Patent Application: US-201414309596-A

Abstract:
non - therapeutic cosmetic or dermatological use of at least one of a compound selected from the group consisting of : gibberellic acids , naringenins , n - acetylaspartic acids , β - aescin , arachadonic acid , quercetin , vitexin and docosahexaenoic acid . suitably , the use is in the treatment of , and / or prevention of , at least one sign of skin ageing or at least one sign of a skin damage condition associated with ageing , wherein the sign of skin ageing or skin damage is present on skin of the face , body or the scalp of a subject .

Description:
the inventors have identified retinoic acid - mimicking compounds which have similar gene - regulating signatures to retinoic acid and which may be used in compositions for cosmetic use . in particular , they have identified ingredients which can help protect against and alleviate the signs of aging . they have demonstrated hitherto unknown biological activity for the identified ingredients using an array of in vitro tests . the in vitro tests explored the influence of the compounds of the invention on key biological markers in the skin which are known to have an influence on skin aging . moreover , and advantageously , the compounds of the invention were found to be non - irritating when applied in pure form to the skin . matrix metalloproteinase [ mmps ] inhibitors — targets wrinkles and sagging skin through inhibition / reduction of collagen degradation uv irradiation increases the expression of matrix metalloproteinase enzymes in the skin ( fisher et al . 1997 ; farage et al . 2008 ). mmps are capable of degrading all kinds of extracellular matrix proteins ( fisher et al . 1997 ). the mmps are initially synthesized as inactive enzymes with a pro - peptide domain that must be removed before the enzyme is active . under normal conditions mmps are involved in the remodeling of the extracellular matrix , for example , in tissue repair , but when induced in excess by uv light or via inflammatory stimulation , mmp activity causes degradation and tissue - remodeling to an extent which results in connective - tissue damage and pre - mature aging ( kahari and saarialho - kere 1997a ; fisher et al . 1997 ). different types of mmp have different substrate preferences . several mmps are involved in the degradation of collagen : mmp - 1 starts by cleaving collagen , which is then further degraded by mmp - 3 and mmp - 9 . also , mmp - 2 , mmp - 8 , and mmp - 13 are involved in the degradation of collagen ( kerkyliet et al . 1999 ). compounds which inhibit mmp expression will prevent or reduce mmp induced collagen loss or destruction . in view of the fact that the compounds of the invention have been shown to inhibit mmp expression ( fig2 & amp ; fig3 ), the compounds can therefore be used to prevent / or alleviate the formation of skin wrinkles and skin laxity associated with collagen lost or destruction . a further advantage being that healthy skin integrity is maintained . pro - collagen 1 — targets wrinkles and sagging skin through promotion of collagen synthesis uv light not only mediates the destruction of collagen it also impairs the ongoing collagen synthesis by inhibiting the gene expression of procollagen ( kahari and saarialho - kere 1997b ). the compounds of the invention , gibberellic acid , naringenin , n - acetylaspartic acid , β - aescin , arachadonic acid , quercetin , vitexin and docosahexaenoic acid ( ethyl ester ) have been shown to promote pro - collagen 1 expression ( fig4 ). in particular , naringenin , gibberellic acid and n - acetylaspartic acid , dee and be have been found to be particularly good pro - collagen expression promoters . the results indicate that the compounds of the invention , can be used to prevent effects of collagen reduction in the skin , that is , prevent or alleviate the formation of skin wrinkles and fine lines , reduce the severity of skin wrinkles and prevent against skin laxity and other signs associated with reduced collagen levels . with aging including photoaging , the skin is known to become more susceptible to inflammation . inflammation is a defence mechanism for the skin against various forms of attack : physical , chemical , and biological . uv light induces collagen degradation through increased mmp production , and inflammation also results in an increased expression of mmp ( bennett et al . 2008 ; kahari and saarialho - kere 1997b ). pro - inflammatory cytokines , including il - 6 , are mediators inducing the expression of several mmp &# 39 ; s , including mmp - 1 , mmp - 2 , mmp - 3 , mmp - 9 ( dasu et al . 2003 ; kossakowska et al . 1999 ; parks et al . 2004 . il - 6 has also been implicated in the uv - induced induction of the collagen degrading mmp - 1 ( wlaschek et al . 