8.5. By adding an additive, phase separation is induced. This produces a protein-rich phase emulsified in the mixture as solvent and additive. On account of surface effects, emulsified protein-rich droplets adopt a round shape. Through the choice of solvent, of additive and of protein concentration, the average diameter of the protein microbeads can be adjusted to values between 0.1 μm and 100 μm.
As additive, all substances can be used which, on the one hand, are miscible with the first solvent and, on the other hand, induce the formation of a protein-rich phase. If the microbead formation is carried out in organic solvents, then organic substances suitable for this purpose are those which have a lower polarity than the solvent, e.g. toluene. In aqueous solutions, salts may be used as additive whose ions have more marked cosmotropic properties than sodium ions and chloride ions (e.g. ammonium sulfate; potassium phosphate). The end concentration of the additive should be between 1% and 50% by weight, based on the protein solution, depending on the type of additive.
The protein-rich droplets are fixed by curing, with retention of the round shape. The fixing is based here on the formation of strong intermolecular interactions. The type of interactions may be noncovalent, e.g. through the formation of intermolecular β-pleated sheet crystals, or covalent, e.g. through chemical crosslinking. The curing can take place through the additive and/or through the addition of a further suitable substance. The curing takes place at temperatures between 0 and 80° C., preferably between 5 and 60° C.
This further substance may be a chemical crosslinker. A chemical crosslinker is understood here as meaning a molecule in which at least two chemically reactive groups are joined together via a linker. Examples thereof are sulfhydryl-reactive groups (e.g. maleimides, pydridyldisulfides, α-haloacetyls, vinylsulfones, sulfatoalkylsulfones (preferably sulfatoethylsulfones)), amine-reactive groups (e.g. succinimidyl esters, carbodiimides, hydroxymethylphosphine, imido esters, PFP esters, aldehydes, isothiocyanates etc.), carboxy-reactive groups (e.g. amines etc.), hydroxyl-reactive groups (e.g. isocyanates etc.), unselective groups (e.g. aryl azides etc.) and photoactivatable groups (e.g. perfluorophenyl azide etc.). These reactive groups can form covalent linkages with amine, thiol, carboxyl or hydroxyl groups present in proteins.
The stabilized microbeads are washed with a suitable further solvent, e.g. water, and then dried by methods known to the person skilled in the art, e.g. by lyophilization or spray-drying. The success of bead formation is checked with the help of scanning electron microscopy.
Suitable proteins for producing protein microbeads are proteins which are present in predominantly intrinsically unfolded form in aqueous solution. This state can be calculated, for example, by an algorithm on which the program IUpred is based (http://iupred.enzim.hu/index.html; The Pairwise Energy Content Estimated from Amino Acid Composition Discriminates between Folded and Intrinsically Unstructured Proteins; Zsuzsanna Dosztányi, Veronika Csizmók, Péter Tompa and István Simon; J. Mol. Biol. (2005) 347, 827-839). A predominantly intrinsically unfolded state is assumed if a value of >0.5 is calculated for more than 50% of the amino acid radicals according to this algorithm (prediction type: long disorder).
Suitable proteins for producing protein microbeads are silk proteins. These are understood below as meaning those proteins which comprise highly repetitive amino acid sequences and are stored in the animal in a liquid form and during their secretion fibers are produced by shearing or spinning. (Craig, C. L. (1997) Evolution of arthropod silks. Annu. Rev. Entomol. 42: 231-67).
Particularly suitable proteins for producing protein microbeads are spider silk proteins which can be isolated in their original form from spiders.
Very particularly suitable proteins are silk proteins which could be isolated from the “Major Ampullate” gland of spiders.
Preferred silk proteins are ADF3 and ADF4 from the “Major Ampullate” gland of Araneus diadematus (Guerette et al., Science 272, 5258:112-5 (1996)).
Likewise suitable proteins for producing protein microbeads are natural or synthetic proteins which are derived from natural silk proteins and which have been produced using genetic engineering methods heterologously in prokaryotic or eukaryotic expression systems. Nonlimiting examples of prokaryotic expression organisms are Escherichia coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum etc. Nonlimiting examples of eukaryotic expression organisms are yeasts, such as Saccharomyces cerevisiae, Pichia pastoris etc., filamentous fungi, such as Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, Acremonium chrysogenum etc., mammal cells, such as Hela cells, COS cells, CHO cells etc., insect cells, such as Sf9 cells, MEL cells etc.
Of particular preference for producing protein microbeads are synthetic proteins which are based on repeat units of natural silk proteins. Besides the synthetic repetitive silk protein sequences, these can additionally comprise one or more natural nonrepetitive silk protein sequences (Winkler and Kaplan, J Biotechnol 74:85-93 (2000)).
Among the synthetic silk proteins, for producing protein microbeads, preference is given to synthetic spider silk proteins which are based on repeat units of natural spider silk proteins. Besides the synthetic repetitive spider silk protein sequences, these can additionally comprise one or more natural nonrepetitive spider silk protein sequences.
Among the synthetic spider silk proteins, the so-called C16-protein is preferably to be specified (Huemmerich et al. Biochemistry, 43(42):13604-13612 (2004)). This protein has the polypeptide sequence shown in SEQ ID NO: 1.
Besides the polypeptide sequence shown in SEQ ID NO:1, functional equivalents, functional derivatives and salts of this sequence in particular are also preferred.
According to the invention, “functional equivalents” are understood in particular as meaning also mutants which have an amino acid other than that specifically given in at least one sequence position of the abovementioned amino acid sequences but nevertheless have one of the abovementioned biological properties. “Functional equivalents” thus comprise the mutants obtainable by one or more amino acid additions, substitutions, deletions and/or inversions, where the specified changes can arise in any sequence position provided they lead to a mutant with the profile of properties according to the invention. Functional equivalence is in particular also present if the reactivity patterns between mutant and unchanged polypeptide are in qualitative agreement. “Functional equivalents” in the above sense are also “precursors” of the described polypeptides, and “functional derivatives” and “salts” of the polypeptides.
Here, “precursors” are natural or synthetic precursors of the polypeptides with or without the desired biological activity.
Examples of suitable amino acid substitutions are given in the table below:
The expression “salts” is understood as meaning either salts of carboxyl groups or acid addition salts of amino groups of the protein molecules according to the invention. Salts of carboxyl groups can be produced in a manner known per se and comprise inorganic salts, such as, for example, sodium, calcium, ammonium, iron and zinc salts, and also salts with organic bases, such as, for example amines, such as triethanolamine, arginine, lysine, piperidine and the like. Acid addition salts, such as, for example, salts with mineral acids, such as hydrochloric acid or sulfuric acid and salts with organic acids, such as acetic acid and oxalic acid, are likewise provided by the invention.
“Functional derivatives” of polypeptides according to the invention can likewise be produced on functional amino acid side groups or on their N- or C-terminal end using known techniques. Such derivatives comprise, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups, prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups, prepared by reaction with acyl groups.
The invention further provides protein microbeads which consist of couplings of a protein (i) and an effector molecule (ii). All of the proteins already specified above are suitable as protein (i). The protein (i) can either itself already be present as protein microbead and then be coupled with an effector molecule (ii), or else the protein (i) is not in the form of a protein microbead and is coupled with the effector molecule (ii) and only then is the coupled molecule converted to a protein microbead or the coupling takes place during phase separation.
Effector molecules (ii) are understood below as meaning molecules which have, a certain predictable effect. These may either be protein-like molecules, such as enzymes, or non-proteinogenic molecules, such as dyes, photoprotective agents, vitamins, provitamins, antioxidants and fatty acids, conditioners or compounds comprising metal ions.
Among the protein-like effector molecules, enzymes and antibodies are preferred. Among the enzymes, the following are preferred as effector molecules (ii): oxidases, peroxidases, proteases, glucanases, mutanase, tyrosinases, laccases, metal-binding enzymes, lactoperoxidase, lysozyme, amyloglycosidase, glucose oxidase, super oxide dismutase, photolyase, T4 endonuclease, katalase, thioredoxin, thioredoxin reductase. For the protein-like, but not enzymatic effector molecules, the following are preferred as effector molecules (ii): antimicrobial peptides, hydrophobins, collagen, proteins binding carotenoid, proteins binding heavy metals, proteins binding odorants, proteins binding cellulose, proteins binding starch, proteins binding keratin.
Highly suitable protein-like effector molecules (ii) are also hydrolyzates of proteins of vegetable and animal sources, for example hydrolyzates of proteins of marine origin.
Among the non-protein-like effector molecules (ii), carotenoids are preferred. According to the invention, carotenoids are understood as meaning the following compounds and their esterified or glycosylated derivatives: β-carotene, lycopene, lutein, astaxanthin, zeaxanthin, cryptoxanthin, citranaxanthin, canthaxanthin, bixin, β-Apo-4-carotenal, β-Apo-8-carotenal, β-Apo-8-carotenoic esters, neurosporene, echinenone, adonirubin, violaxanthin, torulene, torularhodin, individually or as a mixture. Preferably used carotenoids are β-carotene, lycopene, lutein, astaxanthin, zeaxanthin, citranaxanthin and canthaxanthin.
Further preferred effector molecules (ii) are UV photoprotective filters. These are understood as meaning organic substances which are able to absorb ultraviolet rays and release the absorbed energy again in the form of long-wave radiation, e.g. heat. The organic substances may be oil-soluble or water-soluble.
