Cosmetic preparation with aquaporin stimulators and their use

Abstract

Use of glyceryl glycosides to stimulate aquaporin expression.

Description

[0001] The development concerns cosmetic and dermatological preparations containing aquaporin stimulators, in particular glyceryl glycosides, and their use to improve skin moisturizing.

[0002] The skin is the largest organ in the human body. Among its many functions (e.g., thermoregulation and as a sensory organ), its barrier function, which prevents the skin (and thus ultimately the entire organism) from drying out, is arguably the most important. At the same time, the skin acts as a protective barrier against the penetration and absorption of external substances. This barrier function is provided by the epidermis, which, as the outermost layer, forms the actual protective shield against the environment. At about one-tenth of the total thickness, it is also the thinnest layer of the skin.

[0003] The epidermis is a stratified tissue in which the outer layer, the horny layer (stratum corneum), represents the important part for the barrier function. It is subject to wear and tear through contact with the environment and is therefore in a constant renewal process, continuously shedding fine scales to the outside and producing new keratinized cell and lipid material from within.

[0004] Elias' skin model (PM Elias, Structure and Function of the Stratum Corneum Permeability Barrier, Drug Dev. Res. 13, 1988, 97-105), which is now recognized among experts, describes the stratum corneum as a two-component system, similar to a brick wall (brick-and-mortar model). In this model, the corneocytes correspond to the bricks, and the complex lipid membrane in the intercellular spaces corresponds to the mortar. This system essentially represents a physical barrier against hydrophilic substances, but due to its dense, multi-layered structure, lipophilic substances can also only pass through it with difficulty. The special structure of the stratum corneum protects the skin on the one hand, and stabilizes its own flexibility by binding a defined amount of water on the other.

[0005] Mechanical stresses, such as pressure, impact, or shear forces, can also be absorbed to an astonishing degree by the stratum corneum alone or in conjunction with the deeper skin layers. Larger pressure, rotation, or shear forces are transmitted to deeper skin layers via the interlocking of the epidermis with the dermis (papillary structure).

[0006] Regulating water and moisture content is one of the most important functions of the epidermal lipid membrane. However, it not only acts as a barrier against external chemical and physical influences but also contributes to the cohesion of the stratum corneum.

[0007] The lipids of the stratum corneum consist primarily of ceramides, free fatty acids, cholesterol, and cholesterol sulfate and are distributed throughout the entire stratum corneum. The composition of these lipids is crucial for the intact function of the epidermal barrier and thus for the water impermeability of the skin.

[0008] Even cleansing the skin with a simple water bath—without the addition of surfactants—initially causes the stratum corneum to swell. The degree of this swelling depends, among other things, on the duration of the bath and its temperature. At the same time, water-soluble substances are washed away or out, such as water-soluble dirt particles, but also the skin's own substances responsible for the stratum corneum's water-binding capacity. The skin's own surfactants also dissolve and wash out skin oils to a certain extent. After the initial swelling, this causes subsequent drying of the skin, which can be significantly intensified by detergent additives.

[0009] In healthy skin, these processes are generally insignificant, as the skin's protective mechanisms can easily compensate for such minor disturbances in the upper layers. However, even in the case of non-pathological deviations from the normal state, e.g., due to environmental wear and tear or irritation, light damage, aging skin, etc., the protective mechanism on the skin's surface is disrupted.

[0010] In aging skin, for example, regenerative renewal occurs more slowly, with the stratum corneum's water-binding capacity in particular declining. This makes it inflexible, dry, and cracked ("physiologically" dry skin). This results in barrier damage. The skin becomes vulnerable to negative environmental influences such as the invasion of microorganisms, toxins, and allergens. This can even lead to toxic or allergic skin reactions.

[0011] In pathologically dry and sensitive skin, barrier damage is a priori present. Epidermal intercellular lipids are produced defectively or in insufficient quantities or composition. The consequence is increased permeability of the stratum corneum and inadequate protection of the skin against loss of hygroscopic substances and water.

[0012] The skin's barrier function can be quantified by determining transepidermal water loss (TEWL). This is the evaporation of water from the body's interior, excluding water loss through perspiration. Determining the TEWL value has proven extremely informative and can be used to diagnose chapped or cracked skin, determine the compatibility of chemically different surfactants, and so on.

