Patent Grant
9820923
The invention relates generally to new succinate derivatives. Moreover, the invention relates to the use of one or more succinate derivatives in cosmetic or pharmaceutical compositions, which are preferably dermatological compositions and are preferably used topically.
Cellular ageing can be subdivided into two different processes. One is intrinsic ageing, caused by factors derived from inside the cells. The second is extrinsic ageing, caused by factors from the cells environment. The process of ageing leads to a plethora of cellular, metabolic and tissue-derived changes. Among these are—a decline in metabolic activity, a lower rate of cell division, a reduced DNA repair capacity, an accumulation of oxidized biomolecules (DNA, proteins, carbohydrates and lipids), an accumulation of misfolded proteins, cellular stiffening of vessel walls and an impaired peripheral blood circulation. All these aspects are closely interlinked with each other.
In premature skin aging one can observe changes within all the dermal layers: In the upper skin layer, the epidermis, one can usually observe a influence on the epidermal thickness. The epidermis becomes thinner with aging. This is caused by a reduced mitotic activity of keratinocytes within the stratum basale; the consequence is a reduced water-binding capacity, a lowered elasticity, a compromised barrier function and wrinkle formation. In the dermis, the amount of fibroblast decreases and their activity falls. The consequence is a reduced production of tropocollagen, hyaluronic acid and elastin that leads to a loss of elasticity, reduced sebum production and reduced perspiration. Among the most prominent processes within premature skin ageing is the chemical alteration of biomolecules. These modifications target intra- and extracellular proteins, nucleic acids or lipids and sugars. These alterations in turn, can result in diminished biological function. Examples are oxidation, carbonylation by ROS (reactive oxygen species), AGE-modifications (Advanced Glycation Endproducts) or lipidperoxidation.
UVB irradiation is a key factor during extrinsic skin aging and it alters cutaneous structure and function. Repeated exposure to UV radiation from the sun causes premature aging of skin. Under conditions of increased formation of oxidative stress as associated e.g. with UV irradiation, free radicals are formed, that cause damages to the cell contents and induced apoptosis, and aging. In particular, sunbathing and the visit of solarium amplify aging and support the formation of age spots, which result in additional premature skin ageing.
To combat wrinkles, which are visible signs of skin ageing, a lot of anti-ageing cosmetic products have been developed yet, which promote the reduction of wrinkles. Quite a series of facial products with various substances, e.g. vitamin C, vitamin E, Q10, lipoic acid, collagen and hyaluronic acid (HA) have been developed with the promise to be “wrinkles killer”.
Reactive oxygen species (ROS) are a major reason for skin aging. In lower concentrations these molecule are important signaling molecules. In elevated concentrations the effect becomes destructive (“oxidative stress”). The most prominent extrinsic ROS-inducer is the ultraviolet radiation. It leads to the formation of highly reactive hydroxyl radicals, superoxides, peroxides and other reactive species which can oxidize all kinds of biomolecules. Among these is the enzyme complex called proteasome. The proteasome functions as a proteolytic complex, degrading damaged or misfolded proteins. As the proteasome itself is a protein, the increased ROS-level lead to an accumulation of unwanted proteins, which cannot be degraded adequately anymore. This results in a vicious cycle where reactive oxygen damages the proteasome resulting in an impaired activity, resulting in more damaged proteasome. The proteasome degrades damaged, misfolded, oxidized or unnecessary proteins. It is involved in turn over regulation of proteins, cell cycle control, gene expression, (oxidative) stress, immune response and carcinogenesis. The proteasomal main function is the cell clearance of abnormal, denatured or in general damaged and unwanted proteins as well as for the regulated degradation of short-lived proteins. The efficacy of the degradation process is of enormous importance for the cellular homeostasis. The 20S proteasome is a cylindric structure, with a molecular weight of 700 kDa. It has a diameter of 12 nm and a length of 17 nm and consists of 4 rings, forming a tube like structure. The two outer rings are made of 7 different [alpha]-subunits. The other 2 rings build the middle structure of the 20S proteasome and consist of 7 diverse [beta]-subunits. The outer [alpha]-rings control the access of 20S-proteasome substrates into the inner proteolytic chamber. This proteolytic activity could be divided into three different parts: 1. Peptidyl-glutamyl-peptide-hydrolysing activity (caspase like activity); 2. Trypsin like activity and 3. Chymotrypsin like activity. The proteolytic action results in the generation of oligopeptides, generally with a length of 8-10 amino acids. In contrast to the 26S proteasome polyubiquitination is not necessary to degrade proteins. The 20S proteasome is ATP-independent.
In the skin the proteasome has an important role in ageing/photo-ageing. Following UV energy absorption there are different changes in the skin, including production of free radicals, modified proteins and 4-hydroxy-2-nonenal (HNE). These products affect the gene expression in skin cells and lead to an increase of matrix metalloproteinases (MMPs) and a decrease of their inhibitors (TIMPs) which results in a reduction of collagen and therefore in wrinkle formation. A drastic decline in proteasome activity and a simultaneous accumulation of modified and ubiquitinated proteins after UV irradiation in human keratinocytes has been reported. Today it is proven that there is a decline of proteasome activity and expression in human keratinocytes and fibroblasts during ageing. It is evident that this down regulation of proteasome activity is a result of protein aggregation. In a circuit, shrinking of proteasome activity leads to new protein aggregates and non degraded polyubiquitinated proteins, which in turn affect the proteasome again. Based on these findings, skin ageing is linked to proteasome dysfunction and preservation of “normal” proteasome function allays skin ageing. SDS and some fatty acids have been reported to stimulate proteasome activities in the test tube by favoring the open confirmation of the proteasome. Recently, isolated oleuropein, the most abundant of the phenolic compounds in Olea europaea leaf extract, olive oil and olives was shown to have stimulatory impact on proteasome activities. Furthermore, a phaeodactylum algae extract was reported to stimulate proteasome activity (WO 02/080876) as well as Palmitoyl Isoleucin. Besides the stimulation of proteasome activity by stimulation of one or more of the enzymatic activities, the upregulation of the proteasomal proteins itself is a potent method to induce the proteasomal degradation. This is of special interest regarding the fact that not only proteasomal activity declines in elderly people but also the expression of proteasomal subunits. Overexpression of proteasomal subunits in primary human embryonic fibroblasts and the accompanied increased rate of lipolysis were shown to extend the lifespan of these cells by 15-20%. Moreover, it was shown that restoration of the normal level of proteasome subunits in aged human fibroblasts reduces the level of ageing biomarkers. A decline of proteasome activities has been revealed in human primary cultures undergoing replicative senescence, whereas proteasome inhibition in young cells induces premature senescence.
Maintenance of cellular homeostasis influences the rate of ageing and is determined by several factors, including efficient proteolysis of damaged proteins. Protein degradation is predominantly catalyzed by the proteasome. Specifically, the proteasome is responsible for cell clearance of abnormal, denatured or in general damaged proteins as well as for the regulated degradation of short-lived proteins. As the proteasome has an impaired function during ageing, actives that restore its function are needed. Furthermore, UV irradiation impairs proteasomal function, thereby enhancing the cellular deposition of toxic proteins. Importantly, centenarians show a very high activity of the proteasome and are a good example of healthy ageing.
The activation of the proteasome is also linked to age-associated diseases, especially the neurodegenerative diseases in which misfolded proteins aggregate. Proteasome activators enhance the survival of Huntington's disease neuronal model cells and the development of Alzheimer's disease and Parkinson's disease is partly based on an inefficient proteasomal clearance.
The most important intrinsic ROS source is the mitochondrion. This is a cellular organelle with the function to generate ATP, the driving force for nearly every biological function in the cellular metabolism. The mitochondrial electron transport chain does not work with 100% efficiency, thus generating misdirected electrons. These electrons finally lead to the generation of superoxides and therewith oxidative stress.
The strongest intracellular radical scavenging mechanism is the glutathione system. It combines enzymes like catalase and superoxide dismutase (SOD) that can dissipate free radicals. Even small doses of UV or other ROS generating circumstances can override these protective systems. Here, the addition of extrinsic antioxidants becomes important. Among these, there are two main possibilities to supply antioxidant power to a cell. One is the supply with chemical antioxidants. Molecules, those are capable of detoxifying ROS. A second strategy is to supply substances, which are able to induce intrinsic cellular antioxidative systems like Hemoxidase 1 (HO1).
The loss of even skin tone due to an irregular distribution of collagen, haemoglobin and the formation of age spots are one of the most disturbing signs of ageing. Probably accumulated melanin and lipofuscin are responsible for the age spots, which is not clear yet. These age spots appear as invaginations of the basal membrane and a striking increase in the number of dermal papillae combined with irregular arranged melanin clusters was observed. Described for inflammatory response, the formation of age spots goes along with the upregulation of genes. The topical application of substances that bleach melanin, is the oldest approach to lighten age spots. Tyrosinase which is essential for the synthesis of new melanin, can be inhibit to lighten age spots. Reducing the amount of melanin produced, the prevention of melanocyte proliferation can be a useful strategy. In one form or another in melanin metabolism or catabolism Skin-lightening active ingredients intervene. Mostly brown to black melanind pigments are formed in the melanocytes of the skin. Then they are transferred to the keratinocytes and give the skin or hair its colour. Hydroxy-substituted aromatic amino acids such as L-tyrosine and L-DOPA, the yellow to red pheomelanins additionally from sulfur-containing molecules forms the brown-black eumelanins in mammals. For various reasons, skin-lightening agents are used: if for some reason the melanin-forming melanocytes in human skin are not evenly distributed, pigment spots occur which are either lighter or darker than the surrounding skin area. To balance out this pigment spots, skin lightening agents are sold which at least partially help. Aside from many people want to lighten their skin colour or to prevent skin pigmentation—so therefore the need is big of safe effective skin and hair lightening agents.
The therapeutic treatment of melanin-induced pigmentation disorders such as hyperpigmentations is one area of application (e.g. scar hyperpigmentations, post-traumatic drug-induced hyperpigmentations, post-inflammatory hyperpigmentations induced by phototoxic reactions, ephelides). Increasing in Skin pigmentation, caused by UV Light, can be avoided by using UV-absorbing substances. Merely inhibit the increase in skin pigmentation caused by UV light, the UV absorbers do not bring about a true lightening.