1993 ). interleukin - 6 ( il - 6 ) is produced at sites of inflammation ( kessler - becker et al . 2004 ), and the levels of this cytokine are elevated in inflammatory skin conditions such as psoriasis ( grossman et al . 1989 ). expression of il - 6 has also been correlated to the epidermal hyperplasia seen in psoriatica skin . epidermal hyperplasia is one of the features associated with photoaging skin ( rijken and bruijnzeel 2009 ). inflammation also results in the generation of reactive oxygen species ( ros ), which is involved in several destructive processes in the skin , further accelerating the aging process ( meier et al . 1989 ) ( conner and grisham 1996 ). an effective treatment for wrinkles needs to address the damage caused by photo damage , but also the inflammation induced damage arising from pro - inflammatory cytokine production and enhanced mmp production arising from same , and ros generation arising from the inflammatory process . the compounds of the invention , and in particular , gibberellic acid , naringenin , n - acetyl aspartic acid , have been shown to inhibit production of the pro - inflammatory cytokine interleukin - 6 ( fig6 ) in dermal fibroblasts . therefore the compounds of the invention , and particularly , naringenin , gibberellic acid and n - acetylaspartic acid can be used to prevent against and alleviate the symptoms of inflammation and related inflammatory photo - aging symptoms , such as wrinkle formation and skin darkening . skin pigmentation is due to the presence of melanin , ( a pigment produced in the epidermis by the hydroxylation of tyrosine by the enzyme tyrosinase ). melanin pigments are made in specialized cells called melanocytes , packed in organelles called melanosomes that are transferred to keratinocytes to assure proper diffusion throughout the epidermis . the terms “ whitening / pigmentation agents ” refer to active agents that are able to reduce or modulate skin pigmentation by limiting melanin production , inhibiting melanosome maturation and transfer , or by stimulating pigment degradation . skin lightening products are commercially available for cosmetic purposes in order to obtain lighter skin complexion . clinically they are also used for treatment of hyperpigmentary disorders such as melasma , café au lait spot and solar lentigo . all of these target naturally melanin production and many of the commonly used agents are known as competitive inhibitors of tyrosinase , one of the key enzymes in melanogenesis ( hearing and jimenez 1987 ;). however , melanogenesis is not only controlled by tyrosinase , instead there are several factors that play an important role for darkening the skin ( gillbro and olsson 2011 ) such as the transfer of melanosomes from melanocytes to the surrounding keratinocytes or paracrine production of melanogeneic factors from keratinocytes . ( thody and graham 1998 ) ( lerner and mcguire 1961 ; lerner et al . 1954 ; lerner and mcguire 1964 ) ( schauer et al . 1994 ). ( jabbour 2003 ; levine et al . 1991 ) ( abe et al . 1969 ; busca and ballotti 2000 ; hunt et al . 1994 ; im et al . 1998 ). one important mechanism seems to be paracrine production of granulocyte - macrophage colony - stimulating factor ( gm - csf ). keratinocytes responds to exposure of uva and uvb with increased production of gm - csf ( imokawa et al . 1996 ; hirobe et al . 2004 ). melanocytes possess specific binding sites for gm - csf , and once bound gm - csf increases pigment production in melanocytes ( imokawa et al . 1996 ). even thus uva and uvb activate keratinocytes differently both uva and uvb results in the production of gm - csf . thus gm - csf is important for the pigmentation of the epidermis and can be seen as a biomarker for skin pigmentation . gm - csf has been shown to play an important role in inflammation and inhibition of gm - scf has been shown to decrease skin inflammation ( hamilton 2008 ). it has also been suggested that gm - csf plays a part in sustaining inflammatory response , by inhibiting the apoptosis of monocytes , thereby contributing to the transition from acute to chronic inflammatory skin disease ( bratton et al . 1995 ). the compounds of the invention have been shown to inhibit gm - csf ( fig7 & amp ; fig8 ) and in and particular , naringenin , gibberellic acid and n - acetylaspartic acid have been shown to have inhibitory action on gm - csf . the compound of the invention can therefore be used to prevent unwanted skin pigmentation and / or to prevent inflammation - induced redness associated with elevated levels of gm - scf which can arise from aging and uv damage . photo damage and sunburn is a major cause of extrinsic aging . excessive uvb exposure is accompanied by erythema ( redness ), edema ( puffiness ), vasodilatation and angiogenesis as a result of increased vascular permeability . vascular endothelial growth factor ( vegf ) is the main mediator of these changes ( hirakawa et al . 