Oil-soluble UV-B filters which may be used are, for example, the following substances: 3-benzylidenecamphor and derivatives thereof, e.g. 3-(4-methylbenzylidene)camphor;
4-aminobenzoic acid derivatives, preferably 2-ethylhexyl 4-(dimethylamino)benzoate, 2-octyl 4-(dimethylamino)benzoate and amyl 4-(dimethylamino)benzoate;
esters of cinnamic acid, preferably 2-ethylhexyl 4-methoxycinnamate, propyl 4-methoxycinnamate, isoamyl 4-methoxycinnamate, isopentyl 4-methoxycinnamate, 2-ethylhexyl 2-cyano-3-phenylcinnamate (octocrylene);
esters of salicylic acid, preferably 2-ethylhexyl salicylate, 4-isopropylbenzyl salicylate, homomethyl salicylate;
derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4′-methylbenzophenone, 2,2′-dihydroxy-4-methoxy-benzophenone;
esters of benzalmalonic acid, preferably di-2-ethylhexyl 4-methoxybenzmalonate;
triazine derivatives, such as, for example, 2,4,6-trianilino-(p-carbo-2′-ethyl-1′-hexyloxy)-1,3,5-triazine (octyltriazone) and dioctylbutamidotriazone (Uvasorb® HEB);
propane-1,3-diones, such as, for example, 1-(4-tert-butylphenyl)-3-(4′-methoxyphenyl)-propane-1,3-dione,
Suitable water-soluble substances are:
2-phenylbenzimidazole-5-sulfonic acid and the alkali metal, alkaline earth metal, ammonium, alkylammonium, alkanolammonium and glucammonium salts thereof;
sulfonic acid derivatives of benzophenones, preferably 2-hydroxy-4-methoxybenzo-phenone-5-sulfonic acid and its salts;
sulfonic acid derivatives of 3-benzylidenecamphor, such as, for example, 4-(2-oxo-3-bornylidenemethyl)benzenesulfonic acid and 2-methyl-5-(2-oxo-3-bornylidene)-sulfonic acid and salts thereof.
Particular preference is given to the use of esters of cinnamic acid, preferably 2-ethylhexyl 4-methoxycinnamate, isopentyl 4-methoxycinnamate, 2-ethylhexyl 2-cyano-3-phenylcinnamate (octocrylene).
Furthermore, the use of derivatives of benzophenone, in particular 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4′-methylbenzophenone, 2,2′-dihydroxy-4-methoxybenzophenone, and the use of propane-1,3-diones, such as, for example, 1-(4-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione is preferred.
Suitable typical UV-A filters are:
derivatives of benzoylmethane, such as, for example, 1-(4′-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione, 4-tert-butyl-4′-methoxydibenzoylmethane or 1-phenyl-3-(4′-isopropylphenyl)propane-1,3-dione;
aminohydroxy-substituted derivatives of benzophenones, such as, for example, N,N-diethylaminohydroxybenzoyl n-hexylbenzoate.
The UV-A and UV-B filters can of course also be used in mixtures.
Suitable UV filter substances are given in the table below.
Besides the two abovementioned groups of primary photoprotective substances, it is also possible to use secondary photoprotective agents of the antioxidant type which interrupt the photochemical reaction chain which is triggered when UV radiation penetrates into the skin. Typical examples thereof are superoxide dismutase, catalase, tocopherols (vitamin E), coenzyme Q10, ubiquinanes, quinones and ascorbic acid (vitamin C).
A further group are antiirritants which have an antiinflammatory effect on skin damaged by UV light. Such substances are, for example, bisabolol, phytol and phytantriol.
Effector molecules (ii) according to the invention are also inorganic pigments which stop UV rays. Preference is given to pigments based on metal oxides and/or other metal compounds which are insoluble or sparingly soluble in water and chosen from the group of oxides of zinc (ZnO), titanium (TiO2), iron (e.g. Fe2O3), zirconium (ZrO2), silicon (SiO2), manganese (e.g. MnO), aluminum (Al2O3), cerium (e.g. Ce2O3), mixed oxides of the corresponding metals and mixtures of such oxides.
The inorganic pigments can be present here in coated form, i.e. are treated superficially. This surface treatment can consist, for example, in providing the pigments with a thin hydrophobic layer by a method known per se, as described in DE-A-33 14 742.
Further preferred effector molecules (ii) are vitamins, in particular vitamin A and esters thereof.
For the purposes of the present invention, retinoids are understood as meaning vitamin A alcohol (retinol) and its derivatives, such as vitamin A aldehyde (retinal), vitamin A acid (retinoic acid) and vitamin A ester (e.g. retinyl acetate, retinyl propionate and retinyl palmitate). The term retinoic acid here comprises both all-trans retinoic acid and also 13-cis-retinoic acid. The terms retinol and retinal preferably comprise the all-trans compounds. A preferred retinoid used for the suspensions according to the invention is all-trans-retinol, referred to below as retinol.
Further preferred effector molecules (ii) are vitamins, provitamins and vitamin precursors from the groups A, C, E and F, in particular 3,4-didehydroretinol, β-carotene (provitamin of vitamin A), ascorbic acid (vitamin C), and the palmitic esters, glucosides or phosphates of ascorbic acid, tocopherols, in particular α-tocopherol, and its esters, e.g. the acetate, the nicotinate, the phosphate and the succinate; also vitamin F, which is understood as meaning essential fatty acids, particularly linoleic acid, linolenic acid and arachidonic acid.
The vitamins, provitamins or vitamin precursors of the vitamin B group or derivatives thereof and the derivatives of 2-furanone to be used with preference according to the invention include, inter alia:
Vitamin B1, trivial name thiamine, chemical name 3-[(4′-amino-2′-methyl-5′-pyrimidinyl)-methyl]-5-(2-hydroxyethyl)-4-methylthiazolium chloride.
Vitamin B2, trivial name riboflavin, chemical name 7,8-dimethyl-10-(1-D-ribityl)-benzo[g]pteridine-2,4(3H,10H)-dione. In free form, riboflavin occurs, for example, in whey, other riboflavin derivatives can be isolated from bacteria and yeasts. A stereoisomer of riboflavin which is likewise suitable according to the invention is lyxoflavin, which can be isolated from fish meal or liver and bears a D-arabityl radical instead of the D-ribityl radical.
Vitamin B3. The compounds nicotinic acid and nicotinamide (niacinamide) often bear this name. According to the invention, preference is given to nicotinamide.
Vitamin Bs (pantothenic acid and panthenol). Preference is given to using panthenol. Derivatives of panthenol which can be used according to the invention are, in particular, the esters and ethers of panthenol, and cationically derivatized panthenols. In a further preferred embodiment of the invention, derivatives of 2-furanone can also be used in addition to pantothenic acid or panthenol. Particularly preferred derivatives are the also commercially available substances dihydro-3-hydroxy-4,4-dimethyl-2(3H)-furanone with the trivial name pantolactone (Merck), 4-hydroxymethyl-γ-butyrolactone (Merck), 3,3-dimethyl-2-hydroxy-γ-butyrolactone (Aldrich) and 2,5-dihydro-5-methoxy-2-furanone (Merck), with all stereoisomers being expressly included.
These effector molecule compounds advantageously impart moisturizing and skin-calming properties to the protein microbeads (i) according to the invention.
Vitamin B6, which is not understood here as meaning a uniform substance, but the derivatives of 5-hydroxymethyl-2-methylpyridin-3-ol known under the trivial names pyridoxin, pyridoxamine and pyridoxal.
Vitamin B7 (biotin), also referred to as vitamin H or “skin vitamin”. Biotin is (3aS,4S,6aR)-2-oxohexahydrothienol[3,4-d]imidazole-4-valeric acid.
Panthenol, pantolactone, nicotinamide and biotin are very particularly preferred according to the invention.
According to the invention, suitable derivatives (salts, esters, sugars, nucleotides, nucleosides, peptides and lipids) of the specified compounds can be used as effector molecules. As lipophilic, oil-soluble antioxidants from this group, preference is given to tocopherol and derivatives thereof, gallic esters, flavonoids and carotenoids and also butylhydroxytoluene/anisole. Preferred water-soluble antioxidants are amino acids, e.g. tyrosine and cysteine and derivatives thereof, and tannins, in particular those of vegetable origin.
Furthermore, preference is given to so-called peroxide decomposers, i.e. compounds which are able to decompose peroxides, particularly preferably lipid peroxides. These are understood as meaning organic substances, such as, for example, pyridine-2-thiol-3-carboxylic acid, 2-methoxypyrimidinolcarboxylic acids, 2-methoxypyridinecarboxylic acids, 2-dimethylaminopyrimidinolcarboxylic acids, 2-dimethylaminopyridinecarboxylic acids.
Triterpenes, in particular triterpenoic acids, such as ursolic acid, rosmaric acid, betulinic acid, boswellic acid and bryonolic acid.
A further preferred effector molecule (ii) is lipoic acid and suitable derivatives (salts, esters, sugars, nucleotides, nucleosides, peptides and lipids).
Further preferred effector molecules (ii) are fatty acids, in particular saturated fatty acids which carry an alkyl branch, particularly preferably branched elcosanoic acids, such as 18-methyleicosanoic acid.
Further preferred effector molecules (ii) are dyes, for example food dyes, semi-permanent dyes, reactive or oxidation dyes. In the case of the oxidation dyes, it is preferred to link a component as effector molecule (ii) with the protein microbeads (i) and then to couple oxidatively with the second dye component at the site of action, i.e. following application to skin. In the case of oxidation dyes it is also preferred to carry out the coupling of the dye components before the linkage with the protein microbeads (i).
The reactive dyes can also preferably be linked as a component as effector molecule (ii) with the protein microbeads (I) and then be applied to the skin. In addition, those dyes which are linked as effector molecule (ii) with the protein microbeads (i) can be used in decorative cosmetics by application to skin.
Suitable dyes are all customary hair dyes for the molecules according to the invention. Suitable dyes are known to the person skilled in the art from cosmetics handbooks, for example Schrader, Grundlagen and Rezepturen der Kosmetika [Fundamentals and formulations of cosmetics], Hithig Verlag, Heidelberg, 1989, ISBN 3-7785-1491-1.
Particularly advantageous dyes are those specified in the list below. The Colour Index Numbers (CIN) are given in the Rowe Colour Index, 3rd edition, Society of Dyers and Colourists, Bradford, England, 1971.
Food dyes can also be highly suitable as dyes.
The effector molecules (ii) are joined to protein (i). The bond between (i) and (ii) can either be a covalent bond, or be based on ionic or van der Waals' interactions or hydrophobic interactions or hydrogen bridge bonds or adsorption.
Each type of coupling, covalent or noncovalent, of effector molecule (ii) to protein (i) forming the microbead can take place in the dissolved state before phase separation. After the coupling, the formation of microbeads described in Example 1 takes place by phase separation.
Alternatively, a coupling of the effector molecule (ii) can also take place onto the protein microbead (i) already produced by phase separation, or during the phase separation process.