Therefore, object of the present invention was to provide compositions comprising active compounds, which have positive benefits to human skin cell, and thereby provide moisturizing and/or anti-ageing effects. In particular, the object was to provide active compounds which are capable to promote cell repair and regeneration, and thus prolong the health of cell life span of a cell in nucleus and cytoplasm in eukaryotes. The active compounds to be specified should be toxicologically safe, effective already at relatively low concentrations, well tolerated by the skin, stable (in particular in normal cosmetic and/or pharmaceutical formulations), and easy to formulate and economical to produce.
The subject of the invention relates to a compound of formula (I):
wherein R1, R′1, R2, R′2, R3, R′3, R4 R′4, R5, R′5, R6, R7 are independently of one another and denote hydrogen, hydroxyl, C1 to C6-alkyl group or R6 and R7 form together a C5-C6 ring system, and
wherein n=0, 1, 2, 3, or 4.
The term “or C1 to C6-alkyl group” represents alkyl moieties, which maybe branched or linear, and which maybe saturated or unsaturated. Preferred alkyl moieties maybe selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert.-butyl, n-pentyl, 2-methylbutyl, neopentyl, hexyl, methylpentyl, dimethylbutyl, especially preferred are methyl, ethyl and iso-propyl.
The term “R6 and R7 form together a C5-C6 ring system” means that R6 and R7 and the nitrogen atom may form together a saturated or unsaturated ring, which are preferably not further substituted, preferred is that R6 and R7 and the nitrogen atom together form a six ring system, that is preferably a saturated hexyl ring system.
In a preferred embodiment of the invention R1, R′1, R2, R′2, R3, R′3, R4, R′4, R5, R′5 of formula (I) are independently of one another and denote hydrogen, methyl or isopropyl and n=0, 1 or 2.
In a further preferred embodiment R6, R7 of formula (I) are independently of one another and denote hydrogen, methyl, ethyl, isopropyl or R6 and R7 form together with the nitrogen of formula (I) a C6-ring system, which is preferably a saturated hexyl ring system.
In another preferred embodiment of the invention R1, R′1, R2, R′2, R3, R′3, R4, R′4, R5, R′5 of formula (I) are independently of one another and denote hydrogen, methyl or iso-propyl and n=0, 1 or 2 and R6, R7 are independently of one another and denote hydrogen, methyl, ethyl, isopropyl or R6 and R7 form together with the nitrogen of formula (I) a C6-ring system, which is preferably a saturated hexyl ring system.
In a preferred embodiment of the invention the compound of formula (I) is simplified to formula (II), in the case that R′1, R′3 and R′5 denote hydrogen:
wherein R1, R2, R′2, R3, R4 R′4, R5, R6, R7 are independently of one another and denote hydrogen, hydroxyl, C1 to C6-alkyl group or R6 and R7 form together a C5-C6 ring system, and
wherein n=0, 1, 2, 3, or 4.
In a preferred embodiment of the invention R1, R2, R′2, R3, R4, R′4, R5 of formula (II) are independently of one another and denote hydrogen, methyl, or isopropyl and n=0, 1 or 2.
In a further preferred embodiment R6, R7 of formula (II) are independently of one another and denote hydrogen, methyl, ethyl, isopropyl or R6 and R7 form together with the nitrogen of formula (II) a C6-ring system, which is preferably a saturated hexyl ring system.
In another preferred embodiment of the invention R1, R2, R′2, R3, R4, R′4, R5 of formula (II) are independently of one another and denote hydrogen, methyl or isopropyl and n=0, 1 or 2 and R6, R7 are independently of one another and denote hydrogen, methyl, ethyl, isopropyl or R6 and R7 form together with the nitrogen of formula (II) a C6-ring system, which is preferably a saturated hexyl ring system.
In another preferred embodiment the compounds of formula (I) and (II) are selected from the group of following compounds:
In which all enantiomeric and diasteriomeric forms are included, such as:
In which all enantiomeric and diasteriomeric forms are included, such as:
In which all enantiomeric and diasteriomeric forms are included, such as:
In which all enantiomeric and diasteriomeric forms are included, such as:
In which all enantiomeric and diasteriomeric forms are included, such as:
and
and
and
In which all enantiomeric and diasteriomeric forms are included, such as:
In which all enantiomeric and diasteriomeric forms are included, such as:
In which all enantiomeric and diasteriomeric forms are included, such as:
Surprisingly, it was found that compounds of formula (I), respectively formula (II) have the ability to prolong health span of a cell. Especially, cellular repair mechanism in human skin can be activated, antioxidative effects can be observed and apoptosis can be reduced. It has been proved to be particularly advantageous to use a mixture of two compounds of formula (I), e.g. preferred are herewith a combination of trans-3,3,5-trimethylcyclohexyl 4-(dimethylamino)-4-oxobutanoate (S7a) and cis-3,3,5-trimethylcyclohexyl 4-(dimethylamino)-4-oxobutanoate (S7b). In case of enantiomeric mixtures such as described above every ratio between trans and cis isomer for the compounds of formula (I), respectively formula (II) is possible. Especially, preferred is a ratio of trans:cis from 1:200 to 200:1. Also preferred is the ratio from trans:cis from 1:60 to 50:1, more preferably 1:50 to 1:5, most preferably, 1:13 to 1:4. But the effects are also clearly positive when one of the enantiomeric forms is used alone.
Therefore, a preferred embodiment is N,N-Dimethyl-succinamic acid 3,3,5-trimethyl-cyclohexyl ester (S7), which is selected from the enantomeric form of
Another preferred embodiment is N,N-Dimethyl-succinamic acid 3,3,5-trimethyl-cyclohexyl ester (S7), which is selected from the enantomeric form of
Also preferred is a mixture of (1R,2S,5R)-2-isopropyl-5-methyl-cyclohexyl]4-(diisopropylamino)-4-oxo-butanoate (S1) and N,N-diethyl succinic acid amid-3,3,5-trimethylcyclohexylester (S2).
Also preferred is a mixture of N,N-dimethyl succinic acid amid-2,3,6-trimethylcyclohexylester (S3) and 2-(isopropylcyclohexyl)4-(dimethylamino)-4-oxo-butanoate (S5).
Also preferred is an enantiomeric mixture of 3,3,5-trimethylcyclohexyl)3-(dimethylamino)-3-oxo-propanoate (S9), comprises of
In case of enantiomeric mixtures such as described above every ratio between trans and cis isomers for the compounds of formula (I), respectively formula (II) is possible. Especially preferred is a ratio of trans:cis from 1:200 to 200:1. Also preferred is the ratio from trans:cis is from 1:60 to 50:1, more preferably 1:50 to 1:5, most preferably, 1:13 to 1:4.
But in particular it has been proved that the compounds of formula (I), respectively formula (II), especially the compounds S1, S2, S3, S4, S5, S6, S7, S8, S9 and S10 are alone, also in their enantiomeric form are active and benefits to the skin as described above.
In particular, the enantiomeric forms alone, e.g. S7a′ or S7a″ or S7b′ or S7b″ are advantageous, for that the single enantiomeric forms already have the ability to prolong health span of a cell, especially, cellular repair mechanism in human skin can be activated, antioxidative effects can be observed and apoptosis can be reduced.
Therefore, an object of the invention is one or more compounds of formula (I), respectively formula (II) for use in the treatment of cellular ageing and cell life span.
And a further object of the invention is one or more compounds of formula (I), respectively formula (II) for use in the treatment of free-radical activity, in the form that the renewal and differentiation of keratinocytes is regulated, the functional condition of the skin is improved of is kept in good functional condition, for skin protection or treating dry skin.
A concentration of from about 0.001 to 15%, preferred 0.1 to 2%, and most preferred 0.01 to 5.0% by weight of the compound of formula (I), respectively formula (II) in a pharmaceutical or cosmetic composition is preferred, in case of more than two compounds of formula (I), respectively formula (II), the sum of all compounds of formula (I), respectively formula (II) is meant.
It was also found that one or more compounds of formula (I), respectively formula (II) can be used for activation and/or reactivation and/or protection of proteasome in nucleus and cytoplasm in eukaryotes.
The term “one or more compounds of formula (I), respectively formula (II)” means that a mixture of two or more kind of compound of formula (I), respectively formula (II) maybe also possible used.
As proteasome has an impaired function as already described above, during aging it is important to protect, activate or reactivate proteasome, thus positively contributing to affect the anti-ageing.
A further object of the invention is the use, especially the cosmetically use of one or more compounds of formula (I), respectively formula (II) is especially for the treatment of cellular ageing and prolongation of cell life span, which is preferably in the form of anti-aging, delaying aging, toning, moisturizing and/or whitening. In particular, in a preferred embodiment the one or more compounds of formula (I) is used for activation and/or reactivation and/or protection of proteasome in nucleus and cytoplasm in eukaryotes.
In another preferred embodiment the one or more compounds of formula (I), respectively formula (II) are used as active substances in the preparation of a cosmetical or pharmaceutical composition, which are preferably dermatological compositions, preferably for topical use on the skin.
The term “active substances” means that compounds according to formula (I), respectively formula (II) show in an effective amount effects and benefits to the skin that contributes to skin health care, e.g. ageing, antioxidative effects, depigmentation, whitening.
In particular, the one or more compounds of formula (I), respectively formula (II) have an anti-free-radical activity, thus when these active substances are formulated in cosmetic or pharmaceutical compositions, the compositions regulating the renewal and differentiation of the keratinocytes, for improving the functional condition of the skin or keeping them in a good functional condition, for skin protection or treating dry skin. Therefore, in another preferred embodiment the one or more used compounds of formula (I), respectively formula (II) have an anti-free-radical activity, thus regulating the renewal and differentiation of the keratinocytes, for improving the functional condition of the skin or keeping them in a good functional condition, for skin protection or treating dry skin.