2005 ). also moderate levels of uvb light induce the expression of vefg ( brauchle et al . 1996a ). the expression of vegf is also induced by inflammation ( salven et al . 2001 ). vegf is induced by uv irradiation in both skin fibroblasts and keratinocytes ( trompezinski et al . 2001 ) whereas inflammation - induced vegf is mainly expressed in dermal fibroblasts . over expression of vegf from uv - irradiated skin or inflammatory activated skin may contribute to dilated microvasculature , a common feature of aging skin . edema is the cause behind puffiness and results from the increased vascular permeability induced by vegf ( phelps 1990 ; infanger et al . 2004 ). rosacea is a common skin condition characterized by vascular abnormalities in the skin , leading to erythema and telangiectasis ( broken capillaries ). since rosacea is a very visible condition the impact on quality of life for affected persons are large . receptors for vegf are typically expressed in rosacea skin ( smith et al . 2007 ). previous research has showed reduction in the size and number of vessels in an experimental model of psoriasis after vegf blockade ( schonthaler et al . 2009 ). free radical is another threat against younger healthy looking skin . h 2 o 2 is a strongly oxidizing compound and leads the formation of free radicals . it has been shown that vegf is induced also by h 2 o 2 ( brauchle et al . 1996b ). mediators produced during inflammation , such as il - 6 , result in the occurrence of free radicals and h 2 o 2 ( winrow et al . 1993 ), as well as vegf ( cohen et al . 1996 ). reducing the amount of vegf , both from uv irradiation and inflammation , is hence a way to combat redness and puffiness and superficial veins . the compounds of the invention have been shown to inhibit vegf production ( fig9 ) in and particular , naringenin , gibberellic acid and n - acetylaspartic acid have been shown to have inhibitory effect on vegf . therefore the compounds of the invention can be used to combat skin redness caused by uv irradiation or inflammatory action , puffiness around the eyes and broken veins , all of which have an ageing effect on the skin appearance . members of the epidermal growth factor ( egf ) family are important growth factors involved in epithelialization during cutaneous wound healing . heparin - binding egf - like growth factor ( hb - egf ), a member of the egf family , has been shown to play an important role in skin wound healing where it functions by accelerating keratinocyte migration , rather than proliferation ( shirakata et al . 2005 ). the compounds of the invention have been shown ( see table 3 ) to stimulate heparin - binding epidermal growth factor production ( on a gene level ) and therefore can be use can improve the skin wound healing process . table 3 demonstrates gene data for hbegf stimulation by naringenin when topically applied to skin explants , demonstrating skin delivery from topical application on the skin and resulting gene regulation . connective tissue growth factor ( ctgf ) is an ecm - associated heparin - binding protein that binds directly to integrins . it is synthesized by fibroblasts and stimulates proliferation and chemotaxis of these cells . it has also been demonstrated that ctgf is required for reepithelialization in wound healing by promoting cell migration ( igarashi et al . 1993 ). in addition , ctgf is a strong inducer of ecm proteins , such as collagen type i and fibronectin and their integrin receptors (( seeker et al . 2008 ). the compounds of the invention have been shown ( see table 3 ) to stimulate connective tissue growth factor production ( on a gene level ) and thus can be used to improve and / or enhance the skin wound healing process . table 3 demonstrates gene data for ctgf stimulation by the compounds of the invention when topically applied to skin explants , demonstrating skin delivery from topical application on the skin and resulting in gene regulation . autologous fibronectin has a role in promoting healing of chronic skin and corneal ulcers . fibronectin assists in wound healing by contributing to haemostasis , assisting in control of infection and debridement of wounds , and promoting re - epithelialisation , granulation tissue and ultimately a connective tissue of adequate tensile strength to repair the skin defect . the compounds of the invention have been shown to stimulate fibronectin production and particular , arachidonic acid , β - aescin and docosahexaenoic acid ( ethyl ester ) ( fig1 ) have been shown to stimulate higher fibronectin production than retinoic acid . the compounds of the invention can therefore be used to improve the skin wound healing