Preference is given to a noncovalent coupling of the effector molecule (ii) onto protein (i) forming the microbead. This can be based either on ionic or van der Weals' interactions or hydrophobic interactions or hydrogen bridge bonds. Here, during the phase separation, as described in Ex. 4, the effector molecule is incorporated into the protein microbeads (i) or bonded to their surface.
In order to bind effector molecules (ii) non-covalently to the microbead-forming protein (i), the effector molecule (ii) and the protein (i) are dissolved in the same solvent to give a common phase. For this, both components can be brought directly into solution by a solvent or a solvent mixture. Alternatively, the effector molecule (ii) can firstly be dissolved in a solvent other than the microbead-forming protein (i) and then be mixed with the protein solution (i), again giving a common phase. The predissolution of the effector molecule (ii) is especially of advantage if the effector molecule (ii) and the microbead-forming protein (i) can not be dissolved in the same solvent, such as, for example, in the case of aqueous protein solutions (i) and hydrophobic effector molecules (ii). Examples of suitable, water-miscible solvents are alcohols, such as methanol, ethanol and isopropanol, fluorinated alcohols, such as hexafluoroisopropanol and trifluoroethanol, alkanones, such as acetone, and also sulfoxides, such as, for example, dimethyl sulfoxide or formamides such as dimethylformamide. Alternatively, the microbead-forming protein (i) can be dissolved in fluorinated alcohols, such as, for example, hexafluoroisopropanol or trifluoroethanol, and the protein solution can then be mixed with effector molecules (ii) in organic solvents. Suitable solvents which can, for example, be mixed well with hexafluoroisopropanol are, inter glia, alcohols, such as methanol, ethanol and isopropanol, alkanones, such as acetone, sulfoxides, such as, for example, dimethyl sulfoxide, formamides, such as dimethylformamide, haloalkanes, such as methylene chloride, and also further organic solvents, such as tetrahydrofuran.
The noncovalent binding of the effector molecule (ii) to the microbead-forming protein (i) takes place during the assembly of the protein (i) to microbeads, where the assembly can take place as described in Example 1 through induced phase separation into a solid protein phase and a solvent phase. Through the choice of solvent and protein concentration, the average diameter of the protein microbeads can be adjusted to values between 0.1 μm and 100 μm. After the assembly reaction, the morphology of the microbeads (i) should be determined by light and electron microscopic methods.
The binding of the effector molecule can be based on hydrophobic interactions, hydrogen bridges, ionic interaction and van der Waals' interactions or a mixture of these intermolecular forces. Here, the effector molecule can be bound to the surface of the protein microbeads (i), be incorporated into the protein microbeads (i), or be associated with the protein microbeads (i) in both ways.
The binding of the effector molecule to the protein microbeads (i) can be determined through the depletion of the assembly stock of soluble effector molecules (ii). The concentration of the effector molecules (ii) can be measured by a quantitative analysis of the effector molecule properties. For example, the binding of colored effector molecules (ii) can be analyzed, for example, by photometric methods. For this, for example, the coloring of the protein microbeads (i) or the decoloring of the assembly stock are determined by measuring the absorption of the colored effector molecule. Through these methods it is also possible to calculate the charge density of the protein microbeads (i) (effector molecules per protein) and the charge efficiency (% bonded effector molecules).
Alternatively to the noncovalent coupling, a covalent linking of the effector molecule (ii) to the protein microbeads (i) can take place as described in Example 6. This can take place, for example, via the side chains of the polypeptide sequence of the microbead-forming protein (i), in particular via amino functions or hydroxy functions or carboxylate functions or thiol functions. Preference is given to a linking via the amino functions of one or more lysine radicals, via the carboxylate functions of one or more glutamate or aspartate radicals, one or more thiol groups of cysteine radicals or via the N-terminal or C-terminal function of the microbead-forming polypeptide (i). Apart from the amino acid functions occurring in the microbead-forming polypeptide sequence (i), amino acids with suitable functions (e.g. cysteines, lysines, aspartates, glutamates) can also be attached to the sequence or be inserted into the sequence, or amino acids of the microbead-forming polypeptide sequence (i) can be substituted by such amino acid functions.
The linking of the effector molecules (ii) with the microbead-forming protein (i) can either take place directly, i.e. as a covalent linking of two chemical functions already present in (i) and (ii), for example an amino function of (i) is linked with a carboxylate function of (ii) to give the acid amide. The linking can, however, also take place via a so-called linker, i.e. an at least bifunctional molecule which enters into a bond with (i) with one function and is linked to (ii) with one or more other functions.
If the effector molecule (ii) likewise consists of a polypeptide sequence, the linking of (i) and (ii) can take place as a so-called fusion protein, i.e. a general polypeptide sequence which consists of the two part sequences (i) and (ii).
It is also possible for so-called spacer elements to be incorporated between (i) and (ii), for example polypeptide sequences which have a potential cleavage site for a protease, lipase, esterase, phosphatase, hydrolase, or oligo- or polypeptide sequences which permit simple purification of the fusion protein, for example so-called His tags, i.e. oligohistidine radicals.
In addition, the spacer elements can be composed of alkyl chains, ethylene glycol and polyethylene glycols.
Particular preference is given to linker and/or spacer elements which have a potential cleavage site for a protease, lipase, esterase, phosphatase, hydrolase, i.e. are enzymatically cleavable.
Examples of enzymatically cleavable linkers which can be used in the case of the molecules according to the invention are specified, for example, in WO 98/01406, to the entire content of which reference is hereby expressly made.
Particular preference is given to linkers and spacers which are thermocleavable, photocleavable. Corresponding chemical structures are known to the person skilled in the art and are integrated between the molecule parts (i) and (ii).
The linking in the case of a non-protein-like effector molecule with the protein microbeads (i) takes place preferably through functionalizable radicals (side groups, C or N terminus) on the microbead-forming polypeptide (i), which enter into a covalent bond with a chemical function of the effector molecule.
Preference here is given to the binding linkage via an amino, thiol or hydroxy function of the microbead-forming polypeptide (i), which can enter into a corresponding amide, thioester or ester bond, for example with a carboxyl function of the effector molecule (ii), if appropriate following activation.
A further preferred linking of the protein microbeads (i) with an effector molecule (ii) is the use of a tailored linker. Such a linker has two or more so-called anchor groups with which it can link the microbead-forming polypeptide sequence (i) and one or more effector molecules (ii). For example, an anchor group for (i) may be a thiol function by means of which the linker can enter into a disulfide bond with a cysteine radical of the microbead-forming polypeptide (i). An anchor group for (ii) can, for example, be a carboxyl function by means of which the linker can enter into an ester bond with a hydroxyl function of the effector molecule (ii).
The use of such tailored linkers permits the precise matching of the linking to the desired effector molecule. Furthermore, it is thereby possible to link a plurality of effector molecules with a microbead-forming polypeptide sequence (i) in a defined way.
The linker used is governed by the functionality to be coupled. Of suitability are, for example, molecules which couple to microbead-forming polypeptides (i) by means of sulfhydryl-reactive groups, e.g. maleimides, pydridyldisulfides, α-haloacetyls, vinylsulfones, sulfatoalkylsulfones (preferably sulfatoethylsulfones) and to effector molecules (ii) by means of
Alternatively, a direct coupling between effector molecules and the protein microbeads (i) can be carried out, for example, by means of carbodiimides, glutardialdehyde, the abovementioned or other crosslinkers known to the person skilled in the art.
The effector molecules (ii) coupled to protein microbeads (i) covalently or noncovalently may be active in their bonded form. Alternatively, the effector molecules (ii) coupled to protein microbeads (i) can, however, also be released from the protein microbeads (i) or from their surface.
The release of covalently coupled effector molecules (ii) from the protein microbeads (i) can take place through cleavage of specifically introduced cleavable spacers or coupling linkers, which may, for example, be thermocleavable, photocleavable or enzymatically cleavable, but also through proteolytic degradation (e.g. by proteases) as described in Example 5 or through dissolution of the protein microbeads (i) or through mechanical destruction of the protein microbeads (i).
The release of noncovalently coupled effector molecules (ii) from the protein microbeads (i) can take place through desorption in suitable solvents, through degradation of the microbeads (i) by proteases or through dissolution of the protein microbeads (i) or through mechanical destruction of the protein microbeads (i). Suitable solvents for the desorption are all solvents or solvent mixtures in which the effector molecule (ii) can dissolve. Solvents which can dissolve the protein microbeads (i) are, for example, fluorinated alcohols, such as trifluoroethanol and hexafluoroisopropanol and also solutions of chaotropic salts, such as, for example, urea, guanidinium hydrochloride and guanidinium thiocyanate.
Suitable proteases can be added as technical proteases to a suspension of protein microbeads (i) in a targeted way or occur naturally at the desired site of action of the effector molecules (ii), such as, for example, skin proteases or proteases released by microorganisms.
The rate and kinetics of the release of the effector molecules (ii) can be controlled through the charge density with effector molecules (ii) and the average size of the microbeads (i).
For the use according to the invention in cosmetics, the protein microbeads (i) are formulated with customary further active ingredients and auxiliaries used in cosmetics.
Preferably, the protein microbeads (i) according to the invention are used for skin cosmetics. They permit a high concentration and long action time of skincare or skin-protecting effector substances.
Suitable auxiliaries and additives for producing hair cosmetic, nail cosmetic or skin cosmetic preparations are known to the person skilled in the art and can be found in cosmetics handbooks, for example Schrader, Grundlagen and Rezepturen der Kosmetika [Fundamentals and formulations of cosmetics], Hüthig Verlag, Heidelberg, 1989, ISBN 3-7785-1491-1.
The cosmetic compositions according to the invention may be skin cosmetic, nail cosmetic, hair cosmetic, dermatological, hygiene or pharmaceutical compositions.
Preferably, the compositions according to the invention are in the form of a gel, foam, spray, ointment, cream, emulsion, suspension, lotion, milk or paste. If desired, liposomes or microspheres can also be used.
The cosmetically or pharmaceutically active compositions according to the invention can additionally comprise cosmetically and/or dermatologically active ingredients, and auxiliaries.