In a preferred embodiment of the invention the one or more compounds of formula (I), respectively formula (II) is N,N-Dimethyl-succinamic acid 3,3,5-trimethyl-cyclohexyl ester (S7), wherein all enantiomeric and diastereomeic forms are incorporated herewith. Especially, in a preferred embodiment of the invention the used one or more compounds of formula (I), respectively formula (II) is trans-3,3,5-trimethylcyclohexyl 4-(dimethylamino)-4-oxobutanoate (s7a),
and/or
More particularly, the present invention also refers to a non-therapeutic (rather cosmetic) method for activation and/or reactivation and/or protection of proteasome in nucleus and cytoplasmin eukaryotes, wherein one or more compounds of formula (I)
wherein R1, R′1, R2, R′2, R3, R′3, R4 R′4, R5, R′5, R6, R7 are independently of one another and denote hydrogen, hydroxyl, C1 to C6-alkyl group or R6 and R7 form together a C5-C6 ring system, and
wherein n=0, 1, 2, 3, or 4
is administered to human tissue or skin.
In a preferred embodiment of the invention the compound of formula (I) is simplified to formula (II), in the case that R′1, R′3 and R′5 denote hydrogen thus formula (I) can be represent as follows:
wherein R1, R2, R′2, R3, R4 R′4, R5, R6, R7, are independently of one another and denote hydrogen, hydroxyl, C1 to C6-alkyl group or R6 and R7 form together a C5-C6 ring system, and
wherein n=0, 1, 2, 3, or 4.
In one embodiment the present invention refers to a method for affecting a skin care treatment, comprising topically applying to the skin a skin care efficient amount of one or more of a compound of formula (I), respectively formula (II). Skin care treatment is preferably, for whitening or for reducing signs of aging.
In another embodiment the present invention refers to a method for the treatment of the epidermis of human beings suffering from the signs of aging, suffering from dry skin and for the prevention of maladies associated with free radical formation on skin, comprising topically administering an effective amount of one or more compounds of formula (I), respectively formula (II) to the epidermis.
Pharmaceutical Compositions
The compounds of formula (I), respectively formula (II) can easily be incorporated in the given concentrations in common pharmaceutical compositions.
It is specifically contemplated that pharmaceutical compositions may be prepared using a pharmacological concentration of the active compounds or its salts disclosed in the present invention. It is not intended that the present invention be limited by the particular nature of the therapeutic preparation, so long as the preparation comprises the active agent or its salts. These therapeutic preparations can be administered to mammals for veterinary use, such as with domestic animals, and clinical use in humans in a manner similar to other therapeutic agents. In general, the dosage required for therapeutic efficacy will vary according to the type of use and mode of administration, as well as the particularized requirements of individual hosts. A person having ordinary skill in this art would readily be able to deter-mine, without undue experimentation, the appropriate dosages and routes of administration of ascorbic acid of the present invention.
Thus, a further object of the invention is one or more compounds of formula (I), respectively formula (II) as a medicament.
In a preferred embodiment the one or more compounds of formula (I), respectively formula (II) is for use in the treatment of cellular ageing and cell life span, the treatment of free-radical activity, in the form that the renewal and differentiation of keratinocytes is regulated, the functional condition of the skin is improved of is kept in good functional condition, for skin protection or treating dry skin.
A preferred embodiment of the present invention relates to pharmaceutical compositions, comprising
wherein R1, R′1, R2, R′2, R3, R′3, R4 R′4, R5, R′5, R6, R7 are independently of one another and denote hydrogen, hydroxyl, C1 to C6-alkyl group or R6 and R7 form together a C5-C6 ring system, and
wherein n=0, 1, 2, 3, or 4, and
In a preferred embodiment of the invention the compound of formula (I) is simplified to formula (II), in the case that R′1, R′3 and R′5 denote hydrogen as have been already described above.
Cosmetic Compositions
The compounds of formula (I), respectively formula (II) can easily be incorporated in the given concentrations in common cosmetic compositions such as pump sprays, aerosol sprays, creams, ointments, tinctures, lotions and the like without having an odorous, colouring or sensating effect. In this case, it is also possible and in many cases advantageous to combine the compounds of formula (I), respectively formula (II) with further active ingredients.
Therefore another object of the invention is the cosmetical use of one or more compounds of formula (I), respectively formula (II) for the treatment of anti-aging, delaying aging, toning, moisturizing and/or whitening. Especially, the cosmetical use of one or more compounds of formula (I), respectively formula (II) is preferably for activation and/or reactivation and/or protection of proteasome in nucleus and cytoplasm in eukaryotes. Another preferred embodiment is the use of one or more compounds of formula (I), respectively formula (II), wherein the one or more compounds of formula (I) or (II) have an anti-free-radical activity, thus regulating the renewal and differentiation of the keratinocytes, for improving the functional condition of the skin or keeping them in a good functional condition, for skin protection or treating dry skin.
The compositions according to the present invention can be produced by conventional processes known per se, such that one or more compounds of formula (I), respectively formula (II) are incorporated into products particularly for topical application which can have a conventional composition and which in addition to the effects mentioned hereinbefore or hereinafter can also be used for the treatment, care and cleansing of the skin or hair.
A preferred embodiment of the present invention relates to cosmetic compositions, comprising
wherein R1, R′1, R2, R′2, R3, R′3, R4 R′4, R5, R′5, R6, R7 are independently of one another and denote hydrogen, hydroxyl, C1 to C6-alkyl group or R6 and R7 form together a C5-C6 ring system, and
wherein n=0, 1, 2, 3, or 4, and
In a preferred embodiment of the invention the compound of formula (I) is simplified to formula (II), in the case that R′1, R′3 and R′5 denote hydrogen as have been already described above.
The cosmetic or pharmaceutical acceptable carriers are, preferably, selected from the group consisting of water, alcohols containing 2 to 6 carbon atoms, polyols containing 1 to 10 carbon atoms and 2 to 4 hydroxyl groups and oil bodies. Particularly preferably are, besides water, ethanol, isopropyl alcohol, ethylene glycol, propylene glycol, glycerol, trimethylolpropane, pentaerythritol and esters of linear or branched, saturated, and, particularly, unsaturated fatty acids containing 6 to 22, and, preferably, 8 to 18 carbon atoms with alcohols containing 1 to 6 carbon atoms.
The cosmetic or pharmaceutical compositions according to the invention may contain components (a) and (b) in the weight ratio of from 0.1:99 to 99.9:1, preferably, of from 10:90 to 90:10, more preferably of from 25:75 to 75:25, and most preferably of from 40:60 to 60:40. Components (a+b) and (c) may be contained in the weight ratio of from 0.01:99.9 to 2:98, preferably of from 0.5:99.5 to 1.5:98.5, and specifically of about 1:99.
The cosmetic or pharmaceutical compositions according to the invention preferably comprise as active substance of compound (I) and respectively formula (II):
The trans-3,3,5-trimethylcyclohexyl 4-(dimethylamino)-4-oxobutanoate (s7a) may be preferably both Enantiomer (1S,5R)-3,3,5-trimethylcyclohexyl]4-(dimethylamino)-4-oxo-butanoate and/or (1R,5S)-3,3,5-trimethylcyclohexyl]4-(dimethylamino)-4-oxo-butanoate:
and the cis-3,3,5-trimethylcyclohexyl 4-(dimethylamino)-4-oxobutanoate (s7b) is may be preferably both Enantiomer (1R,5R)-3,3,5-trimethylcyclohexyl]4-(dimethylamino)-4-oxo-butanoate and/or (1S,5S)-3,3,5-trimethylcyclohexyl]4-(dimethylamino)-4-oxo-butanoate:
It has been shown that such a mixture of trans-3,3,5-trimethylcyclohexyl 4-(dimethylamino)-4-oxobutanoate and cis-3,3,5-trimethylcyclohexyl 4-(dimethylamino)-4-oxobutanoate has antioxidative properties, which benefits to skin healthcare. Further, the mixture shows a good activation and/or reactivation and/or protection effect for proteasom, which benefits to cell life span.
But a mixture of only single enantiomers in a pharmaceutical or cosmetic composition is also as active as a mixture of enantiomeric forms. Thus in a preferred embodiment the pharmaceutical or cosmetic composition comprises one or more compounds of formula (I), respectively formula (II), wherein the compounds of formula (I), respectively formula (II) is selected from the group of consisting of compounds S1, S2, S6, S7, S8, S9, S10 and wherein the compounds are selected individual, such that each enantiomeric form of S1, S2, S6, S7, S8, S9, S10 as described above can be selected solely. Preferred is a cosmetic or pharmaceutical composition which comprises one or more compounds of formula (I), respectively formula (II) selected from S7a′ and/or 7a″ and/or S7b′ and/or S7b″.
The cosmetic or pharmaceutical compositions according to the invention may comprises further additives which are selected from the group consisting of surfactants, oil bodies, emulsifiers, superfatting agents, pearlising waxes, consistency factors, polymers, silicone compounds, waxes, stabilizers, antidandruff agents, film formers, swelling agents, hydrotropes, preservatives, solubilizers, complexing agents, reducing agents, alkalising agents, perfume oils, dyes, thickeners, fats, lecithins, phospholipids, UV protection factors, moisturizers, biogenic agents, antioxidants, deodorants, antiperspirants, insect repellants, self-tanning agents, tyrosine inhibitors (depigmenting agents), and the like as additional auxiliaries and additives.
More particularly, the additives are selected from the group consisting of
A cosmetic composition, preferably a topical preparation according to the invention containing
In the context of the invention, anti-ageing or biogenic agents are, for example antioxidants, primary or second dary sun protection factors, matrix-metalloproteinase inhibitors (MMPI), skin moisturizing agents, glycosaminglycan stimulators, anti-inflammatory agents and TRPV1 antagonists.
In case vitamin A and/or a derivative of vitamin A is used as component (b1) or as constituent of component (b1), the total amount thereof preferably is in the range of from 0.1 to 3% b.w., based on the total weight of the preparation.
In case vitamin E and/or a derivative of vitamin E is used as component (b1) or as constituent(s) of component (b1), the total amount thereof preferably is in the range of from 0.1 to 2% b.w., based on the total weight of the preparation.