[0013] The water content in the top layer of the skin is of utmost importance for the beauty, well-groomedness, and natural function of the skin. It can be positively influenced to a limited extent by incorporating moisture regulators (moisturizers), such as glycerin, into cosmetic formulations.

[0014] Anionic surfactants, which are commonly found in cleansing preparations, can cause a long-lasting pH increase in the stratum corneum, severely impairing regenerative processes that serve to restore and renew the skin's barrier function. In this case, a new, often highly unfavorable equilibrium develops in the stratum corneum between regeneration and the loss of essential substances through regular extraction, which significantly impairs the skin's external appearance and the physiological functioning of the stratum corneum.

[0015] Products for the care, treatment, and cleansing of dry and damaged skin are well known. However, their contribution to the regeneration of a physiologically intact, hydrated, and smooth stratum corneum is limited in scope and duration.

[0016] The effect of ointments and creams on the barrier function and hydration of the stratum corneum is essentially based on the covering (occlusion) of the treated skin areas. The ointment or cream represents a (second) artificial barrier, so to speak, designed to prevent water loss from the skin. This physical barrier can be easily removed—for example, with cleansers—returning the skin to its original, impaired condition. Furthermore, the skincare effect can diminish with regular treatment. Furthermore, a moisturizer is usually added to cosmetic formulations. Moisturizers are skin-compatible, hygroscopic substances (e.g., glycerin, urea, or amino acids) designed to retain water evaporating from the skin. After discontinuing the product, the skin very quickly returns to its pre-treatment condition.Certain products may even temporarily worsen the condition of the skin. Therefore, a lasting product effect is generally not achieved, or only to a limited extent.

[0017] Water transport across cell membranes is a fundamental process of life that has received considerable attention over the past century. Awareness of its physiological and clinical significance increased dramatically following the discovery of a specific water channel in red blood cells by Peter Agre, who was awarded the Nobel Prize in Chemistry in 2003.

[0018] Water is crucial for the proper functioning of the skin. In addition to maintaining all transport functions and physiological functions in the living layers of the epidermis (e.g., stratum basale, s. spinosum, s. granulosum), water is also of great importance in the stratum corneum. The enzymes active there can only adequately perform their functions if the stratum corneum is sufficiently hydrated. The correct pH is a prerequisite for enzyme activity.

[0019] When applied exogenously, glycerin is an inexpensive moisturizer. Moisturizers (humectants) are not humectants in the true sense of the word, but rather substances or mixtures of substances that give cosmetic products the ability to increase the moisture content of the stratum corneum after being gently massaged into the skin. Recommended moisturizers include: arginine pyroglutamate, chondroitin sulfate, hyaluronic acid, inositol, lactic acid (sodium lactate), sodium acrylate-vinyl alcohol copolymers, sodium isostearyl-2-lactate, oligopeptides, polysiloxanes, pyroglutamic acid, 2-pyrrolidone, and uronic acids. Unlike petrolatum, which also increases moisture, moisturizers do not have an occlusive effect. The effectiveness of a moisturizer can be determined by measuring the TransEpidermal Water Loss (TEWL).

[0020] Glycerin acts as a moisturizer and also improves the hydration of the stratum corneum through its water-binding properties.

[0021] Endogenously, glycerin is not only a moisturizer but also a metabolite important for triglyceride synthesis. Glycerin also represents an energy source in cellular metabolism.

[0022] Aquaporins are a group of structurally related proteins found in plant and animal cell membranes that form channels (pores) for polar substances of low molecular weight, especially water.