Preferably, the cosmetic compositions according to the invention comprise at least one protein microbead, and at least one constituent different therefrom which is chosen from cosmetically active ingredients, emulsifiers, surfactants, preservatives, perfume oils, thickeners, hair polymers, hair and skin conditioners, graft polymers, water-soluble or dispersible silicone-containing polymers, photoprotective agents, bleaches, gel formers, care agents, colorants, tinting agents, tanning agents, dyes, pigments, consistency regulators, humectants, refatting agents, collagen, protein hydrolyzates, lipids, antioxidants, antifoams, antistats, emollients and softeners. The protein microbeads (i) can also be present in the cosmetic preparations in encapsulated form.
Advantageously, the antioxidants are chosen from the group consisting of amino acids (e.g. glycine, histidine, tyrosine, tryptophan) and derivatives thereof, imidazoles (e.g. urocanic acid) and derivatives thereof, peptides such as D,L-carnosine, D-carnosine, L-carnosine and derivatives thereof (e.g. anserine), carotenoids, carotenes (e.g. β-carotene, lycopene) and derivatives thereof, chlorogenic acid and derivatives thereof, lipoic acid and derivatives thereof (e.g. dihydrolipoic acid), aurothioglucose, propyl-thiouracil and other thiols (e.g. thioredoxin, glutathione, cysteine, cystine, cystamine and the glycosyl, N-acetyl, methyl, ethyl, propyl, amyl, butyl and lauryl, palmitoyl, oleyl, γ-linoleyl, cholesteryl and glyceryl esters thereof) and salts thereof, dilauryl thin-dipropionate, distearyl thiodipropionate, thiodipropionic acid and derivatives thereof (esters, ethers, peptides, lipids, nucleotides, nucleosides and salts), and sulfoximine compounds (e.g. buthionine sulfoximines, homocysteine sulfoximines, buthionine sulfones, penta-, hexa-, heptathionine sulfoximine) in very low tolerated doses (e.g. pmol to μmol/kg), also (metal) chelating agents (e.g. α-hydroxy fatty acids, palmitic acid, phytic acid, lactoferrin), α-hydroxy acids (e.g. citric acid, lactic acid, malic acid), humic acid, bile acid, bile extracts, bilirubin, biliverdin, EDTA and derivatives thereof, unsaturated fatty acids and derivatives thereof (e.g. γ-linolenic acid, linoleic acid, oleic acid), folic acid and derivatives thereof, ubiquinone and ubiquinol and derivatives thereof, vitamin C and derivatives thereof (e.g. sodium ascorbate, ascorbyl palmitate, Mg ascorbyl phosphate, ascorbyl acetate), tocopherol and derivatives (e.g. vitamin E acetate, tocotrienol), vitamin A and derivatives (vitamin A palmitate), and coniferyl benzoate of benzoin resin, rutinic acid and derivatives thereof, α-glycosylrutin, ferulic acid, furfurylideneglucitol, carnosine, butylhydroxytoluene, butylhydroxyanisole, nordihydroguaiacic acid, nordihydroguaiaretic acid, trihydroxybutyrophenone, uric acid and derivatives thereof, mannose and derivatives thereof, zinc and derivatives thereof (e.g. ZnO, ZnSO4), selenium and derivatives thereof (e.g. selenomethionine), stilbenes and derivatives thereof (e.g. stilbene oxide, trans-stilbene oxide).
Also advantageous are so-called peroxide decomposers, i.e. compounds which are able to decompose peroxides, particularly preferably lipid peroxides. These are understood as meaning organic substances, such as, for example, pyridine-2-thiol-3-carboxylic acid, 2-methoxypyrimidinolcarboxylic acids, 2-methoxypyridinecarboxylic acids, 2-dimethylaminopyrimidinolcarboxylic acids, 2-dimethylaminopyridinecarboxylic acids.
Customary thickeners in such formulations are crosslinked polyacrylic acids and derivatives thereof, polysaccharides and derivatives thereof, such as xanthan gum, agar-agar, alginates or tyloses, cellulose derivatives, e.g. carboxymethylcellulose or hydroxycarboxymethylcellulose, fatty alcohols, monoglycerides and fatty acids, polyvinyl alcohol and polyvinylpyrrolidone. Preference is given to using nonionic thickeners.
Suitable cosmetically and/or dermocosmetically active ingredients are, for example, coloring active ingredients, skin and hair pigmentation agents, tinting agents, tanning agents, bleaches, keratin-hardening substances, antimicrobial active ingredients, photofilter active ingredients, repellent active ingredients, hyperemic substances, keratolytically and keratoplastically effective substances, antidandruff active ingredients, antiphlogistics, keratinizing substances, antioxidative active ingredients and/or active ingredients which act as free-radical scavengers, skin moisturizing or humectant substances, refatting active ingredients, antierythematous or antiallergic active ingredients, branched fatty acids, such as 18-methyleicosanoic acid, and mixtures thereof.
Artificially skin-tanning active ingredients which are suitable for tanning the skin without natural or artificial irradiation with UV rays are, for example, dihydroxyacetone, alloxan and walnut shell extract. Suitable keratin-hardening substances are usually active ingredients, as are also used in antiperspirants, such as, for example, potassium aluminum sulfate, aluminum hydroxychloride, aluminum lactate, etc.
Antimicrobial active ingredients are used to destroy microorganisms or to inhibit their growth and thus serve both as preservative and as deodorizing substance which reduces the formation or the intensity of body odor. These include, for example, customary preservatives known to the person skilled in the art, such as p-hydroxy-benzoic esters, imidazolidinylurea, formaldehyde, sorbic acid, benzoic acid, salicylic acid, etc. Such deodorizing substances are, for example, zinc ricinoleate, triclosan, undecylenic acid alkylolamides, triethyl citrate, chlorhexidine etc.
Suitable preservatives, which are listed below with their E number, are to be used advantageously according to the invention.
Also suitable according to the invention are preservatives or preservative auxiliaries customary in cosmetics dibromodicyanobutane (2-bromo-2-bromomethyl-glutarodinitrile), 3-iodo-2-propynyl butylcarbamate, 2-bromo-2-nitropropane-1,3-diol, imidazolidinylurea, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-chloroacetamide, benzalkonium chloride, benzyl alcohol, formaldehyde cleavers.
Also suitable as preservatives are phenyl hydroxyalkyl ethers, in particular the compound known under the name phenoxyethanol on account of its bactericidal and fungicidal effects on a number of microorganisms.
Other antimicrobial agents are likewise suitable for being incorporated into the preparations according to the invention. Advantageous substances are, for example, 2,4,4′-trichloro-2′-hydroxydiphenyl ether (irgasan), 1,6-di(4-chlorophenylbiguanido)-hexane (chiorhexidine), 3,4,4′-trichlorocarbanilide, quaternary ammonium compounds, oil of cloves, mint oil, thyme oil, triethyl citrate, farnesol (3,7,11-trimethyl-2,6,10-dodecatrien-1-ol), and the active ingredients or active ingredient combinations described in the patent laid-open specifications DE-37 40 186, DE-39 38 140, DE-42 04 321, DE-42 29 707, DE-43 09 372, DE-44 11 664, DE-195 41 967, DE-195 43 695, DE-195 43 696, DE-195 47 160, DE-196 02 108, DE-196 02 110, DE-196 02 111, DE-196 31 003, DE-196 31 004 and DE-196 34 019 and the patent specifications DE-42 29 737, DE-42 37 081, DE-43 24 219, DE-44 29 467, DE-44 23 410 and DE-195 16 705. Sodium hydrogencarbonate is also to be used advantageously. Antimicrobial polypeptides can also likewise be used.
Suitable photofilter active ingredients are substances which absorb UV rays in the UV-B and/or UV-A region. Suitable UV filters are, for example, 2,4,6-triaryl-1,3,5-triazines in which the aryl groups can each carry at least one substituent which is preferably chosen from hydroxy, alkoxy, specifically methoxy, alkoxycarbonyl, specifically methoxycarbonyl and ethoxycarbonyl and mixtures thereof. Also suitable are p-aminobenzoic esters, cinnamic esters, benzophenones, camphor derivatives, and pigments which stop UV rays, such as titanium dioxide, talc and zinc oxide.
Suitable UV filter substances are any UV-A and UV-B filter substances. Examples to be mentioned are:
The cosmetic and dermatological preparations according to the invention can advantageously also comprise inorganic pigments which stop UV rays and are based on metal oxides and/or other metal compounds which are insoluble or sparingly soluble in water, chosen from the group of oxides of zinc (ZnO), titanium (TiO2), iron (e.g. Fe2O3), zirconium (ZrO2), silicon (SiO2), manganese (e.g. MnO), aluminum (Al2O3), cerium (e.g. Ce2O3), mixed oxides of the corresponding metals and mixtures of such oxides.
The inorganic pigments can be present here in coated form, i.e. are treated superficially. This surface treatment can consist, for example, in providing the pigments with a thin hydrophobic layer by a method known per se, as described in DE-A-33 14 742.
Suitable repellent active ingredients are compounds which are able to repel or drive away certain animals, in particular insects, from humans. These include, for example, 2-ethyl-1,3-hexanediol, N,N-diethyl-m-toluamide etc. Suitable hyperemic substances, which stimulate the flow of blood through the skin, are e.g. essential oils, such as dwarf pine extract, lavender extract, rosemary extract, juniperberry extract, horse chestnut extract, birch leaf extract, hayflower extract, ethyl acetate, camphor, menthol, peppermint oil, rosemary extract, eucalyptus oil, etc. Suitable keratolytic and keratoplastic substances are, for example, salicylic acid, calcium thioglycolate, thioglycolic acid and its salts, sulfur, etc. Suitable antidandruff active ingredients are, for example, sulfur, sulfur polyethylene glycol sorbitan monooleate, sulfur ricinol polyethoxylate, zinc pyrithione, aluminum pyrithione, etc. Suitable antiphlogistics, which counteract skin irritations, are, for example, allantoin, bisabolol, dragosantol, camomile extract, panthenol, etc.
The cosmetic compositions according to the invention can comprise, as cosmetic and/or pharmaceutical active ingredient (also, if appropriate, as auxiliary), at least one cosmetically or pharmaceutically acceptable polymer. These include, quite generally, cationic, amphoteric and neutral polymers.