In case vitamin C and/or a derivative of vitamin C is used as component (b1) or as constituent of component (b-1), the total amount thereof preferably is in the range of from 0.01 to 3% b.w., based on the total weight of the preparation.
In case ubiquinone is used as component (b1) or as constituent of component (b1), the total amount thereof preferably is in the range of from 0.001 to 0.1% b.w., based on the total weight of the preparation.
In case hyaluronic acid and/or a derivative or salt of hyaluronic acid is used as component (b5) or as constituent of component (b5), the total amount thereof preferably is in the range of from 0.01 to 3% b.w., based on the total weight of the preparation.
In case Retinol and/or a derivative of retinol is used as component (b5) or as constituent of component (b5), the total amount thereof preferably is in the range of from 0.01 to 1% b.w., based on the total weight of the preparation.
In case alpha-bisabolol (natural or synthetic) is used as component (b5) or as constituent of component (b5), the total amount thereof preferably is in the range of from 0.01 to 0.5% b.w., based on the total weight of the preparation.
In case oat glucan is used as component (b5) or as constituent of component (b5), the total amount thereof preferably is in the range of from 0.01 to 1% b.w., based on the total weight of the preparation.
In case Echinacea purpurea extract is used as component (b5) or as constituent of component (b5), the total amount thereof preferably is in the range of from 0.1 to 0.6% b.w., based on the total weight of the preparation.
In case Alpinia galanga leaf extract is used as component (b5) or as constituent of component (b5), the total amount thereof preferably is in the range of from 0.1 to 5% b.w., based on the total weight of the preparation.
In case Sinorhizobium Meliloti Ferment Filtrate is used as component (b5) or as constituent of component (b-5), the total amount thereof preferably is in the range of from 0.1 to 5% b.w., based on the total weight of the preparation.
In case Tetradecyl Aminobutyroylvalylaminobutyric Urea Trifluoroacetate is used as component (b5) or as constituent of component (b5), the total amount thereof preferably is in the range of from 0.1 to 3% b.w., based on the total weight of the preparation.
In case Retinyl palmitate is used as component (b3) or as constituent of component (b-3), the total amount thereof preferably is in the range of from 1 to 3% b.w., based on the total weight of the preparation.
In case Ursolic acid is used as component (b3) or as constituent of component (b-3), the total amount thereof preferably is in the range of from 0.01 to 1% b.w., based on the total weight of the preparation.
In case one or more than one extract from the leaves of the Rosaceae family, sub-family Rosoideae is used as component (b3) or as constituent of component (b-3), the total amount thereof preferably is in the range of from 0.01 to 3% b.w., based on the total weight of the preparation.
In case Genistein and/or Daidzein is used as component (b3) or as constituent of component (b3), the total amount thereof preferably is in the range of from 0.01 to 2% b.w., based on the total weight of the preparation.
Additional Additives
A cosmetic composition, preferably a topical preparation according to the invention containing
As mentioned above, proteasom, can be significantly degraded by free radicals. The combination of one or more compounds of formula (I), respectively formula (II) and one or more antioxidants of component (b1) is particularly beneficial because antioxidants additionally protect proteasom from reactive oxygen species.
The combination of one or more compounds of formula (I), respectively formula (II) and one or more sun protection factors of component (b2) is particularly beneficial because UV light is one major source for reactive oxygen species. Furthermore as mentioned above, collagen fragments in the dermis resulting from UVB-induced collagen degradation and strongly reduced proteasom in human skin. Particular advantageous are therefore cosmetic, dermatological and/or pharmaceutical preparations according to the invention which additionally include one or more UV filters (UV absorbers) and which thus act as compositions with proteasom stimulating activity and additionally as a sunscreen, overall resulting in a higher, improved proteasom level.
The combination of one or more compounds of formula (I), respectively formula (II) and one or more agents selected from the group consisting of matrix-metalloproteinase (MMP) inhibitor (b3) is particularly beneficial resulting in an overall higher, improved proteasom level.
The combination of one or more compounds of formula (I), respectively formula (II) and one or more skin moisturizing agents (b4) is particularly beneficial because skin moisturizing agents additionally improve the moisture status of the skin.
The combination of one or more compounds of formula (I) and one or more agents selected from the group consisting of glycosaminoglycans (GAGs) and further substances stimulating the synthesis of glycosaminoglycans (b5) is particularly beneficial resulting in an overall higher, improved GAG level.
It has been found rather advantageous to add the anti-ageing actives forming components (b1) to (b7) to the respective compositions in the following amounts:
Other preferred auxiliaries and additives are anionic and/or amphoteric or zwitterionic surfactants. Typical examples of anionic surfactants are soaps, alkyl benzenesulfonates, alkanesulfonates, olefin sulfonates, alkylether sulfonates, glycerol ether sulfonates, methyl ester sulfonates, sulfofatty acids, alkyl sulfates, fatty alcohol ether sulfates, glycerol ether sulfates, fatty acid ether sulfates, hydroxy mixed ether sulfates, monoglyceride (ether) sulfates, fatty acid amide (ether) sulfates, mono- and dialkyl sulfosuccinates, mono- and dialkyl sulfosuccinamates, sulfotriglycerides, amide soaps, ether carboxylic acids and salts thereof, fatty acid isethionates, fatty acid sarcosinates, fatty acid taurides, N-acylamino acids such as, for example, acyl lactylates, acyl tartrates, acyl glutamates and acyl aspartates, alkyl oligoglucoside sulfates, protein fatty acid condensates (particularly wheat-based vegetable products) and alkyl (ether) phosphates. If the anionic surfactants contain polyglycol ether chains, they may have a conventional homolog distribution although they preferably have a narrow-range homolog distribution. Typical examples of amphoteric or zwitterionic surfactants are alkylbetaines, alkylamidobetaines, aminopropionates, aminoglycinates, imidazolinium betaines and sulfobetaines. The surfactants mentioned are all known compounds. Information on their structure and production can be found in relevant synoptic works, cf. for example J. Falbe (ed.), “Surfactants in Consumer Products”, Springer Verlag, Berlin, 1987, pages 54 to 124 or J. Falbe (ed.), “Katalysatoren, Tenside and Mineralöladditive (Catalysts, Surfactants and Mineral Oil Additives)”, Thieme Verlag, Stuttgart, 1978, pages 123-217. The percentage content of surfactants in the preparations may be from 0.1 to 10% by weight and is preferably from 0.5 to 5% by weight, based on the preparation.
2. Oil Bodies
Suitable oil bodies, which form constituents of the O/W emulsions, are, for example, Guerbet alcohols based on fatty alcohols having 6 to 18, preferably 8 to 10, carbon atoms, esters of linear C6-C22-fatty acids with linear or branched C6-C22-fatty alcohols or esters of branched C6-C13-carboxylic acids with linear or branched C6-C22-fatty alcohols, such as, for example, myristyl myristate, myristyl palmitate, myristyl stearate, myristyl isostearate, myristyl oleate, myristyl behenate, myristyl erucate, cetyl myristate, cetyl palmitate, cetyl stearate, cetyl isostearate, cetyl oleate, cetyl behenate, cetyl erucate, stearyl myristate, stearyl palmitate, stearyl stearate, stearyl isostearate, stearyl oleate, stearyl behenate, stearyl erucate, isostearyl myristate, isostearyl palmitate, isostearyl stearate, isostearyl isostearate, isostearyl oleate, isostearyl behenate, isostearyl oleate, oleyl myristate, oleyl palmitate, oleyl stearate, oleyl isostearate, oleyl oleate, oleyl behenate, oleyl erucate, behenyl myristate, behenyl palmitate, behenyl stearate, behenyl isostearate, behenyl oleate, behenyl behenate, behenyl erucate, erucyl myristate, erucyl palmitate, erucyl stearate, erucyl isostearate, erucyl oleate, erucyl behenate and erucyl erucate. Also suitable are esters of linear C6-C22-fatty acids with branched alcohols, in particular 2-ethylhexanol, esters of C18-C38-alkylhydroxy carboxylic acids with linear or branched C6-C22-fatty alcohols, in particular Dioctyl Malate, esters of linear and/or branched fatty acids with polyhydric alcohols (such as, for example, propylene glycol, dimerdiol or trimertriol) and/or Guerbet alcohols, triglycerides based on C6-C10-fatty acids, liquid mono-/di-/triglyceride mixtures based on C6-C18-fatty acids, esters of C6-C22-fatty alcohols and/or Guerbet alcohols with aromatic carboxylic acids, in particular benzoic acid, esters of C2-C12-dicarboxylic acids with linear or branched alcohols having 1 to 22 carbon atoms or polyols having 2 to 10 carbon atoms and 2 to 6 hydroxyl groups, vegetable oils, branched primary alcohols, substituted cyclohexanes, linear and branched C6-C22-fatty alcohol carbonates, such as, for example, Dicaprylyl Carbonate (Cetiol® CC), Guerbet carbonates, based on fatty alcohols having 6 to 18, preferably 8 to 10, carbon atoms, esters of benzoic acid with linear and/or branched C6-C22-alcohols (e.g. Finsolv® TN), linear or branched, symmetrical or asymmetrical dialkyl ethers having 6 to 22 carbon atoms per alkyl group, such as, for example, dicaprylyl ether (Cetiol® OE), ring-opening products of epoxidized fatty acid esters with polyols, silicone oils (cyclomethicones, silicone methicone grades, etc.) and/or aliphatic or naphthenic hydrocarbons, such as, for example, squalane, squalene or dialkylcyclohexanes.
3. Emulsifiers
Other surfactants may also be added to the preparations as emulsifiers, including for example:
The addition products of ethylene oxide and/or propylene oxide onto fatty alcohols, fatty acids, alkylphenols, glycerol mono- and diesters and sorbitan mono- and diesters of fatty acids or onto castor oil are known commercially available products. They are homologue mixtures of which the average degree of alkoxylation corresponds to the ratio between the quantities of ethylene oxide and/or propylene oxide and substrate with which the addition reaction is carried out. C12/18 fatty acid monoesters and diesters of addition products of ethylene oxide onto glycerol are known as lipid layer enhancers for cosmetic formulations. The preferred emulsifiers are described in more detail as follows:
Superfatting agents may be selected from such substances as, for example, lanolin and lecithin and also polyethoxylated or acylated lanolin and lecithin derivatives, polyol fatty acid esters, monoglycerides and fatty acid alkanolamides, the fatty acid alkanolamides also serving as foam stabilizers.