[0023] Aquaporins allow the rapid exchange of large amounts of water and glycerol across the plasma membrane and intracellular membranes, e.g., in erythrocytes, epithelial cells, or growing plant cells. In contrast to uncatalyzed, purely physical diffusion through the lipid layer, aquaporin-mediated transport of water across the plasma membrane in erythrocytes is characterized by lower sensitivity to low temperatures and inhibitability by inhibitors (e.g., HgCl2). From a functional perspective, the aquaporin group includes the TIP proteins (TIP = tonoplast intrinsic protein) and PIP proteins (PIP = plasma membrane intrinsic protein) from plant cells, as well as the CHIP proteins (CHIP = channel-forming integral protein) from the plasma membrane of animal cells.Expression of cDNAs of the TIP, PIP, or CHIP genes in Xenopus oocytes (amphibian oocytes, Xenopus oocyte expression system) significantly increases water exchange through the plasma membrane of these cells—providing strong support for the water transport function of these proteins. From a genetic perspective, the TIP, PIP, and CHIP proteins belong to an evolutionarily ancient family of channel-forming membrane proteins, the MIP proteins (MIP = major intrinsic protein), and possess six membrane-spanning domains. They exist in a tetrameric form within the membrane.

[0024] In many organs, aquaporins play a key role in regulating water balance. They prevent cells from bursting, for example, due to a change in the salt concentration in the environment (osmotic regulation). Primary urine secretion and secondary urine formation in the kidneys are mediated by aquaporins. Aquaporins also play a crucial role in the secretion production of some exocrine glands (salivary glands, lacrimal glands).

[0025] DE 199 44 625 describes antiperspirant preparations containing aquaporin modulators. However, the function and effect of the aquaporin modulators are not explained. However, since these are antiperspirant preparations, i.e., preparations intended to reduce or prevent the leakage of fluid from the skin pores (sweating), it can be assumed that the modulation involves the control of water transport between cells and not the stimulation of aquaporin expression, i.e., an increase in the number of aquaporins.

[0026] Combining aquaporins from plants, bacteria, amphibians, etc., there are more than 150 isoforms. The functional classification of aquaporins has so far included two groups: a) pure water pores (aquaporins: AQP-0, 1, -2, -4, -5, -6 and -8) and b) Pores that allow the passage of small uncharged molecules such as glycerol and urea in addition to water (aquaglyceroporins: AQP-3, -7, -9 and -10).

[0027] In AQP-3-deficient mice, it was demonstrated that the glycerol content of the skin is reduced (Hara, Ma, and Verkmann in J Biol. Chem. 277, 46616-46621), leading to inadequate hydration of the stratum corneum. Furthermore, skin elasticity is reduced in these mice, and barrier repair after stratum corneum damage is slowed. The water content in the stratum corneum of AQP-3-deficient mice is reduced by a factor of three, which correlates with the reduced glycerol content (also by a factor of three). This is clear evidence that the water-binding capacity of glycerol is essential for stratum corneum hydration.

[0028] The skin is able to slowly adapt to dry environmental conditions (e.g., winter climates, air conditioning) through increased ceramide synthesis and thus counteract dehydration. However, modern living conditions (e.g., artificial indoor climates, extensive body cleansing) can dramatically limit the functionality of this natural mechanism.

[0029] It is known that the use of sea minerals (bathing in the Dead Sea) leads to an improvement in the skin's condition and that the application of cosmetic formulations containing sea minerals leads to a strengthening of the skin's lipid barrier. In vitro cell culture models have shown that osmotic stress, caused by increased salt content of the culture medium and thus increased osmolarity, causes an increase in AQP-3 expression. This increase in aquaglyceroporins suggests a protective / rebalancing response of the skin as a "countermeasure" to this "in vitro" simulated dryness and ultimately leads to better hydration of the skin from within and improved absorption of the glycerin and water offered in a cosmetic or dermatological preparation.

[0030] Dry skin, in particular, suffers from insufficient water and glycerol content in the upper epidermal layers, including the stratum corneum. Dry skin is often caused by exogenous factors such as stress conditions (UV radiation, winter climate, dry indoor climate, e.g., due to air conditioning) or by endogenous factors such as skin aging and atopy.

[0031] Important enzymes, such as those required for regulated exfoliation of the cornea, only function to the required extent when the skin is sufficiently hydrated and at a specific pH level. In this case, insufficient enzyme activity could result in flaky, itchy, and visually disturbed skin.

[0032] Water transport from the deeper layers of the skin upwards is limited. Water and glycerol transport must occur through the cell membranes, which is the responsibility of aquaporins. The number of aquaporins present in the skin's cell membranes is limited and varies depending on the skin type and skin region.