Suitable polymers are, for example, cationic polymers with the INCI name Polyquaternium, e.g. copolymers of vinylpyrrolidone/N-vinylimidazolium salts (Luviquat FC, Luviquat HM, Luviquat MS, Luviquat&commat, Care), copolymers of N-vinyl-pyrrolidone/dimethylaminoethyl methacrylate, quaternized with diethyl sulfate (Luviquat PQ 11), copolymers of N-vinylcaprolactam/N-vinylpyrrolidone/N-vinylimidazolium salts (Luviquat E Hold), cationic cellulose derivatives (Polyquaternium-4 and -10), acrylamide copolymers (Polyquaternium-7) and chitosan.
Suitable cationic (quaternized) polymers are also Merquat (polymer based on dimethyldiallylammonium chloride), Gafquat (quaternary polymers which are formed by reacting polyvinylpyrrolidone with quaternary ammonium compounds), polymer JR (hydroxyethylcellulose with cationic groups) and plant-based cationic polymers, e.g. guar polymers, such as the Jaguar grades from Rhodia.
Further suitable polymers are also neutral polymers, such as polyvinylpyrrolidones, copolymers of N-vinylpyrrolidone and vinyl acetate and/or vinyl propionate, poly-siloxanes, polyvinylcaprolactam and other copolymers with N-vinylpyrrolidone, polyethyleneimines and salts thereof, polyvinylamines and salts thereof, cellulose derivatives, polyaspartic acid salts and derivatives. These include, for example, Luviflex 0 Swing (partially hydrolyzed copolymer of polyvinyl acetate and polyethylene glycol, BASF).
Suitable polymers are also nonionic, water-soluble or water-dispersible polymers or oligomers, such as polyvinylcaprolactam, e.g. Luviskol 0 Plus (BASF), or polyvinyl-pyrrolidone and copolymers thereof, in particular with vinyl esters, such as vinyl acetate, e.g. Luviskol 0 VA 37 (BASF), polyamides, e.g. based on itaconic acid and aliphatic diamines, as are described, for example, in DE-A-43 33 238.
Suitable polymers are also amphoteric or zwitterionic polymers, such as the octylacryl-amide/methyl methacrylate/tert-butylaminoethyl methacrylate-hydroxypropyl methacrylate copolymers obtainable under the names Amphomer (National Starch), and zwitterionic polymers, as are disclosed, for example, in the German patent applications DE39 29 973, DE 21 50 557, DE28 17 369 and DE 3708 451. Acrylamido-propyltrimethylammonium chloride/acrylic acid or methacrylic acid copolymers and alkali metal and ammonium salts thereof are preferred zwitterionic polymers. Further suitable zwitterionic polymers are methacroylethylbetaine/methacrylate copolymers, which are commercially available under the name Amersette (AMERCHOL), and copolymers of hydroxyethyl methacrylate, methyl methacrylate, N,N-dimethylamino-ethyl methacrylate and acrylic acid (Jordapon (D)).
Suitable polymers are also nonionic, siloxane-containing, water-soluble or -dispersible polymers, e.g. polyether siloxanes, such as Tegopren 0 (Goldschmidt) or Besi&commat (Wacker).
The formulation base of cosmetic compositions according to the invention preferably comprises cosmetically and/or pharmaceutically acceptable auxiliaries. Pharmaceutically acceptable auxiliaries are the auxiliaries which are known for use in the field of pharmacy, food technology and related fields, in particular the auxiliaries listed in the relevant pharmacopeia (e.g. DAB Ph. Eur. BP NF), and other auxiliaries whose properties do not preclude a physiological application.
Suitable auxiliaries may be: glidants, wetting agents, emulsifying and suspending agents, preservatives, antioxidants, antiirritatives, chelating agents, emulsion stabilizers, film formers, gel formers, odor masking agents, resins, hydrocolloids, solvents, solubility promoters, neutralizing agents, permeation accelerators, pigments, quaternary ammonium compounds, refatting and superfatting agents, ointment, cream or oil base substances, silicone derivatives, stabilizers, sterilizing agents, propellants, drying agents, opacifiers, thickeners, waxes, softeners, white oil. An embodiment in this regard is based on specialist knowledge, as shown, for example, in Fiedler, H. P. Lexikon der Hilfsstoffe für Pharmazie, Kosmetik and angrenzende Gebiete [Lexicon of auxiliaries for pharmacy, cosmetics and related fields], 4th edition, Aulendorf: ECV-Editio-Kantor-Verlag, 1996.
To produce the dermocosmetic compositions according to the invention, the active ingredients can be mixed or diluted with a suitable auxiliary (excipient). Excipients may be solid, semisolid or liquid materials which can serve as vehicles, carriers or medium for the active ingredient. The admixing of further auxiliaries takes place, if desired, in the manner known to the person skilled in the art. In addition, the polymers and dispersions are suitable as auxiliaries in pharmacy, preferably as or in (a) coating composition(s) or binder(s) for solid drug forms. They can also be used in creams and as tablet coatings and tablet binders.
According to a preferred embodiment, the compositions according to the invention are a skin cleansing composition.
Preferred skin cleansing compositions are soaps of liquid to gel-like consistency, such as transparent soaps, luxury soaps, deodorant soaps, cream soaps, baby soaps, skin protection soaps, abrasive soaps and syndets, pasty soaps, soft soaps and washing pastes, exfoliating soaps, moisture wipes, liquid washing, showering and bathing preparations, such as washing lotions, shower baths and gels, foam baths, oil baths and scrub preparations, shaving foams, lotions and creams.
According to a further preferred embodiment, the compositions according to the invention are cosmetic compositions for the care and protection of the skin and hair, nailcare compositions or preparations for decorative cosmetics.
Suitable skin cosmetic compositions are, for example, face tonics, face masks, deodorants and other cosmetic lotions. Compositions for use in decorative cosmetics include, for example, concealing sticks, stage make-up, mascara and eye shadows, lipsticks, kohl pencils, eyeliners, blushers, powders and eyebrow pencils.
Furthermore, the dermatological compositions according to the invention can be used in nose strips for pore cleansing, in antiacne compositions, repellents, shaving compositions, aftershave and preshave care compositions, aftersun care compositions, hair removal compositions, hair colorants, intimate care compositions, footcare compositions, and in baby care.
The skincare compositions according to the invention are, in particular, W/O or O/W skin creams, day creams and night creams, eye creams, face creams, antiwrinkle creams, sunscreen creams, moisturizing creams, bleaching creams, self-tanning creams, vitamin creams, skin lotions, care lotions and moisturizing lotions.
Skin cosmetic and dermatological compositions based on the protein microbeads described above display advantageous effects. The protein microbeads (i) can, inter alia, contribute to the moisturization and conditioning of the skin and to an improvement in the feel of the skin. The protein microbeads (i) can also act as thickeners in the formulations. By adding the protein microbeads (i) according to the invention it is possible, in certain formulations, to achieve a considerable improvement in skin compatibility.
Skin cosmetic and dermatological compositions preferably comprise at least one protein microbead (i) in an amount of from about 0.001 to 30% by weight, preferably 0.01 to 20% by weight, very particularly preferably 0.1 to 12% by weight, based on the total weight of the composition.
Particularly photoprotective compositions based on the protein microbeads (i) have the property of increasing the residence time of the UV-absorbing ingredients compared to customary auxiliaries such as polyvinylpyrrolidone.
Depending on the field of use, the compositions according to the invention can be applied in a form suitable for skincare, such as, for example, as cream, foam, gel, stick, mousse, milk, spray (pump spray or propellant-containing spray) or lotion.
Besides the protein microbeads (i) and suitable carriers, the skin cosmetic preparations can also comprise further active ingredients and auxiliaries customary in skin cosmetics, as described above. These include, preferably, emulsifiers, preservatives, perfume oils, cosmetic active ingredients, such as phytantriol, vitamin A, E and C, retinol, bisabolol, panthenol, photoprotective agents, bleaches, colorants, tinting agents, tanning agents, collagen, enzymes, protein hydrolyzates, stabilizers, pH regulators, dyes, salts, thickeners, gel formers, consistency regulators, silicones, humectants, refatting agents and/or further customary additives.
Preferred oil and fat components of the skin cosmetic and dermatological compositions are the abovementioned mineral and synthetic oils, such as, for example, paraffins, silicone oils and aliphatic hydrocarbons having more than 8 carbon atoms, animal and vegetable oils, such as, for example, sunflower oil, coconut oil, avocado oil, olive oil, lanolin, or waxes, fatty acids, fatty acid esters, such as, for example, triglycerides of C6-C30 fatty acids, wax esters, such as, for example, jojoba oil, fatty alcohols, vaseline, hydrogenated lanolin and acetylated lanolin, and mixtures thereof.
The protein microbeads (i) according to the invention can also be mixed with conventional polymers if specific properties are to be established.
To establish certain properties, such as, for example, improving the feel to the touch, the spreading behavior, the water resistance and/or the binding of active ingredients and auxiliaries such as pigments, the skin cosmetic and dermatological preparations can additionally also comprise conditioning substances based on silicone compounds.
Suitable silicone compounds are, for example, polyalkylsiloxanes, polyarylsiloxanes, polyarylalkylsiloxanes, polyether siloxanes or silicone resins.
The cosmetic or dermocosmetic preparations are produced by customary methods known to the person skilled in the art.
Preferably, the cosmetic and dermocosmetic compositions are present in the form of emulsions, in particular as water-in-oil (W/O) or oil-in-water (O/W) emulsions.
However, it is also possible to choose other types of formulation, for example gels, oils, oleogels, multiple emulsions, for example in the form of W/O/W or O/W/O emulsions, anhydrous ointments or ointment bases, etc. Emulsifier-free formulations, such as hydrodispersions, hydrogels or a Pickering emulsion are also advantageous embodiments.
Emulsions are produced by known methods. Besides at least one protein microbead (i), the emulsions usually comprise customary constituents, such as fatty alcohols, fatty acid esters and, in particular, fatty acid triglycerides, fatty acids, lanolin and derivatives thereof, natural or synthetic oils or waxes and emulsifiers in the presence of water. The choice of additives specific to the type of emulsion and the production of suitable emulsions is described, for example, in Schrader, Grundlagen and Rezepturen der Kosmetika [Fundamentals and formulations of cosmetics], Hüthig Buch Verlag, Heidelberg, 2nd edition, 1989, third part, to which reference is hereby expressly made.