process . fibroblast growth factor ( fgf ) is one of the growth factors involves in the wound healing process and has been showed to help restore deficiencies in impaired wound healing and it has been suggested as a treatment for patients with deficient wound repair . also plasminogen activator ( plat ) is activated by all - trans retinoic acid , triple a and ga . the activation of the plasminogen activator system may be one mechanism by which all - trans retinoic acid exerts beneficial effects in cutaneous wound healing . the compounds of the invention , and in particular , naringenin , gibberellic acid and n - acetylaspartic acid have been shown to stimulate fibroblast growth factor production ( fig1 ). naringenin , gibberellic acid and n - acetylaspartic acid have been shown to result in higher fgf production than retinoic acid . therefore these compounds used improve the skin wound healing process . skin renewal begins with the generation of new keratinocytes from stem cells residing in the stratum basale , the inner layer of the epidermis . as new cells keep forming , keratinocytes migrate upward and differentiate into corneocytes . keratin , which is a highly fibrous protein , is produced during the differentiation process and causes cell walls to harden , forming the stratum corneum ( sc ). the terminal differentiation of keratinocytes ultimately results in cell death . old corneocytes are shed from the sc through desquamation . aging is associated with reduced epidermal proliferation . kallikreins are serine proteases and thought to be responsible for the proteolysis of desmosomes during desquamation ( komatsu et al . 2005 ). kallikrein 8 ( klk8 ) is involved both in epidermal proliferation and corneocyte shedding ( kishibe et al . 2007 ) both directly , and through the induction of klk 6 and klk 7 . although ra is very effective stimulating the desquamation process by inducing high production of kallikrein it also known to be somewhat pro - inflammatory which is activating other processes involved in the aging of the skin negatively . the compounds of the invention , arachidonic acid , quercetin , n - acetyl aspartic acid , docosahexaenoic acid ( ethyl ester ), gibberellic acid , naringenin and β - aescin have been shown to induce production of kallikrein 8 ( fig1 ). in particular , naringenin , gibberellic acid and n - acetylaspartic acid have good inducing effects . the compounds of the invention can therefore be used to enhance skin desquamation , improve skin hydration , skin luster and brightness . skin is composed of two layer , the outer epidermis and the inner dermis ( proksch et al . 2008 ) keratinocytes compose the outermost layer of the epidermis , the stratum corneum , which provides a physical and chemical bather towards the environment , preventing the entry of foreign substances and the loss of water and important constituents of the skin and body ( borgono et al . 2007 ). the keratinocytes in the inner layer of the epidermis , the basal layer , are proliferating and undifferentiated . when they start to differentiate they exit the cell cycle and start moving upward in the epidermis towards the surface of the skin ( fuchs 2007 ). the keratinocytes are linked to each other by cohesive mechanical junctions named desmosomes ( sandjeu and haftek 2009 ). keratin 1 and keratin 10 are early markers of keratinocyte differentiation . keratin 1 and keratin 10 work as a dimer ( keratin 1 / 10 ) and are expressed together . as the differentiation process continues the expression of keratin 1 and keratin 10 is downregulated again and is replaced by other molecules i . e . involucrin and filaggrin ( lu et al . 1994 ). there is evidence suggesting that increased levels of keratin 1 / 10 are associated with reduced tumorigenesis ( tiano et al . 2002 ).] with differentiation , keratinocytes move upward and became granular keratinocytes ( gks ). gks produce almost all of the proteins and lipids required for the protective bather function undergoing a specially programmed cell death called cornification ( toulza et al . 2007 ). the cornification process gives rise to corneocytes , they are devoid of cell organelles and mainly consist of keratin . eventually the corneocytes at the outermost layer of the stratum corneum are gradually shed and replaced by newly - keratinised cells in a process called desquamation ( hara et al . 