[0033] It is therefore necessary to treat certain skin areas, especially the stratum corneum, with moisturizing cosmetic and dermatological preparations. Conventional cosmetics only counteract the water loss caused by this process through occlusion and the supply of lipids to improve the stratum corneum's barrier, as well as the administration of moisturizers such as glycerin or urea. The resulting effect is therefore usually only short-term, as no deep penetration, i.e., no moisturizing of deeper skin layers, is achieved.

[0034] Currently, increasing aquaporin expression is only possible through the use of steroids. Steroids are known, such as the ecdysteroid from Ajuga Turkestanica, which induces the formation of aquaporins in cell membranes through hormonal stimulation of cellular metabolism.

[0035] However, steroids are unsuitable for cosmetic products due to their numerous side effects. Therefore, the invention uses aquaporin stimulators that are not steroids, i.e., they do not contain a cyclopentanoperhydrophenanthrene (gonane) scaffold.

[0036] The state of the art therefore lacks preparations that promote or positively influence the body's own improvement in the hydration of the stratum corneum without harmful side effects.

[0037] Based on this known state of the art, the object of the invention is to positively influence the moisture balance of the skin.

[0038] It was not foreseeable for the expert that a promotion and stimulation of aquaporin expression and thus an increase in the body's own or endogenous and exogenous supply of the skin with water and moisturizers, such as glycerol, is possible through cosmetic and dermatological preparations containing glyceryl glycosides.

[0039] This increased absorption and greater bioavailability of moisture is released from the cells over time, leading to improved hydration and physiological function of the upper epidermal layers. These improvements are characterized by, among other things: - improved homeostasis (enzyme activities, nutrient supply, waste removal), - improved elasticity (wrinkle reduction), - improved protection against infections, - improved skin feeling (reduced tension, cracking, itching) and - improved energy supply

[0040] The object of the present invention was also to provide skin care preparations which maintain or restore the barrier properties of the skin, especially when the natural hydration, especially of dry skin, is insufficient.

[0041] They should also be suitable for the prevention of damage resulting from skin dehydration, such as fissures, inflammatory or allergic processes, or even atopic dermatitis. The present invention also aims to provide stable skin-care cosmetic and / or dermatological agents that protect the skin from environmental influences such as sun and wind. In particular, the effect of the preparations should be rapid and lasting.

[0042] AQP stimulators can act in various ways. Preferred aquaporin stimulators according to the invention enhance the expression of aquaporins AQP3, AQP5, AQP7, and AQP9; significant increases are possible, particularly for AQP3, with the preparations according to the invention.

[0043] By quantifying the mRNA for AQP-3 and Western blotting, it can be demonstrated that the number of aquaporins in the epidermis increases significantly upon application of preparations containing aquaporin stimulators according to the invention.

[0044] In Western blotting, proteins from skin lysates are electrophoretically separated by molecular weight in gels and then transferred to a nitrocellulose membrane, where they are immobilized. When the proteins on the membrane are incubated in an antibody solution specific for AQP, the AQP is selectively labeled and can be qualitatively and quantitatively determined using subsequent detection and staining steps.

[0045] When quantifying the mRNA level of a protein, the copy number of the genetic material (DNA) for the protein in a cell is determined. The mRNA copies serve as a template for protein synthesis at the cellular level and, as a quantifiable quantity, are directly upstream of the finished protein.

[0046] According to the invention, aquaporin stimulators from the group of • Glyceryl glycosides, especially hexosyl glycerides and / or (hexosyl)hexosyl glycerides • cAMP analogues, • PKA (adenylic cyclase) activators and • Phosphodiesterase inhibitors, especially caffeine, theophylline.

[0047] According to the use according to the invention, the cosmetic and dermatological preparations are characterized in that the cosmetically or pharmaceutically acceptable aquaporin modulator(s) is / are present in concentrations of 0.0001 - 20.00 wt.%, preferably 0.0005 - 10.00 wt.%, particularly preferably 0.001 - 5.00 wt.%, in each case based on the total weight of the composition.

[0048] Modulators for the formation of the aquaporins AQP3 and AQP5 are particularly preferred.