A suitable emulsion in the form of a W/O emulsion, e.g. for a skin cream etc., generally comprises an aqueous phase which is emulsified in an oil or fatty phase using a suitable emulsifier system. A polyelectrolyte complex can be used for the provision of the aqueous phase.
Preferred fatty components which may be present in the fatty phase of the emulsions are: hydrocarbon oils, such as paraffin oil, purcellin oil, perhydrosqualene and solutions of microcrystalline waxes in these oils; animal or vegetable oils, such as sweet almond oil, avocado oil, calophyllum oil, lanolin and derivatives thereof, castor oil, sesame oil, olive oil, jojoba oil, karite oil, hoplostethus oil, mineral oils whose distillation start-point under atmospheric pressure is at about 250° C. and whose distillation end-point is at 410° C., such as, for example, Vaseline oil, esters of saturated or unsaturated fatty acids, such as alkyl myristates, e.g. isopropyl myristate, butyl myristate or cetyl myristate, hexadecyl stearate, ethyl or isopropyl palmitate, octanoic or decanoic acid triglycerides and cetyl ricinoleate.
The fatty phase can also comprise silicone oils which are soluble in other oils, such as dimethylpolysiloxane, methylphenylpolysiloxane and the silicone glycol copolymer, fatty acids and fatty alcohols.
Besides the protein microbeads (i), waxes can also be used, such as, for example, carnauba wax, candelilia wax, beeswax, microcrystalline wax, ozokerite wax and Ca, Mg and Al oleates, myristates, linoleates and stearates.
In addition, an emulsion according to the invention may be in the form of an O/W emulsion. Such an emulsion usually comprises an oil phase, emulsifiers which stabilize the oil phase in the water phase, and an aqueous phase, which is usually present in thickened form. Suitable emulsifiers are preferably O/W emulsifiers, such as polyglycerol esters, sorbitan esters or partially esterified glycerides.
According to a further preferred embodiment, the compositions according to the invention are a shower gel, a shampoo formulation or a bathing preparation.
Such formulations comprise at least one protein microbead (i) and usually anionic surfactants as base surfactants and amphoteric and/or nonionic surfactants as cosurfactants. Further suitable active ingredients and/or auxiliaries are generally chosen from lipids, perfume oils, dyes, organic acids, preservatives and antioxidants, and thickeners/gel formers, skin conditioning agents and humectants.
These formulations advantageously comprise 2 to 50% by weight, preferably 5 to 40% by weight, particularly preferably 8 to 30% by weight, of surfactants, based on the total weight of the formulation.
In the washing, shower and bath preparations, all of the anionic, neutral, amphoteric or cationic surfactants customarily used in body-cleansing compositions can be used.
Suitable anionic surfactants are, for example, alkyl sulfates, alkyl ether sulfates, alkylsulfonates, alkylarylsulfonates, alkyl succinates, alkyl sulfosuccinates, N-alkoyl sarcosinates, acyl taurates, acyl isethionates, alkyl phosphates, alkyl ether phosphates, alkyl ether carboxylates, alpha-olefinsulfonates, in particular the alkali metal and alkaline earth metal salts, e.g. sodium, potassium, magnesium, calcium, and ammonium and triethanolamine salts. The alkyl ether sulfates, alkyl ether phosphates and alkyl ether carboxylates can have between 1 and 10 ethylene oxide or propylene oxide units, preferably 1 to 3 ethylene oxide units, in the molecule.
These include, for example, sodium lauryl sulfate, ammonium lauryl sulfate, sodium lauryl ether sulfate, ammonium lauryl ether sulfate, sodium lauryl sarcosinate, sodium oleyl succinate, ammonium lauryl sulfosuccinate, sodium dodecylbenzenesulfonate, triethanolamine dodecylbenzenesulfonate.
Suitable amphoteric surfactants are, for example, alkylbetaines, alkylamidopropyl-betaines, alkylsulfobetaines, alkyl glycinates, alkyl carboxyglycinates, alkyl amphoacetates or -propionates, alkyl amphodiacetates or -dipropionates.
For example, cocodimethylsulfopropylbetaine, laurylbetaine, cocamidopropylbetaine or sodium cocamphopropionate can be used.
Suitable nonionic surfactants are, for example, the reaction products of aliphatic alcohols or alkylphenols having 6 to 20 carbon atoms in the alkyl chain, which may be linear or branched, with ethylene oxide and/or propylene oxide. The amount of alkylene oxide is about 6 to 60 mol per mole of alcohol. In addition, alkylamine oxides, mono- or dialkylalkanolamides, fatty acid esters of polyethylene glycols, ethoxylated fatty acid amides, alkyl polyglycosides or sorbitan ether esters are suitable.
Furthermore, the washing, shower and bath preparations can comprise customary cationic surfactants, such as, for example, quaternary ammonium compounds, for example cetyltrimethylammonium chloride.
In addition, the shower gel/shampoo formulations can comprise thickeners, such as, for example, sodium chloride, PEG-55, propylene glycol oleate, PEG-120 methylglucose dioleate and others, and also preservatives, further active ingredients and auxiliaries and water.
According to a further preferred embodiment, the compositions according to the invention are a hair-treatment composition.
Hair-treatment compositions according to the invention preferably comprise at least one protein microbead (i) in an amount in the range from about 0.01 to 30% by weight, preferably 0.5 to 20% by weight, based on the total weight of the composition.
Preferably, the hair treatment compositions according to the invention are in the form of a setting foam, hair mousse, hair gel, shampoo, hair spray, hair foam, end fluid, neutralizer for permanent waves, hair colorant and bleach or hot-oil treatment. Depending on the field of use, the hair cosmetic preparations can be applied as (aerosol) spray, (aerosol) foam, gel, gel spray, cream, lotion or wax. Hair sprays include here both aerosol sprays and also pump sprays without propellant gas. Hair foams include both aerosol foams and also pump foams without propellant gas. Hair sprays and hair foams preferably include predominantly or exclusively water-soluble or water-dispersible components. If the compounds used in the hair sprays and hair foams according to the invention are dispersible in water, they can be applied in the form of aqueous microdispersions with particle diameters of usually 1 to 350 nm, preferably 1 to 250 nm. The solids contents of these preparations are here usually in a range from about 0.5 to 20% by weight. These microdispersions do not usually require emulsifiers or surfactants for their stabilization.
The hair cosmetic formulations according to the invention comprise, in a preferred embodiment, a) 0.01 to 30% by weight of at least one protein microbead (i), b) 20 to 99.95% by weight of water and/or alcohol, c) 0 to 50% by weight of at least one propellant gas, d) 0 to 5% by weight of at least one emulsifier, e) 0 to 3% by weight of at least one thickener, and up to 25% by weight of further constituents.
Alcohol is understood as meaning all alcohols customary in cosmetics, e.g. ethanol, isopropanol, n-propanol.
Further constituents are to be understood as meaning the additives customary in cosmetics, for example propellants, antifoams, interface-active compounds, i.e. surfactants, emulsifiers, foam formers and solubilizers. The interface-active compounds used may be anionic, cationic, amphoteric or neutral. Further customary constituents may also be, for example, preservatives, perfume oils, opacifiers, active ingredients, UV filters, care substances, such as panthenol, collagen, vitamins, protein hydrolyzates, alpha- and beta-hydroxycarboxylic acids, stabilizers, pH regulators, dyes, viscosity regulators, gel formers, salts, humectants, refatting agents, complexing agents and further customary additives.
Also included here are all styling and conditioner polymers known in cosmetics which can be used in combination with the protein microbeads (i) according to the invention if quite specific properties are to be established.
Suitable conventional hair cosmetics polymers are, for example, the above-mentioned cationic, anionic, neutral, nonionic and amphoteric polymers, to which reference is made here.
To establish certain properties, the preparations can additionally also comprise conditioning substances based on silicone compounds. Suitable silicone compounds are, for example, polyalkylsiloxanes, polyarylsiloxanes, polyarylalkylsiloxanes, polyether siloxanes, silicone resins or dimethicone copolyols (CTFA) and amino-functional silicone compounds, such as amodimethicones (CTFA).
The polymers according to the invention are particularly suitable as setting compositions in hair styling preparations, in particular hair sprays (aerosol sprays and pump sprays without propellant gas) and hair foams (as aerosol foams and pump foams without propellant gas).
In a preferred embodiment, spray preparations comprise a) 0.01 to 30% by weight of at least one protein microbead (i), b) 20 to 99.9% by weight of water and/or alcohol, c) to 70% by weight of at least one propellant, d) 0 to 20% by weight of further constituents.
Propellants are the propellants customarily used for hair sprays or aerosol foams. Preference is given to mixtures of propane/butane, pentane, dimethyl ether, 1,1-difluoroethane (HFC-152 a), carbon dioxide, nitrogen or compressed air.
A formulation preferred according to the invention for aerosol hair foams comprises a) 0.01 to 30% by weight of at least one protein microbead (i), b) 55 to 99.8% by weight of water and/or alcohol, c) 5 to 20% by weight of a propellant, d) 0.1 to 5% by weight of an emulsifier, e) 0 to 10% by weight of further constituents.
Emulsifiers which can be used are all emulsifiers customarily used in hair foams. Suitable emulsifiers may be nonionic, cationic or anionic or amphoteric.
Examples of nonionic emulsifiers (INCI nomenclature) are laureths, e.g. laureth-4; ceteths, e.g. ceteth-1, polyethylene glycol cetyl ether, ceteareths, e.g. ceteareth-25, polyglycol fatty acid glycerides, hydroxylated lecithin, lactyl esters of fatty acids, alkyl polyglycosides.
Examples of cationic emulsifiers are cetyldimethyl-2-hydroxyethylammonium dihydrogenphosphate, cetyltrimonium chloride, cetyltrimonium bromide, cocotrimonium methyl sulfate, Quaternium-1 to x (INCI).