2011 ). to maintain homeostasis and a functional bather the delicate balance between proliferating and differentiating keratinocytes need to be tightly regulated . the compounds of the invention , arachidonic acid , quercetin , n - acetyl aspartic acid , docosahexaenoic acid ( ethyl ester ), gibberellic acid , naringenin and β - aescin have been shown have been shown to stimulate production of keratin 1 / 10 . the compounds of the invention can therefore be used to maintain healthy skin condition and hydration , skin luster and brightness . connectivity mapping approach identifies compounds with retinoic acid - like gene regulation behaviour a connectivity mapping ( cmap ) approach was used to identify retinoid - like compounds . gene expression profiling was performed on skin biopsies from healthy female subjects which were topically applied with tretinoin ( all - trans retinoic acid ) in a concentration of 0 . 025 % ( retin - a ® ex . johnson & amp ; johnson ) for one week . gene signatures obtained from the tretinoin treated skin were queried in the cmap database . compounds identified which ranked highly in the cmap were topically applied on skin explants received from breast plastic surgery . gene arrays were repeated on the treated samples 24 hours after topical application . skin biopsies were taken from sun - exposed forearms of 8 healthy caucasian female subjects , aged 50 + years . patients were treated with tretinoin ( retin - a ® concentration 0 . 025 %) for one week . at the end of the week 3 mm punch biopsies were taken from each site of treatment . the study was vehicle controlled . two biopsies were taken per treatment , per arm , for sufficient rna concentration . biopsies were immediately stored under controlled conditions at 4 ° c . for 24 hours prior to rna extraction . subcutaneous fat from full thickness skin , obtained from surgical waste material from breast reduction procedures , was removed with a scalpel . three healthy caucasian female donors were used for this study , aged 23 , 41 and 60 years . the female donors differed from those of the skin - treatment study outlined previously 8 mm punch biopsies were taken from the skin ( and these biopsies are now referred to as skin explants ). skin explants were put on milipore cell culture inserts ( 12 mm diameter ). inserts containing skin explants were put in 6 - well plates ( one insert / well ) and 1 ml of supplemented keratinocyte medium ( ex cascade biologics ) was added to each well . a tretinoin - containing cream ( aberela ® ex . janssen ): 0 . 05 % tretinoin in the cream , was applied topically to each explant ( 5 μl / cm 2 ) using a positive displacement pipette . a placebo cream without tretinoin was used as a control . the skin explants were incubated with tretinoin for 24 hours at 37 ° c . in 5 % co 2 - humidified air . viability of explants was controlled using parallel alamar blue test using suppliers protocol ( ex . invitrogen ) a hand - held qiagen tissue ruptor mini kit was used for rma extraction from skin biopsies as per the supplier protocol . qiagen tissue ruptor was used to disrupt the tissue in qiazol ( ex . qiagen ) according to the supplier protocol . qiagen minikit was used for rna extraction according to the manufacturer &# 39 ; s protocol . rna was stored in − 80 ° c . prior to cdna conversion . rna concentration was measured with nd - 1000 spectrophotometer ( ex . nanodrop technologies ) and rna quality was evaluated using the agilent 2100 bioanalyzer system ( ex . agilent technologies ). 250 nanograms of total rna from each skin or skin explant sample was used to prepare biotinylated fragmented crna according to the genechip ® 3 ′ ivt express kit manual ( pn702646 rev 1 , ex . affymetrix inc .). affymetrix genechip ® expression arrays ( human genome u133 plus 2 . 0 ) were hybridized for 16 hours in a 45 ° c . incubator , rotated at 60 rpm . according to the genechip expression wash , stain and scan manual ( pn 702731 rev 2 , ex . affymetrix inc .) the arrays were washed and stained using the fluidics station 450 and scanned using the genechip ® scanner 3000 7g . analysis of the gene expression data was carried out in the statistical computing language r ( http :// www . r - project . org ) using packages available from the bioconductor project ( www . bioconductor . org ). the raw data was normalized using the robust multi - array average ( rma ) method . in order to search for differentially expressed genes between the x samples and the y samples group an empirical bayes moderated t - test was then applied using the ‘ limma ’ package . the findings from the cmap analysis connected the gene signature of retinoic acid - treated human skin to novel potential compounds with the ability to mimic the activity of retinoic acid . many genes were individually shown to be regulated by all - trans retinoic acid . due to the intervariable differences in the patient material from the explants study and clinical study , separate approaches were taken . the gene expression profiles in biopsies after all - trans retinoic acid treatment compared with vehicle - treated controls were examined by dna microarray analysis . two separate studies on gene