[0049] Particularly advantageous according to the invention are the D- and / or L-hexosylglycerides that induce the formation of aquaporin-3 proteins. They activate the protein kinases present in the cells, particularly protein kinase A, which stimulates aquaporin expression. Experiments on cell cultures have shown that adding aquaporin stimulators according to the invention to the culture medium can lead to a tripling of the AQP-3 number (see Example 1).

[0050] Mitogen-activated kinases (Galcheva-Gorgova et al., Science 1994), which are catalyzed by glycosylglycerides, are then able to phosphorylate certain serine and threonine sites on many other intracellular proteins, thus activating them in turn. This includes some transcription factors that are necessary for the creation of mRNA copies of the genetic material. These activated transcription factors can then enter the cell nucleus and trigger the mRNA copies of the gene segment - here: AQP-3 - whereupon more aquaporin-3 is produced in protein form in the cell.

[0051] Glyceryl glycoside (glucosylglyceride) is preferred for stimulating aquaporin expression.

[0052] The hexoses underlying the hexosylglycerides used according to the invention are preferably selected from the group of aldohexoses, usually in their pyranoid form, i.e. allo(pyrano)se, altro(pyrano)se, gluco(pyrano)se, manno(pyrano)se, gulo(pyrano)se, ido(pyrano)se, galacto(pyrano)se and talo(pyrano)se.

[0053] The (hexosyl)hexoses underlying the (hexosyl)hexosylglycerides according to the invention can be selected from the group of pyranosylpyranoses and furanosylpyranoses with 1,4-glycosidic or 1,6-glycosidic bonds. They are preferably selected from the group of maltose, leucrose, and lactose.

[0054] Accordingly, the hexosylglycerides according to the invention can be characterized by the general structural formulas and the (hexosyl)hexosylglycerides according to the invention can be characterized by the general structural formulas and and respectively.

[0055] It is advantageous to use D-hexosylglycosides, but L-hexosylglycosides can also be used with high efficacy in the sense of the present invention.

[0056] Hexosylglycerides based on D- or L-ketohexoses, i.e. psicose, fructose, sorbose or tagatose, usually in their furanoid form, can also be used advantageously in the context of the present invention.

[0057] Glucosylglycerides of the general formula and / or the general formula and / or the general formula and / or the general formula are preferred according to the invention.

[0058] The particularly preferred hexosylglyceride is D-glucosylglycerol.

[0059] It is particularly advantageous if hexosylglycerides of natural origin are used.

[0060] It was not foreseeable for the person skilled in the art that the glycosylglycerides according to the invention or cosmetic or dermatological preparations containing them - act better as a moisturizing agent and - are more effective against skin aging than the active ingredients, combinations of active ingredients and preparations of the state of the art.

[0061] According to the invention, the preparations contain 0.001 to 15% by weight of glycosylglycerides, in particular 0.01 to 9.5% by weight, very particularly preferably 0.1 to 5% by weight.

[0062] According to the invention, the cosmetic preparations may contain, in addition to the aquaporin stimulators, substances that cause osmotic stress on the treated skin areas and thus achieve a further increase in hydration.

[0063] According to the invention, advantageous substances for triggering osmotic stress are: - Inorganic salts (especially alkaline earth and alkali metal salts containing a chloride, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, linear and / or cyclic oligophosphate, carbonate or hydrogen carbonate anion, especially NaCl, NaBr, NaI, Na2B4O7, Na2SiO3, Na2CO3, NaHCO3, Na3PO4, Na2HPO4, NaH2PO4, KCl, KI, LiCl, NH4Cl, ZnCl2, Al2SO3, MgCl and MgSO4) - Salts of acids naturally occurring in the skin (e.g. those involved in energy metabolism such as sodium lipoate, sodium citrate, ammonium lactate, sodium lactate, sodium bicarbonate, sodium citrate) or weak carboxylic acids (e.g. sodium propionate) - Natural mixtures of salts, especially sea minerals - Sugars with a molecular weight up to 600 g / mol, in particular sorbitol, mannitol, sucrose, glucose - Amino acids, especially glycine, alanine and / or asparagine.