Anionic emulsifiers can be chosen, for example, from the group of alkyl sulfates, alkyl ether sulfates, alkylsulfonates, alkylarylsulfonates, alkyl succinates, alkyl sulfosuccinates, N-alkoyl sarcosinates, acyl taurates, acyl isethionates, alkyl phosphates, alkyl ether phosphates, alkyl ether carboxylates, alpha-olefinsulfonates, in particular the alkali metal and alkaline earth metal salts, e.g. sodium, potassium, magnesium, calcium, and ammonium and triethanolamine salts. The alkyl ether sulfates, alkyl ether phosphates and alkyl ether carboxylates can have between 1 and 10 ethylene oxide or propylene oxide units, preferably 1 to 3 ethylene oxide units, in the molecule.
A preparation suitable according to the invention for styling gels can, for example, have the following composition: a) 0.01 to 30% by weight of at least one protein microbead (1), b) 80 to 99.85% by weight of water and/or alcohol, c) 0 to 3% by weight, preferably 0.05 to 2% by weight, of a gel former, d) 0 to 20% by weight of further constituents. The use of gel formers may be advantageous in order to establish specific rheological or other application-related properties of the gels. Gel formers which can be used are all gel formers customary in cosmetics. These include slightly crosslinked polyacrylic acid, for example Carbomer (INCI), cellulose derivatives, e.g. hydroxypropylcellulose, hydroxyethylcellulose, cationically modified celluloses, polysaccharides, e.g. xanthan gum, caprylic/capric triglyceride, sodium acrylate copolymers, polyquaternium-32 (and) Paraffinum Liquidum (INCI), sodium acrylate copolymers (and) paraffinum liquidum (and) PPG-1 trideceth-6, acrylamidopropyltrimonium chloride/acrylamide copolymers, steareth-10 allyl ether, acrylate copolymers, polyquaternium-37 (and) paraffinum liquidum (and) PPG-1 trideceth-6, polyquaternium 37 (and) propylene glycol dicaprate dicaprylate (and) PPG-1 trideceth-6, polyquaternium-7, polyquaternium-44.
The protein microbeads (i) according to the invention can be used as conditioners in cosmetic preparations.
A preparation comprising the protein microbeads (i) according to the invention can preferably be used in shampoo formulations as setting and/or conditioning compositions. Preferred shampoo formulations comprise a) 0.01 to 30% by weight of at least one protein microbead (i), b) 25 to 94.95% by weight of water, c) 5 to 50% by weight of surfactants, c) 0 to 5% by weight of a further conditioning agent, d) 0 to 10% by weight of further cosmetic constituents.
In the shampoo formulations, all of the anionic, neutral, amphoteric or cationic surfactants customarily used in shampoos can be used.
Suitable anionic surfactants are, for example, alkyl sulfates, alkyl ether sulfates, alkylsulfonates, alkylarylsulfonates, alkyl succinates, alkyl sulfosuccinates, N-alkoyl sarcosinates, acyl taurates, acyl isethionates, alkyl phosphates, alkyl ether phosphates, alkyl ether carboxylates, alpha-olefinsulfonates, in particular the alkali metal and alkaline earth metal salts, e.g. sodium, potassium, magnesium, calcium, and ammonium and triethanolamine salts. The alkyl ether sulfates, alkyl ether phosphates and alkyl ether carboxylates can have between 1 and 10 ethylene oxide or propylene oxide units, preferably 1 to 3 ethylene oxide units, in the molecule.
Of suitability are, for example, sodium lauryl sulfate, ammonium lauryl sulfate, sodium lauryl ether sulfate, ammonium lauryl ether sulfate, sodium lauroyl sarcosinate, sodium oleyl succinate, ammonium lauryl sulfosuccinate, sodium dodecylbenzenesulfonate, triethanolamine dodecylbenzenesulfonate.
Suitable amphoteric surfactants are, for example, alkylbetaines, alkylamidopropyl-betaines, alkylsulfobetaines, alkyl glycinates, alkyl carboxyglycinates, alkyl amphoacetates or -propionates, alkyl amphodiacetates or -dipropionates.
For example, cocodimethylsulfopropylbetaine, laurylbetaine, cocamidopropylbetaine or sodium cocamphopropionate can be used.
Suitable nonionic surfactants are, for example, the reaction products of aliphatic alcohols or alkylphenols having 6 to 20 carbon atoms in the alkyl chain, which may be linear or branched, with ethylene oxide and/or propylene oxide. The amount of alkylene oxide is about 6 to 60 mol per mole of alcohol. In addition, alkylamine oxides, mono- or dialkylalkanolamides, fatty acid esters of polyethylene glycols, alkyl polyglycosides or sorbitan ether esters are suitable.
Furthermore, the shampoo formulations can comprise customary cationic surfactants, such as, for example, quaternary ammonium compounds, for example cetyltrimethylammonium chloride.
In the shampoo formulations, in order to achieve certain effects, customary conditioning agents can be used in combination with the protein microbeads (i).
These include, for example, the abovementioned cationic polymers with the INCI name Polyquaternium, in particular copolymers of vinylpyrrolidone/N-vinylimidazolium salts (Luviquat FC, Luviquat&commat, HM, Luviquat MS, Luviquat Care), copolymers of N-vinylpyrrolidone/dimethylaminoethyl methacrylate, quaternized with diethyl sulfate (Luviquat D PQ 11), copolymers of N-vinylcaprolactam/N-vinylpyrrolidone/N-vinyl-imidazolium salts (Luviquat D Hold), cationic cellulose derivatives (Polyquaternium-4 and -10), acrylamide copolymers (Polyquaternium-7). In addition, protein hydrolyzates can be used, and also conditioning substances based on silicone compounds, for example polyalkylsiloxanes, polyarylsiloxanes, polyarylalkylsiloxanes, polyether siloxanes or silicone resins. Further suitable silicone compounds are dimethicone copolyols (CTFA) and amino-functional silicone compounds, such as amodimethicones (CTFA). In addition, cationic guar derivatives, such as Guar Hydroxypropyltrimonium Chloride (INCI) can be used.
An aqueous C16 solution is prepared. For this, lyophilized C16 is dissolved in 6 M guanidinium thiocyanate (GdmSCN) with an end concentration of 0.1-10 mg/ml. The GdmSCN is then removed by dialysis against 5 mM potassium phosphate pH 8.0.
The formation of β-pleated-sheet-rich C16-microbeads is induced at room temperature by rapidly adding 500 mM potassium phosphate pH 8.0 or 800 mM ammonium sulfate (end concentration) to the protein solution, followed by brief mixing (e.g. swirling the reaction vessel). The mean particle diameter is dependent on the protein concentration used and can be varied between 0.5 μm and 15 μm. The particles are then washed with water and lyophilized. The success of the microbead formation is checked using scanning electron microscopy.
The thermal stability of C16-microbeads was determined using the “Thermogravimetric Analyser TGA 7” apparatus from Perkin-Elmer. Here, aluminum sample pans with a volume of 40 μl were used. The flushing gas used for the balance was 4 I/h of nitrogen, and the flushing gas used for the sample space was 1.6 I/h of nitrogen or air (in each case following measurement). The sample was heated from 30 to 500° C. at a heating rate of 5 K/min. During the measurement, the sample weight and the oven temperature were recorded.
C16-Microbeads can absorb and release water from the air and thus serve as moisture-regulating substance in cosmetic applications. In order to demonstrate this, 0.4 g-0.5 g of C16-microbeads, which have been stored beforehand at −20° C., were dried for 16 h under reduced pressure. As a result of this treatment, the microbeads lost 12% of their weight (based on the dry weight). Subsequent storage at room temperature and 100% atmospheric humidity led to a slow weight increase of 25% over the course of 19 days (see
In order to show that the C16-microbeads are suitable as carriers and formulation auxiliaries for effector molecules, canthaxanthin was bound as example to C16-microbeads.
For this, 500 μl of a solution of 20 mg/ml of C16 in 5 mM potassium phosphate (pH 8.0) were mixed with 50 μl of a solution of 2 mg/ml of canthaxanthin in DMSO after the insoluble microcrystals have been removed by centrifugation. Subsequently, the formation of C16-microbeads was induced by adding 500 μl of a 1 M potassium phosphate solution (pH 8.0).
During the formation of the C16-microbeads, the canthaxanthin was removed quantitatively from the solution, as could be shown by determining the absorbance in the supernatant after centrifuging off the C16-microbeads (
In order to test how strong the bonding of the canthaxanthin to the C16-microbeads was, the charged microbeads were washed with 5 mM potassium phosphate (pH 8.0), 5 mM potassium phosphate (pH 8.0)+5% DMSO and pure DMSO. For this, the C16-microbeads were incubated with 1 ml of each of the solutions for about 1 min. Centrifugation was then carried out and the canthaxanthin content in the supernatant was measured by determining the absorbance. Whereas washing without and with only 5% DMSO was not able to bring any canthaxanthin into solution, single washing with pure DMSO resulted in the majority of the bound canthaxanthin being brought into solution (
As control, 500 μl of a solution of 5 mM potassium phosphate (pH 8.0) were mixed with 50 μl of the canthaxanthin solution in DMSO. In the absence of C16, the solution remained clear even after adding 500 μl of a 1 M potassium phosphate solution (pH 8.0). The canthaxanthin was not able, under the conditions of assembly, to form a solid phase on its own.
In order to test whether the canthaxanthin is essentially adsorbed to the surface of the C16-microbeads or whether the canthaxanthin has also been incorporated into the C16-microbeads, 500 μl of a solution of 5 mM potassium phosphate (pH 8.0) were mixed with 50 μl of the canthaxanthin solution in DMSO and then 10 mg of uncharged, white C16-microbeads were added. 500 μl of the 1 M potassium phosphate solution (pH 8.0) were then added to the suspension. After centrifuging off the C16-microbeads, the majority of the canthaxanthin used was found in the supernatant, only a small part was bound to the microbeads (
Besides washing out canthaxanthin using solvents such as, for example, DMSO (
The supernatants were removed and the C16-microbeads charged with canthaxanthin were taken up in 1 ml of a 5 mM potassium phosphate solution (pH 8.0). The canthaxanthin content in the supernatants was determined photometrically by reference to the canthaxanthin absorbance (supernatant after C16 precipitation). Whereas as in the control without C16, the total canthaxanthin remained in the supernatant, the canthaxanthin in the two batches with C16 was bound quantitatively by the C16-microbeads (
In order to degrade the microbeads, 3 U of Proteinase K (Roche) were added next to the C16-microbeads from batch (a) and incubated together with the control without protease (batch b) at 37° C. At the end of incubation, centrifugation was carried out and the supernatants were measured photometrically. Whereas in the case of the control without protease (batch b), no canthaxanthin could be measured in the supernatant, in the batch with protease (a), canthaxanthin was released quantitatively from the C16-microbeads. Overall, through the experiment it becomes clear that canthaxanthin can be released through proteolytic degradation of the microbeads.