The consistency factors mainly used are fatty alcohols or hydroxyfatty alcohols containing 12 to 22 and preferably 16 to 18 carbon atoms and also partial glycerides, fatty acids or hydroxyfatty acids. A combination of these substances with alkyl oligoglucosides and/or fatty acid N-methyl glucamides of the same chain length and/or polyglycerol poly-12-hydroxystearates is preferably used.
5. Thickening Agents and Rheology Additives
Suitable thickeners are polymeric thickeners, such as Aerosil® types (hydrophilic silicas), polysaccharides, more especially xanthan gum, guar-guar, agar-agar, alginates and tyloses, carboxymethyl cellulose and hydroxyethyl cellulose, also relatively high molecular weight polyethylene glycol monoesters and diesters of fatty acids, polyacrylates (for example Carbopols® [Goodrich] or Synthalens® [Sigma]), polyacrylamides, polyvinyl alcohol and polyvinyl pyrrolidone, surfactants such as, for example, ethoxylated fatty acid glycerides, esters of fatty acids with polyols, for example pentaerythritol or trimethylol propane, narrow-range fatty alcohol ethoxylates and electrolytes, such as sodium chloride and ammonium chloride.
6. Polymers
Suitable cationic polymers are, for example, cationic cellulose derivatives such as, for example, the quaternized hydroxyethyl cellulose obtainable from Amerchol under the name of Polymer JR 400®, cationic starch, copolymers of diallyl ammonium salts and acrylamides, quaternized vinyl pyrrolidone/vinyl imidazole polymers such as, for example, Luviquat® (BASF), condensation products of polyglycols and amines, quaternized collagen polypeptides such as, for example, Lauryldimonium Hydroxypropyl Hydrolyzed Collagen (Lamequat® L, Grünau), quaternized wheat polypeptides, polyethyleneimine, cationic silicone polymers such as, for example, amodimethicone, copolymers of adipic acid and dimethylaminohydroxypropyl diethylenetriamine (Cartaretine®, Sandoz), copolymers of acrylic acid with dimethyl diallyl ammonium chloride (Merquat® 550, Chemviron), polyaminopolyamides and crosslinked water-soluble polymers thereof, cationic chitin derivatives such as, for example, quaternized chitosan, optionally in microcrystalline distribution, condensation products of dihaloalkyls, for example dibromobutane, with bis-dialkylamines, for example bis-dimethylamino-1,3-propane, cationic guar gum such as, for example, Jaguar® CBS, Jaguar® C-17, Jaguar® C-16 of Celanese, quaternized ammonium salt polymers such as, for example, Mirapol® A-15, Mirapol® AD-1, Mirapol® AZ-1 of Miranol and the various polyquaternium types (for example 6, 7, 32 or 37) which can be found in the market under the tradenames Rheocare® CC or Ultragel® 300.
Suitable anionic, zwitterionic, amphoteric and nonionic polymers are, for example, vinyl ace-tate/crotonic acid copolymers, vinyl pyrrolidone/vinyl acrylate copolymers, vinyl acetate/butyl maleate/isobornyl acrylate copolymers, methyl vinylether/maleic anhydride copolymers and esters thereof, uncrosslinked and polyol-crosslinked polyacrylic acids, acrylamidopropyl trimethylammonium chloride/acrylate copolymers, octylacrylamide/methyl methacrylate/tert.-butylaminoethyl methacrylate/2-hydroxypropyl methacrylate copolymers, polyvinyl pyrrolidone, vinyl pyrrolidone/vinyl acetate copolymers, vinyl pyrrolidone/dimethylaminoethyl methacrylate/vinyl caprolactam terpolymers and optionally derivatized cellulose ethers and silicones.
7. Pearlising Waxes
Suitable pearlising waxes are, for example, alkylene glycol esters, especially ethylene glycol distearate; fatty acid alkanolamides, especially cocofatty acid diethanolamide; partial glycerides, especially stearic acid monoglyceride; esters of polybasic, optionally hydroxy-substituted carboxylic acids with fatty alcohols containing 6 to 22 carbon atoms, especially long-chain esters of tartaric acid; fatty compounds, such as for example fatty alcohols, fatty ketones, fatty aldehydes, fatty ethers and fatty carbonates which contain in all at least 24 carbon atoms, especially laurone and distearylether; fatty acids, such as stearic acid, hydroxystearic acid or behenic acid, ring opening products of olefin epoxides containing 12 to 22 carbon atoms with fatty alcohols containing 12 to 22 carbon atoms and/or polyols containing 2 to 15 carbon atoms and 2 to 10 hydroxyl groups and mixtures thereof.
8. Silicones
Suitable silicone compounds are, for example, dimethyl polysiloxanes, methylphenyl polysiloxanes, cyclic silicones and amino-, fatty acid-, alcohol-, polyether-, epoxy-, fluorine-, glycoside- and/or alkyl-modified silicone compounds which may be both liquid and resin-like at room temperature. Other suitable silicone compounds are simethicones which are mixtures of dimethicones with an average chain length of 200 to 300 dimethylsiloxane units and hydrogenated silicates.
9. Waxes and Stabilizers
Besides natural oils used, waxes may also be present in the preparations, more especially natural waxes such as, for example, candelilla wax, carnauba wax, Japan wax, espartograss wax, cork wax, guaruma wax, rice oil wax, sugar cane wax, ouricury wax, montan wax, beeswax, shellac wax, spermaceti, lanolin (wool wax), uropygial fat, ceresine, ozocerite (earth wax), petrolatum, paraffin waxes and microwaxes; chemically modified waxes (hard waxes) such as, for example, montan ester waxes, sasol waxes, hydrogenated jojoba waxes and synthetic waxes such as, for example, polyalkylene waxes and polyethylene glycol waxes.
Metal salts of fatty acids such as, for example, magnesium, aluminium and/or zinc stearate or ricinoleate may be used as stabilizers.
10. Cooling Agents
The compositions may also contain one or more substances with a physiological cooling effect (cooling agents), which are preferably selected here from the following list: menthol and menthol derivatives (for example L-menthol, D-menthol, racemic menthol, isomenthol, neoisomenthol, neomenthol) menthylethers (for example (I-menthoxy)-1,2-propandiol, (1-menthoxy)-2-methyl-1,2-propandiol, 1-menthyl-methylether), menthylesters (for example menthylformiate, menthylacetate, menthylisobutyrate, menthyllactates, L-menthyl-L-lactate, L-menthyl-D-lactate, menthyl-(2-methoxy)acetate, menthyl-(2-methoxyethoxy-)acetate, menthylpyroglutamate), menthylcarbonates (for example menthylpropylenegly-colcarbonate, menthylethyleneglycolcarbonate, menthylglycerolcarbonate or mixtures thereof), the semi-esters of menthols with a dicarboxylic acid or derivatives thereof (for example mono-menthylsuccinate, mono-menthylglutarate, mono-menthylmalonate, O-menthyl succinic acid ester-N,N-(dimethyl)amide, O-menthyl succinic acid ester amide), menthanecarboxylic acid amides (in this case preferably menthanecarboxylic acid-N-ethylamide [WS3] or Nα-(menthanecarbonyl)glycinethylester [WS5], as described in U.S. Pat. No. 4,150,052, menthanecarboxylic acid-N-(4-cyanophenyl)amide or menthanecarboxylic acid-N-(4-cyanomethylphenyl)amide as described in WO 2005 049553 A1, methanecarboxylic acid-N-(alkoxyalkyl)amides), menthone and menthone derivatives (for example L-menthone glycerol ketal), 2,3-dimethyl-2-(2-propyl)-butyric acid derivatives (for example 2,3-dimethyl-2-(2-propyl)-butyric acid-N-methylamide [WS23]), isopulegol or its esters (I-(−)-isopulegol, I-(−)-isopulegolacetate), menthane derivatives (for example p-menthane-3,8-diol), cubebol or synthetic or natural mixtures, containing cubebol, pyrrolidone derivatives of cycloalkyldione derivatives (for example 3-methyl-2(1-pyrrolidinyl)-2-cyclopentene-1-one) or tetrahydropyrimidine-2-one (for example iciline or related compounds, as described in WO 2004/026840), further carboxamides (for example N-(2-(pyridin-2-yl)ethyl)-3-p-menthanecarboxamide or related compounds), (1R,2S,5R)—N-(4-Methoxyphenyl)-5-methyl-2-(1-isopropyl)cyclohexane-carboxamide [WS12], oxamates (preferably those described in EP 2033688 A2).
11. Anti-microbial Agents
Suitable anti-microbial agents are, in principle, all substances effective against Gram-positive bacteria, such as, for example, 4-hydroxybenzoic acid and its salts and esters, N-(4-chlorophenyl)-N′-(3,4-dichlorophenyl)urea, 2,4,4′-trichloro-2′-hydroxy-diphenyl ether (triclosan), 4-chloro-3,5-dimethyl-phenol, 2,2′-methylenebis(6-bromo-4-chlorophenol), 3-methyl-4-(1-methylethyl)phenol, 2-benzyl-4-chloro-phenol, 3-(4-chlorophenoxy)-1,2-propanediol, 3-iodo-2-propynyl butylcarbamate, chlorhexidine, 3,4,4′-trichlorocarbanilide (TTC), antibacterial fragrances, thymol, thyme oil, eugenol, oil of cloves, menthol, mint oil, farnesol, phenoxyethanol, glycerol monocaprate, glycerol monocaprylate, glycerol monolaurate (GML), diglycerol monocaprate (DMC), salicylic acid N-alkylamides, such as, for example, n-octylsalicylamide or n-decylsalicylamide.