[0064] It is advantageous to formulate cosmetic preparations in such a way that, in addition to a water and / or oil phase, they contain other cosmetically beneficial ingredients. Antioxidants, moisturizers, sunscreens, anti-inflammatory agents, and pigments, in particular, exhibit additional synergistic effects. Example 1: Effect of glyceryl glycosides on AQP-3 expression

[0065] Fig. shows the expression levels of aquaporin-3 mRNA in human keratinocytes relative to a constitutively (non-modulatable) expressed gene, the 18S rRNA. Glyceryl glucoside has a better stimulating effect than conventional glycerol or glucose alone, or a 1:1 mixture of glycerol and glyceryl glucoside.

[0066] For this purpose, human keratinocytes in cell culture (37°C, medium Cambrex No. CC-3158 incl. supplement kit No. CC-4152; + 0.1 mM CaCl2) were treated for 24 h in triplicates as follows: - Untreated control (corresponds to 330 mOsm, isosmolar culture medium) - Addition of 1% w / v glycerol to the culture medium (corresponds to 450mOsm, osmotic stress) - Addition of 1.5% w / v glycerol glucoside and 0.5% w / v glycerol to the culture medium (corresponds to 450 mOsm osmotic stress, mixing ratio regarding particle number 1:1) - Addition of 3% w / v glycerol glucoside to the culture medium (corresponds to 450mOsm, osmotic stress) - Addition of 2.25% w / v glucose to the culture medium (corresponds to 450mOsm, osmotic stress)

[0067] The different amounts used are due to the different molecular weights of the substances. The contribution to the increase in osmolarity in the culture medium depends solely on the number of particles added, which is the same in all experiments.

[0068] After harvesting and lysis, total RNA was isolated from the cells and the relative aquaporin-3 mRNA content was determined by quantitative RT-PCR.

[0069] The concentration w / v means mass per volume, where 1.0% w / v corresponds to one gram of substance in 100 ml of solution.

Claims

[1] Use of glyceryl glycosides to stimulate aquaporin expression. [2] Use according to claim 1 for stimulating the expression of AQP-3. [3] Use according to claim 1 for stimulating the expression of AQP-5 and / or AQP-7. [4] Use of cosmetic preparations containing one or more glyceryl glycoside(s) to improve the water and / or moisturizer transport into the skin. [5] Use according to claim 4 for improving the transport of glycerol into the skin. [6] Use according to at least one of claims 4 to 5, characterized by , which contains the cosmetic preparation of at least one substance that induces osmotic stress, which is chosen from the group: - Inorganic salts, - Salts of acids naturally occurring in the skin, - Natural mixtures of salts, - Sugars with a molecular weight up to 600 g / mol and / or - Amino acids. [7] Use according to claim 6, wherein - the inorganic salts are selected from alkaline earth and alkali metal salts which have a chloride, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, linear and / or cyclic oligophosphate, carbonate or hydrogen carbonate anion, - the salts of acids naturally occurring in the skin are selected from salts of energy metabolism, - the natural mixtures of salts are selected from marine minerals, - the sugars with a molecular weight of up to 600 g / mol are selected from glyceryl glucose, sorbitol, mannitol, sucrose and glucose, and / or - the amino acids are selected from glycine, alanine and / or asparagine. [8] Use of at least one of claims 6 to 7, wherein - the inorganic salts are selected from NaCl, NaBr, NaI, Na2B4O7, Na2SiO3, Na2CO3, NaHCO3, Na3PO4, Na2HPO4, NaH2PO4, KCl, KI, LiCl, NH4Cl, ZnCl2, Al2SO3, MgCl and MgSO4, and / or - the salts of acids naturally occurring in the skin are selected from sodium liponate, sodium citrate, ammonium lactate, sodium lactate, sodium bicarbonate, sodium citrate, weak carboxylic acids, sodium propionate. [9] Use according to at least one of claims 4 to 8 for strengthening the barrier function of the skin. [10] Use according to at least one of claims 4 to 8 for improving the water and / or moisturizer transport from the deeper layers of the skin (stratum basale, stratum spinosum or stratum granulosum) to the skin surface and / or into the stratum corneum.