The fact that effector molecules can be bonded covalently to C16 and then incorporated into microbeads was shown using fluorescein as an example. Firstly, an aqueous C16 solution was prepared. For this, lyophilized C16 was dissolved in 6 M guanidinium thiocyanate (GdmSCN) with an end concentration of 10 mg/ml. The GdmSCN was then removed by dialysis against 5 mM potassium phosphate pH 8.0. The carboxyl groups of the C16 were activated in order then to react with ethylenediamine to form an amide (
The stock solutions of EDC and NHS were each prepared shortly prior to use.
For the activation of the C16 carboxyl groups, the following batch was incubated for 15 min at room temperature:
The coupling of ethylenediamine to the activated C16 carboxyl groups was carried out by adding 500 mM ethylenediamine and incubating the batch for two hours at room temperature. The batch was then dialyzed against 5 mM potassium phosphate pH 8.0.
In a second step, fluorescein isothiocyanate was bound to the free amino group of the ethylenediamine coupled to C16 (
The FITC stock solution was prepared in each case shortly prior to use.
The FITC coupling was carried out according to the following protocol:
The precipitation of the soluble RTC-labeled C16 with potassium phosphate produces, as in the case of untreated C16-protein, round microbeads although, in contrast to untreated C16-microbeads, these exhibit significant fluorescence (
The efficiency of the coupling of fluorescein onto C16 was determined photometrically. For this, a 5 mg/ml suspension of the lyophilized modified C16 protein was digested proteolytically (100 mM tris(hydroxymethyl)aminomethane (Tris) pH 8.0; 0.1% sodium dodecylsulfate (SDS); 50 μg/ml of Proteinase K (Roche); incubation for 1 h at 37° C.). The concentration of the fluorescein in the digested solution was calculated using the molar absorbance coefficient ε494nm=77000 cm−1M−1. The coupling efficiency could be calculated from the calculated fluorescein concentration and the known used amount of protein.
In this way, in the C16-microbeads, an average coupling of 12.8 fluorescein molecules onto a C16 molecule was established. As control, a sample was treated according to the same protocol, replacing the EDC and NHS during the activation with water. The coupling efficiency of this sample was less than one fluorescein molecule per C16 molecule. It can be concluded from this that the binding of the fluorescein to C16 follows the mechanism postulated in
Dermocosmetic preparations according to the invention comprising the C16-microbeads prepared according to Example 1 or C16-microbead-canthaxanthin produced according to Example 4 are described below.
Preparation: Heat phases A and B separately from one another to about 80° C. Stir phase B into phase A and homogenize. Stir phase C into the combined phases A and B and homogenize again. Cool with stirring to about 40° C., add phase D, adjust the pH to about 6.5 with phase E, homogenize and cool to room temperature with stirring.
Note: The formulation is prepared without protective gas. Bottling must take place in oxygen-impermeable packagings, e.g. aluminum tubes.
Preparation: Heat phases A and B separately from one another to about 80° C. Stir phase B into phase A and homogenize. Incorporate phase C into the combined phases A and B and homogenize. Cool to about 40° C. with stirring. Add phase D, adjust the pH to about 6.5 with phase E and homogenize. Cool to room temperature with stirring.
Preparation: Dissolve phase A. Stir phase B into phase A, incorporate phase C into the combined phases A and B. Dissolve phase D, stir into the combined phases A, B and C and homogenize. After-stir for 15 min.
Preparation: Weigh in the components of phase A and dissolve to give a clear solution.
Preparation: Dissolve phase A to give a clear solution. Allow phase B to swell and neutralize with phase C. Stir phase A into the homogenized phase B and homogenize.
Preparation: Mix the components of phase A. Dissolve phase B, incorporate into phase A and homogenize.
Preparation: Mix the Components of Phase A. Stir Phase B into Phase A with homogenization. Neutralize with phase C and homogenize again.
Preparation: Heat the components of phase A and B separately from one another to about 80° C. Stir phase B into phase A and homogenize. Heat phase C to about 80° C. and stir into the combined phases A and B with homogenization. Cool to about 40° C. with stirring, add phase D and homogenize again.
Preparation: Heat phase A to about 80° C., stir in phase B and homogenize for 3 min. Heat phase C likewise to 80° C. and stir into the combined phases A and B with homogenization. Cool to about 40° C., stir in phase D and homogenize again.
Preparation: Heat phase A to about 80° C., stir in phase B and homogenize for 3 min. Likewise heat phase C to 80° C. and stir into the combined phases A and B with homogenization. Cool to about 40° C., stir in phase D and homogenize again.
Preparation: Heat the components of phases A and B separately from one another to about 80° C. Stir phase B into phase A with homogenization. Cool to about 40° C. with stirring, add phases C and D and briefly after-homogenize. Cool to room temperature with stirring.
Preparation: Heat phases A and B separately from one another to about 85° C. Stir phase B into phase A and homogenize. Cool to about 40° C. with stirring, add phase C and briefly homogenize again. Cool to room temperature with stirring.
Preparation: Weigh the components of phase A together, stir until everything has dissolved and bottle.
Preparation: Weigh the components of phase A together, stir until everything has dissolved to give a clear solution and bottle.
Preparation: Weigh the components of phase A together, stir until everything has dissolved to give a clear solution and bottle.
Preparation: Mix the components of phase A. Add the components of phase B one after the other and dissolve. Bottle with phase C.
Preparation: Mix the components of phase A. Add the components of phase B one after the other and dissolve. Bottle with phase C.
Preparation: Mix the components of phase A. Dissolve the components of phase B to give a clear solution, then stir phase B into phase A. Adjust the pH to 6-7, bottle with phase C.
Preparation: Mix the components of phase A. Add the components of phase B one after the other and dissolve. Dissolve phase C in the mixture with A and B, then adjust the pH to 6-7. Bottle with phase D.
Preparation: Mix the components of phase A. Add the components of phase B one after the other and dissolve. Dissolve phase C in the mixture with A and B, then adjust the pH to 6-7. Bottle with phase D.
Preparation: Solubilize phase A. Weigh phase B into phase A and dissolve to give a clear solution. Adjust the pH to 6-7, bottle with phase C.
Preparation: Solubilize phase A. Weigh phase B into phase A and dissolve to give a clear solution. Adjust the pH to 6-7, bottle with phase C.
Preparation: Solubilize phase A. Weigh phase B into phase A and dissolve to give a clear solution. Adjust the pH to 6-7, bottle with phase C.
Preparation: Mix the components of phase A. Add the components of phase B one after the other and dissolve. Bottle with phase C.
Preparation: Mix the components of phase A and dissolve. Adjust the pH to 6-7 with citric acid.
Preparation: Mix the components of phase A and dissolve. Adjust the pH to 6-7 with citric acid.
Preparation: Mix the components of phase A and dissolve. Adjust the pH to 6-7 with citric acid.
Preparation: Weigh in the components of phase A and dissolve. Adjust the pH to 6-7. Add phase B and heat to about 50° C. Cool to room temperature with stirring.
Simmondsia Chinensis (Jojoba) Seed Oil
Simmondsia Chinensis (Jojoba) Seed Oil
Preparation: Heat phases A and B separately to about 80° C. Briefly prehomogenize phase B, then stir phase B into phase A and homogenize again. Cool to about 40° C., add phase C and homogenize well again. Adjust the pH to 6-7 with citric acid.
Preparation: Heat phases A and B separately to about 80° C. Stir phase B into phase A and homogenize. Cool to about 40° C. with stirring, add phase C and homogenize again. Allow to cool to room temperature with stirring.
Preparation: Heat phases A and B separately to about 80° C. Stir phase B into phase A and homogenize. Cool to about 40° C. with stirring, add phases C and D and thoroughly homogenize again. Allow to cool to room temperature with stirring.
Dermocosmetic preparations according to the invention are described below, comprising the C16-microbead prepared according to Examples 1, or C16-microbead-canthaxanthin prepared according to Example 4. The following data are parts by weight of an aqueous solution.
Butyrospermum Parkii (Shea
Glycine Soja (Soybean) Oil
Butyrospermum Parkii (Shea Butter)
Glycine Soja (Soybean) Oil
Butyrospermum Parkii (Shea Butter)
Glycine Soja (Soybean) Oil
Copernicia Cerifera
Buxux Chinensis (Jojoba) Oil
Ricinus Communis (Castor) Oil
Butyrospermum Parkii
Butyrospermum Parkii
Glycine Soja (Soybean) Oil
Butyrospermum Parkii
Glycine Soja (Soybean) Oil
Butyrospermum Parkii (Shea Butter)
Glycine Soja (Soybean) Oil
Glycine Soja (Soybean) Oil
Butyrospermum Parkii (Shea Butter)
Glycine Soja (Soybean) Oil
Copernicia Cerifera
Cera Alba
Buxux Chinensis (Jojoba) Oil
Ricinus Communis (Castor) Oil
Butyrospermum Parkii
Buxus Chinensis (Jojoba) Oil
Ricinus Communis (Castor) Oil
The formulations below describe cosmetic sunscreen preparations comprising a combination of at least one inorganic pigment, preferably zinc oxide and/or titanium dioxide and organic UV-A and UV-B filters.
The formulations specified below are prepared in a customary manner known to a person skilled in the art.
The content of C16-microbead prepared according to Example 1 or C16-microbead-canthaxanthin prepared according to Example 4 refers to 100% active ingredient. The active ingredients according to the invention can be used either in pure form or in the form of an aqueous solution. In the case of the aqueous solution, the content of water dem. in the particular formulation must be adjusted.
| Number | Date | Country | Kind |
|---|---|---|---|
| 06100671.4 | Jan 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP07/50517 | 1/19/2007 | WO | 00 | 7/18/2008 |