12. Enzyme Inhibitors
Suitable enzyme inhibitors are, for example, esterase inhibitors. These are preferably trialkyl citrates, such as trimethyl citrate, tripropyl citrate, triisopropyl citrate, tributyl citrate and, in particular, triethyl citrate (Hydagen CAT). The substances inhibit enzyme activity, thereby reducing the formation of odour. Other substances which are suitable esterase inhibitors are sterol sulfates or phosphates, such as, for example, lanosterol, cholesterol, campesterol, stigmasterol and sitosterol sulfate or phosphate, dicarboxylic acids and esters thereof, such as, for example, glutaric acid, monoethyl glutarate, diethyl glutarate, adipic acid, monoethyl adipate, diethyl adipate, malonic acid and diethyl malonate, hydroxycarboxylic acids and esters thereof, such as, for example, citric acid, malic acid, tartaric acid or diethyl tartrate, and zinc glycinate.
13. Odour Absorbers and Antiperspirant Active Agents
Suitable odour absorbers are substances which are able to absorb and largely retain odour-forming compounds. They lower the partial pressure of the individual components, thus also reducing their rate of diffusion. It is important that perfumes must remain unimpaired in this process. Odour absorbers are not effective against bacteria. They comprise, for example, as main constituent, a complex zinc salt of ricinoleic acid or specific, largely odour-neutral fragrances which are known to the person skilled in the art as “fixatives”, such as, for example, extracts of labdanum or styrax or certain abietic acid derivatives. The odour masking agents are fragrances or perfume oils, which, in addition to their function as odour masking agents, give the deodorants their respective fragrance note. Perfume oils which may be mentioned are, for example, mixtures of natural and synthetic fragrances. Natural fragrances are extracts from flowers, stems and leaves, fruits, fruit peels, roots, woods, herbs and grasses, needles and branches, and resins and balsams. Also suitable are animal products, such as, for example, civet and castoreum. Typical synthetic fragrance compounds are products of the ester, ether, aldehyde, ketone, alcohol, and hydrocarbon type. Fragrance compounds of the ester type are, for example, benzyl acetate, p-tert-butylcyclohexyl acetate, linalyl acetate, phenylethyl acetate, linalyl benzoate, benzyl formate, allyl cyclohexylpropionate, styrallyl propionate and benzyl salicylate. The ethers include, for example, benzyl ethyl ether, and the aldehydes include, for example, the linear alkanals having 8 to 18 carbon atoms, citral, citronellal, citronellyloxyacetaldehyde, cyclamen aldehyde, hydroxycitronellal, lilial and bourgeonal, the ketones include, for example, the ionones and methyl cedryl ketone, the alcohols include anethole, citronellol, eugenol, isoeugenol, geraniol, linaool, phenylethyl alcohol and terpineol, and the hydrocarbons include mainly the terpenes and balsams. Preference is, however, given to using mixtures of different fragrances which together produce a pleasing fragrance note. Essential oils of relatively low volatility, which are mostly used as aroma components, are also suitable as perfume oils, e.g. sage oil, camomile oil, oil of cloves, melissa oil, mint oil, cinnamon leaf oil, linden flower oil, juniperberry oil, vetiver oil, olibanum oil, galbanum oil, labdanum oil and lavandin oil. Preference is given to using bergamot oil, dihydromyrcenol, lilial, lyral, citronellol, phenylethyl alcohol, α-hexylcinnamaldehyde, geraniol, benzylacetone, cyclamen aldehyde, linalool, boisambrene forte, ambroxan, indole, hedione, sandelice, lemon oil, mandarin oil, orange oil, allyl amyl glycolate, cyclovertal, lavandin oil, clary sage oil, β-damascone, geranium oil bourbon, cyclohexyl salicylate, Vertofix coeur, iso-E-super, Fixolide NP, evernyl, iraldein gamma, phenylacetic acid, geranyl acetate, benzyl acetate, rose oxide, romilat, irotyl and floramat alone or in mixtures.
Suitable astringent antiperspirant active ingredients are primarily salts of aluminium, zirconium or of zinc. Such suitable antihydrotic active ingredients are, for example, aluminium chloride, aluminium chlorohydrate, aluminium dichlorohydrate, aluminium sesquichlorohydrate and complex compounds thereof, e.g. with 1,2-propylene glycol, aluminium hydroxy-allantoinate, aluminium chloride tartrate, aluminium zirconium trichlorohydrate, aluminium zirconium tetrachlorohydrate, aluminium zirconium pentachlorohydrate and complex compounds thereof, e.g. with amino acids, such as glycine.
14. Film Formers and Anti-dandruff Agents
Standard film formers are, for example, chitosan, microcrystalline chitosan, quaternized chitosan, polyvinyl pyrrolidone, vinyl pyrrolidone/vinyl acetate copolymers, polymers of the acrylic acid series, quaternary cellulose derivatives, collagen, hyaluronic acid and salts thereof and similar compounds.
Suitable antidandruff agents are Pirocton Olamin (1-hydroxy-4-methyl-6-(2,4,4-trimethyl-pentyl)-2-(1H)-pyridinone monoethanolamine salt), Baypival® (Climbazole), Ketoconazol® (4-acetyl-1-{4-[2-(2,4-dichlorophenyl) r-2-(1H-imidazol-1-ylmethyl)-1,3-dioxylan-c-4-ylmethoxy-phenyl}-piperazine, ketoconazole, elubiol, selenium disulfide, colloidal sulfur, sulfur polyethylene glycol sorbitan monooleate, sulfur ricinol polyethoxylate, sulfur tar distillate, salicylic acid (or in combination with hexachlorophene), undecylenic acid, monoethanolamide sulfosuccinate Na salt, Lamepon® UD (protein/undecylenic acid condensate), zinc pyrithione, aluminium pyrithione and magnesium pyrithione/dipyrithione magnesium sulfate.
15. Carriers and Hydrotropes
Preferred cosmetics carrier materials are solid or liquid at 25° C. and 1013 mbar (including highly viscous substances) as for example glycerol, 1,2-propylene glycol, 1,2-butylene glycol, 1,3-propylene glycol, 1,3-butylene glycol, ethanol, water and mixtures of two or more of said liquid carrier materials with water. Optionally, these preparations according to the invention may be produced using preservatives or solubilizers. Other preferred liquid carrier substances, which may be a component of a preparation according to the invention are selected from the group consisting of oils such as vegetable oil, neutral oil and mineral oil.
Preferred solid carrier materials, which may be a component of a preparation according to the invention are hydrocolloids, such as starches, degraded starches, chemically or physically modified starches, dextrins, (powdery) maltodextrins (preferably with a dextrose equivalent value of 5 to 25, preferably of 10-20), lactose, silicon dioxide, glucose, modified celluloses, gum arabic, ghatti gum, traganth, karaya, carrageenan, pullulan, curdlan, xanthan gum, gellan gum, guar flour, carob bean flour, alginates, agar, pectin and inulin and mixtures of two or more of these solids, in particular maltodextrins (preferably with a dextrose equivalent value of 15-20), lactose, silicon dioxide and/or glucose.
In addition, hydrotropes, for example ethanol, isopropyl alcohol or polyols, may be used to improve flow behaviour. Suitable polyols preferably contain 2 to 15 carbon atoms and at least two hydroxyl groups. The polyols may contain other functional groups, more especially amino groups, or may be modified with nitrogen. Typical examples are
Suitable preservatives are, for example, phenoxyethanol, formaldehyde solution, parabens, pentanediol or sorbic acid and the other classes of compounds listed in Appendix 6, Parts A and B of the Kosmetikverordnung (“Cosmetics Directive”).
17. Perfume Oils and Fragrances
Suitable perfume oils are mixtures of natural and synthetic perfumes. Natural perfumes include the extracts of blossoms (lily, lavender, rose, jasmine, neroli, ylang-ylang), stems and leaves (geranium, patchouli, petitgrain), fruits (anise, coriander, caraway, juniper), fruit peel (bergamot, lemon, orange), roots (nutmeg, angelica, celery, cardamom, costus, iris, calmus), woods (pinewood, sandalwood, guaiac wood, cedarwood, rosewood), herbs and grasses (tarragon, lemon grass, sage, thyme), needles and branches (spruce, fir, pine, dwarf pine), resins and balsams (galbanum, elemi, benzoin, myrrh, olibanum, opoponax). Animal raw materials, for example civet and beaver, may also be used. Typical synthetic perfume compounds are products of the ester, ether, aldehyde, ketone, alcohol and hydrocarbon type. Examples of perfume compounds of the ester type are benzyl acetate, phenoxyethyl isobutyrate, p-tert.butyl cyclohexylacetate, linalyl acetate, dimethyl benzyl carbinyl acetate, phenyl ethyl acetate, linalyl benzoate, benzyl formate, ethylmethyl phenyl glycinate, allyl cyclohexyl propionate, styrallyl propionate and benzyl salicylate. Ethers include, for example, benzyl ethyl ether while aldehydes include, for example, the linear alkanals containing 8 to 18 carbon atoms, citral, citronellal, citronellyloxyacetaldehyde, cyclamen aldehyde, hydroxycitronellal, lilial and bourgeonal. Examples of suitable ketones are the ionones, •-isomethylionone and methyl cedryl ketone. Suitable alcohols are anethol, citronellol, eugenol, isoeugenol, geraniol, linalool, phenylethyl alcohol and terpineol. The hydrocarbons mainly include the terpenes and balsams. However, it is preferred to use mixtures of different perfume compounds which, together, produce an agreeable perfume. Other suitable perfume oils are essential oils of relatively low volatility which are mostly used as aroma components. Examples are sage oil, camomile oil, clove oil, melissa oil, mint oil, cinnamon leaf oil, lime-blossom oil, juniper berry oil, vetiver oil, olibanum oil, galbanum oil, ladanum oil and lavendin oil. The following are preferably used either individually or in the form of mixtures: bergamot oil, dihydromyrcenol, lilial, lyral, citronellol, phenylethyl alcohol, hexylcinnamaldehyde, geraniol, benzyl acetone, cyclamen aldehyde, linalool, Boisambrene Forte, Ambroxan, indole, hedione, sandelice, citrus oil, mandarin oil, orange oil, allylamyl glycolate, cyclovertal, lavendin oil, clary oil, damascone, geranium oil bourbon, cyclohexyl salicylate, Vertofix Coeur, Iso-E-Super, Fixolide NP, evernyl, iraldein gamma, phenylacetic acid, geranyl acetate, benzyl acetate, rose oxide, romillat, irotyl and floramat.
18. Dyes
Suitable dyes are any of the substances suitable and approved for cosmetic purposes as listed, for example, in the publication “Kosmetische Färbemittel” of the Farbstoff-kommission der Deutschen Forschungsgemeinschaft, Verlag Chemie, Weinheim, 1984, pages 81 to 106. Examples include cochineal red A (C.I. 16255), patent blue V (C.I. 42051), indigotin (C.I. 73015), chlorophyllin (C.I. 75810), quinoline yellow (C.I. 47005), titanium dioxide (C.I. 77891), indanthrene blue RS (C.I. 69800) and madder lake (C.I. 58000). Luminol may also be present as a luminescent dye. Advantageous coloured pigments are for example titanium dioxide, mica, iron oxides (e.g. Fe2O3 Fe3O4, FeO(OH)) and/or tin oxide. Advantageous dyes are for example carmine, Berlin blue, chromium oxide green, ultramarine blue and/or manganese violet.
Preferred compositions according to the present inventions are selected from the group of products for treatment, protecting, care and cleansing of the skin and/or hair or as a make-up product, preferably as a leave-on product (meaning that the one or more compounds of formula (I) stay on the skin and/or hair for a longer period of time, compared to rinse-off products, so that the moisturizing and/or anti-ageing and/or wound healing promoting action thereof is more pronounced).
The formulations according to the invention are preferably in the form of an emulsion, e.g. W/O (water-in-oil), O/W (oil-in-water), W/O/W (water-in-oil-in-water), O/W/O (oil-in-water-in-oil) emulsion, PIT emulsion, Pickering emulsion, emulsion with a low oil content, micro- or nanoemulsion, a solution, e.g. in oil (fatty oils or fatty acid esters, in particular C6-C32 fatty acid C2-C30 esters) or silicone oil, dispersion, suspension, creme, lotion or milk, depending on the production method and ingredients, a gel (including hydrogel, hydrodispersion gel, oleogel), spray (e.g. pump spray or spray with propellant) or a foam or an impregnating solution for cosmetic wipes, a detergent, e.g. soap, synthetic detergent, liquid washing, shower and bath preparation, bath product (capsule, oil, tablet, salt, bath salt, soap, etc.), effervescent preparation, a skin care product such as e.g. an emulsion (as described above), ointment, paste, gel (as described above), oil, balsam, serum, powder (e.g. face powder, body powder), a mask, a pencil, stick, roll-on, pump, aerosol (foaming, non-foaming or post-foaming), a deodorant and/or antiperspirant, mouthwash and mouth rinse, a foot care product (including keratolytic, deodorant), an insect repellent, a sunscreen, aftersun preparation, a shaving product, aftershave balm, pre- and aftershave lotion, a depilatory agent, a hair care product such as e.g. shampoo (including 2-in-1 shampoo, anti-dandruff shampoo, baby shampoo, shampoo for dry scalps, concentrated shampoo), conditioner, hair tonic, hair water, hair rinse, styling creme, pomade, perm and setting lotion, hair spray, styling aid (e.g. gel or wax), hair smoothing agent (detangling agent, relaxer), hair dye such as e.g. temporary direct-dyeing hair dye, semi-permanent hair dye, permanent hair dye, hair conditioner, hair mousse, eye care product, make-up, make-up remover or baby product.
The formulations according to the invention are particularly preferably in the form of an emulsion, in particular in the form of a W/O, O/W, W/O/W, O/W/O emulsion, PIT emulsion, Pickering emulsion, emulsion with a low oil content, micro- or nanoemulsion, a gel (including hydrogel, hydrodispersion gel, oleogel), a solution e.g. in oil (fatty oils or fatty acid esters, in particular C6-C32 fatty acid C2-C30 esters)) or silicone oil, or a spray (e.g. pump spray or spray with propellant).
Auxiliary substances and additives can be included in quantities of 5 to 99% b.w., preferably 10 to 80% b.w., based on the total weight of the formulation. The amounts of cosmetic or dermatological auxiliary agents and additives and perfume to be used in each case can easily be determined by the person skilled in the art by simple trial and error, depending on the nature of the particular product.
The preparations can also contain water in a quantity of up to 99% b.w., preferably 5 to 80% b.w., based on the total weight of the preparation. It should be noted that the information on additives and their ranges for cosmetic compositions are also valid for pharmaceutical or dermatalogical formulations.
Encapsulation
Although one preferred embodiment of the present invention relates to topical application, the compositions may also be administered orally, preferably in the form of a capsule. The compositions are typically encapsulated by means of a solid covering material, which is preferably selected from starches, degraded or chemically or physically modified starches (in particular dextrins and maltodextrins), gelatins, gum arabic, agar-agar, ghatti gum, gellan gum, modified and non-modified celluloses, pullulan, curdlan, carrageenans, alginic acid, alginates, pectin, inulin, xanthan gum and mixtures of two or more of said substances.
The solid covering material is preferably selected from gelatin (preferred are pork, beef, chicken and/or fish gelatins and mixtures thereof, preferably comprising at least one gelatin with a bloom value of greater than or equal to 200, preferably with a bloom value of greater than or equal to 240), maltodextrin (preferably obtained from maize (corn), wheat, tapioca or potato, preferred maltodextrins have a DE value of 10-20), modified cellulose (for example cellulose ether), alginates (for example Na-alginate), carrageenan (beta-, iota-, lambda- and/or kappa carrageenan), gum arabic, curdlan and/or agar-agar. Gelatin is preferably used, in particular, because of its good availability in different bloom values. Particularly preferred, especially for oral use are seamless gelatin or alginate capsules, the covering of which dissolves very rapidly in the mouth or bursts when chewing. Production may take place, for example, as described in EP 0389700 A1, U.S. Pat. Nos. 4,251,195, 6,214,376, WO 2003 055587 or WO 2004 050069 A1.
The capsules, however, may also represent micro-capsules. “Microcapsules” are understood to be spherical aggregates with a diameter of about 0.1 to about 5 mm which contain at least one solid or liquid core surrounded by at least one continuous membrane. More precisely, they are finely dispersed liquid or solid phases coated with film-forming polymers, in the production of which the polymers are deposited onto the material to be encapsulated after emulsification and coacervation or interfacial polymerization. In another process, liquid active principles are absorbed in a matrix (“microsponge”) and, as microparticles, may be additionally coated with film-forming polymers. The microscopically small capsules, also known as nanocapsules, can be dried in the same way as powders. Besides single-core microcapsules, there are also multiple-core aggregates, also known as microspheres, which contain two or more cores distributed in the continuous membrane material. In addition, single-core or multiple-core microcapsules may be surrounded by an additional second, third etc. membrane. The membrane may consist of natural, semisynthetic or synthetic materials. Natural membrane materials are, for example, gum arabic, agar agar, agarose, maltodextrins, alginic acid and salts thereof, for example sodium or calcium alginate, fats and fatty acids, cetyl alcohol, collagen, chitosan, lecithins, gelatin, albumin, shellac, polysaccharides, such as starch or dextran, polypeptides, protein hydrolyzates, sucrose and waxes. Semisynthetic membrane materials are inter alia chemically modified celluloses, more particularly cellulose esters and ethers, for example cellulose acetate, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose and carboxymethyl cellulose, and starch derivatives, more particularly starch ethers and esters. Synthetic membrane materials are, for example, polymers, such as polyacrylates, polyamides, polyvinyl alcohol or polyvinyl pyrrolidone.
Examples of known microcapsules are the following commercial products (the membrane material is shown in brackets) Hallcrest Microcapsules (gelatin, gum arabic), Coletica Thalaspheres (maritime collagen), Lipotec Millicapseln (alginic acid, agar agar), Induchem Unispheres (lactose, microcrystalline cellulose, hydroxypropylmethyl cellulose), Unicetin C30 (lactose, microcrystalline cellulose, hydroxypropylmethyl cellulose), Kobo Glycospheres (modified starch, fatty acid esters, phospholipids), Softspheres (modified agar agar) and Kuhs Probiol Nanospheres (phospholipids).
The active principles are released from the microcapsules by mechanical, thermal, chemical or enzymatic destruction of the membrane, normally during the use of the preparations containing the microcapsules. Despite the fact that the state of the art a huge range of possibilities for the encapsulation of actives, methods according to which a shell is obtained by coazervation, precipitation or polycondensation of anionic and cationic polymers has been quite suitable for the formation of stable capsules. Particularly, a preferred process for the encapsulation of active principles according to the present invention is characterised in that it comprises the steps of
Of course, anionic and cationic polymers in steps (a) and (c) can be exchanged.
In a preferred embodiment of the invention a 1 to 10 and preferably 2 to 5% by weight aqueous solution of the gel former, preferably agar agar, is normally prepared and heated under reflux. A second aqueous solution containing the cationic polymer, preferably chitosan, in quantities of 0.1 to 2 and preferably 0.25 to 0.5% by weight and the active principle in quantities of 0.1 to 25 and preferably 0.25 to 10% by weight is added in the boiling heat, preferably at 80 to 100° C.; this mixture is called the matrix. Accordingly, the charging of the microcapsules with active principles may also comprise 0.1 to 25% by weight, based on the weight of the capsules. If desired, water-insoluble constituents, for example inorganic pigments, may also be added at this stage to adjust viscosity, generally in the form of aqueous or aqueous/alcoholic dispersions. In addition, to emulsify or disperse the active principles, it can be useful to add emulsifiers and/or solubilisers to the matrix. After its preparation from gel former, cationic polymer and active principle, the matrix optionally is very finely dispersed in an oil phase with intensive shearing in order to produce small particles in the subsequent encapsulation process. It has proved to be particularly advantageous in this regard to heat the matrix to temperatures in the range from 40 to 60° C. while the oil phase is cooled to 10 to 20° C. The actual encapsulation, i.e. formation of the membrane by contacting the cationic polymer in the matrix with the anionic polymers, takes place in the third step. To this end, it is advisable to wash the matrix—dispersed in the oil phase—with an aqueous ca. 0.1 to 3 and preferably 0.25 to 0.5% by weight aqueous solution of the anionic polymer, preferably the alginate, at a temperature in the range from 40 to 100 and preferably 50 to 60° C. and, at the same time, to remove the oil phase if present. The resulting aqueous preparations generally have a microcapsule content of 1 to 10% by weight. In some cases, it can be of advantage for the solution of the polymers to contain other ingredients, for example emulsifiers or preservatives. After filtration, microcapsules with a mean diameter of preferably 1 to 3 mm are obtained. It is advisable to sieve the capsules to ensure a uniform size distribution. The microcapsules thus obtained may have any shape within production-related limits, but are preferably substantially spherical.
The pharmaceutical and cosmetic compositions of the present invention are preferably dermatological compositions, which are preferably administered topically to the skin. Therefore, an object of the invention is a method for cosmetically affecting a skin care treatment comprising topically applying to the skin a skin care efficient amount of one or more of a compound of formula (I), respectively formula (II).
Another object of the invention is a method for the cosmetically treatment of the epidermis of human beings suffering from the signs of aging, suffering from dry skin and for the prevention of free radical formation on skin, comprising topically administering an effective amount of one or more compounds of formula (I)), respectively formula (II) to the epidermis.
In a further embodiment the present invention also refers to a process for preparing a cream, especially intended for topically treatment of skin to reduce signs of aging, reduce dry skin and maladies associated with free radical formation on skin, comprising blending one or more compounds of formula (I), respectively formula (II) with a cream base.
The term “cream base” means a basic formulation comprising ingredients, which are mentioned above, thus the formulation, can be used as cream or lotion or ointment.
Further, a process is preferred for the cosmetical or dermatological treatment of the skin, comprising applying a cosmetic or pharmaceutical composition of the present invention to the skin.
Manufacturing of the Succinate Derivates
Preparation according to method 1 with diethylamine, Yield: 50%.
Preparation according to method 1 with 2,3,6-trimethylcyclohexanol, Yield: 60%.
Preparation according to method 1 with ethylamine hydrochloride, Yield: 40%.
Preparation according to method 1 with 2-isopropylcyclohexanol, Yield: 55%.
Preparation according to method 1 with ammonium hydrochloride, Yield: 50%.
Preparation according to method 2 with piperidin, Yield: 55%.
In analogy to above-mentioned procedure ethyl malonyl chloride reacts with dimethylamine in THF and the resulting intermediate is transferred through transesterification with homomenthol under catalysis of sodium methylate to the end-product. Yield: 40%.
Preparation according to method 1 with glutaric anhydride. Yield: 60%.
NHDF (normal human dermal fibroblasts) cells are disseminated in a 96-well microtiter plate in a concentration of 1×105 cells/well (DMEM, 10% FCS). After cultivation for 24 h at 37° C. and 5% CO2 in DMEM medium, enriched with 10% FCS, various concentrations of the test substances and are added and incubated for a further 72 h. The maximum concentration of the test substances used corresponds from 0.3 times of the IC20 value of the cytotoxicity assay. The 20S Proteasome Assay (AAT Bioquest, Inc.) is performed according to the manufacturer's protocol. The Amplite™ Flourometric 20S Proteasome Assay Kit is a homogenous fluorescent assay that measures the chymotrypsin-like protease activity associated with the proteasome complex. This kit uses LLVY_R110 as a flourogenic indicator for proteasome activity. Cleavage of LLVY-R110 by proteasome generates the strongly green fluorescent R110 that is monitored fluorometrically. The fluorescence of the blank wells with growth medium was subtracted from the value for those wells with the cells. The results of the cells with treatment with different substances were compared with untreated cells.
HHFSC (Human Hair Follicle Stem Cells) isolated from hair follicle bulge (Celprogen) were cultivated in culture ware pre-coated with Human Hair Follicle Stem Cell Extra-cellular Matrix (Celprogen). The cells were incubated 2 h prior to and 16 h after UVB irradiation with test compounds. Cells were irradiated with 25 mJ/cm2 UVB in the presence of buffer solution. Apoptosis induction was evaluated by caspase 3/7 protein expression (Caspase-Glo 3/7, Promega) and quantified by chemiluminescence measurement.
The inhibition of apoptosis induction in the presence of test substances was calculated according to the following equation:
The abbreviations have the following meanings:
DCF Assay (in vitro)
Primary human dermal fibroblasts (Lonza) are disseminated in a 96-well microtiter plate in a concentration of 1×104 cells/well (DMEM, 10% FKS). After cultivation in fully enriched medium (DMEM, 10% FCS) for 24 h at 37° C. and 5% CO2, the serum content was reduced to 0.1% for further 24 h to synchronize the cell cycle. Confluence was supposed to be around 70% at the time, the incubation with the test substances began. Various concentrations of the test substances, two backgrounds (untreated cells), control (possible with solvent) and Trolox as internal standard (1000 μM) were applied to the cells in DMEM and incubated for further 18-20 h. After 24 h of incubation, 100 μl H2DCF-DA-solution (2,7-Dichlorfluorescein, 10 μM) incl. DAPI (1:1000) was added to all samples (excluded the background-control) and incubated for one hour to de-esterify the H2DCF-DA by cellular esterases. The resulting H2DCF was thereby trapped inside the cell. After the incubation, the cells were washed and the prooxidant challenge was set (1 mM, 1 h). The resulting fluorescence was read at Ex 504 nm; Em 524 nm. An increased level of ROS (reactive oxygen species) led to an increased amount of fluorescence.
Results are mean values from 2 independent experiments with quadruple determinations each.
The inhibition of the oxidation in the presence of test substances was calculated according to the following equation:
The abbreviations have the following meanings:
NHDF (normal human dermal fibroblasts) cells were disseminated in a 6-well plate in a concentration of 2×105 cells/well (DMEM, 10% FCS). After cultivation in fully enriched medium (DMEM, 10% FCS) for 72 h at 37° C. and 5% CO2, the serum content was reduced to 0.1% to synchronize the cell cycle. Various concentrations of the test substances, the negative control (untreated cells) and tert. butylhydrochinon as positive control, are added and incubated for a further 24 h. The maximum concentration of the test substances used corresponds to 0.2 times the value of the IC20 of the cytotoxicity assay. After cell lysis, the protein amount was determined using the Biorad BCA assay. All samples were adjusted to the same protein level before application on a fast Criterion Gel (Biorad) to perform electrophoresis for 20 minutes at 300 V. Thereafter the proteins were transferred to a PVDF membrane on a semi-dry blotter (30 minutes, 25 V). The blotted membrane is blocked for 4 hours in a 5% milk powder solution in TBST at 4° C. After washing, the membrane is incubated with the first antibody solution HO-1 from abcam (1:500 in 1% milk powder in TBST) over night. After this, washing is repeated and the membrane has to be incubated for 1 h in the second antibody solution (goat anti mouse coupled to HRP; 1:800 in 1% milk powder). After washing, the membrane is exposed to chemiluminescence HRP-substrate solution for 5 minutes. The resulting band pattern is detected with a chemiluminescence sensitive camera system (Vil-ber Loumat). The quantification was done by densitometry, using the Image J freeware software.
NHDF (normal human dermal fibroblasts) cells were disseminated in a 96-well microtiter plate in a concentration of 2×104 cells/well (DMEM, 10% FCS). After cultivation in fully enriched medium (DMEM, 10% FCS) for 72 h at 37° C. and 5% CO2, the serum content was reduced to 0.1% to synchronize the cell cycle and incubated for further 24 h. Various concentrations of the test substances and TGF-beta1 as internal standard are added and incubated for 72 h. The maximum concentration of the test substances used corresponds to 0.2 times the value of the IC20 of the cytotoxicity assay. Hyaluronic acid is quantified by a competitive ELISA (TECOmedical TE1017).
The percent bound for each standard and unknown substance is calculated using the following equation:
The abbreviations have the following meanings
RFU (standard or test substance): Absorption of the wells with standard or test substance
m and b values from the standard curve y=mx+b
A standard curve is generated by plotting the aborptions of the given concentrations in a linear proportion. The gradient of the line is determined and the value of the substances calculated as follows:
B16V mouse melanoma cells are disseminated in a 96-well micro-titre plate in a concentration of 7.5×103 cells/well. After cultivation for 24 h at 37° C. and 5% CO2 in RPMI medium, enriched with 10% foetal calf serum, various concentrations of the test substances and 0.6 mM tyrosine and 10 nM-MSH (-melanocyte stimulating hormone) are added and incubated for a further 96 h. The maximum concentration of the test substances used corresponds to 0.1 times the value of the IC20 value of the cytotoxicity assay. Only tyrosine and -MSH are added to the controls. After incubation, sodium hydroxide solution (final concentrations: 1 M) is added to the culture medium and the absorption (A) is measured after 3 h at 400 nm.
The inhibition of pigmentation in the presence of the test compounds was calculated using the following equation:
Inhibition of pigmentation (%)=100−[(Atest compound/Acontrol)×100]
wherein
From the inhibition of pigmentation (%) in a series of dilutions of test compounds, the IC50 for each test compound is calculated. This is the concentration of a test compound at which pigmentation is inhibited by 50%.
Aloe Vera Gel
Aloe
Barbadensis
Persea
Gratissima
Triticum
Vulgare
Guarana
Paullinia
Cupana Seed
Hamamelis
Virginiana
Hamamelis
Virginiana
Rosmarinus
officinalis
Vesiculosus
Macadamia
Ternifoia Seed
Glycine Soja
Butyrospermum
Parkii
Helianthus
Annuus
Prunus dulcis
Rubus
Fruticosus
Zingiber
Officinale
Brassica
Campestris
Aloe
Barbadensis
Rubus Idaeus
| Number | Date | Country | Kind |
|---|---|---|---|
| 13171161 | Jun 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/061561 | 6/4/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/198602 | 12/18/2014 | WO | A |
| Number | Date | Country |
|---|---|---|
| 1 493 336 | Jan 2005 | EP |
| 2010112710 | Oct 2010 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20160128923 A1 | May 2016 | US |