expression profiles in human skin after topical application of all - trans retinoic acid were conducted . the first study was performed ex vivo by topically applying all - trans retinoic acid for 24 hours on skin explants from 3 donors prior microarray conduction . the 2 nd study was a clinical in vivo study , topically applying all - trans retinoic acid for 1 week on 8 healthy volunteers . the genes that were upregulated more than 2 fold in the explants study and 1 , 4 - 2 fold in the clinical study were queried in cmap . to find structures that would putatively be able to mimic the activity of retinoic acid on the human skin , the gene signature from retinoic acid that was obtained by the previously explained explant study was queried in the cmap database . the details of the cmap database have been described by . lamb et al . 2006 ). in this method , the similarity between each researcher &# 39 ; s microarray results ( the query signature ) and 453 microarray results for 1309 compounds ( reference signatures ) in the cmap database was evaluated using the kolmogorov - smirnov statistic , a nonparametric , rank - based pattern - matching strategy . each reference signature in the database was scored according to its similarity with the query signature , and the extent of the similarity is described as the “ connectivity score .” the connectivity score ranges from — 1 to — 1 ; nearer to — 1 means higher similarity , 0 means no similarity , and nearer to — 1 means opposite similarity . for the query , the probe id defined by the affymetrix genechip human genome u133a array ( affymetrix , santa clara , calif ., usa ) was used . here , the probe id of u133a corresponding to the signatures of 24 hours in explants and 1 week stimulation in an in vivo clinical study queried in the cmap database via the internet ( http :// www . broad . mit . edu / cmap ). “ permutated results ” results ,” which consist of the arithmetic means of the connectivity scores and the statistical significance of the replicates ( calculated as “ enrichment ” and “ permutation value ” at the cmap ), were used to evaluate the significance of the scores . the the top 100 high ranked compounds that resulted for the query of the two studies described earlier ( explants study and clinical study ) were further analyzed following distinct selection criteria i . e . an enrichment value & gt ; 0 . 495 . the resulting suggestions of structures were searched for compounds of natural origin . agents with reported drug use such as atropin , digoxigenin or oleandomycin , substances with known toxicity such as solanin or tomatidin as well as compounds derived from animal origin were dismissed . resulting from this virtual exercise a total of 8 agents was selected to be taken into further investigation as potential retinoic acid like acting ingredients ( table 1 ). importantly , in both study results all - trans retinoic acid ( tretinoin ) appeared as suggested compound with very high ranking ( cmap ranking number 4 in the explants study , and cmap ranking number 1 in the clinical study ) which gives a confirmation for the suitability of the model as a tool to find active agents with desired gene signature related activity . the in vitro skin permeation experiments were developed and validated according to the organization for economic co - operation and development adopted guideline 428 ( oecd guideline for the testing of chemicals . skin absorption : in vitro method . guideline 428 ( paris , april 2004 , updated january 2011 )). for this investigation , static franz glass diffusion cells ( permegear , usa ) were used . these cells consist of donor and receptor chambers in between which a piece of porcine skin was positioned ( thickness 800 μm ). about 20 mg / cm 2 of each formulation were applied on the skin surface through the donor compartment in triplicate for each formulation . the pig skin samples were equilibrated for 30 minutes , and air bubbles were removed . the cells were kept at constant temperature with a circulating water flow at 37 ° c . and the receptor compartment content ( ethanol / water 50 : 50 ) was continuously agitated by magnetic stirrers . after an exposure time of 24 hours , the diffusion cells were dismounted and each active was analysed in each compartment . the non absorbable dose : the skin surface was washed with 10 . 0 ml of methanol and one tape - strip was made in order to remove the residual donor samples . the potentially absorbable dose : the tape stripping procedure was used in order to separate the stratum corneum from the skin sample . for this purpose , the sc of the treated area was removed by 14 successive tape - stripping using d - squam ™ ( φ = 14 mm , ex . cuderm ) with a constant pressure ( 225 g · cm 2 ) for 5 s . the strips were placed in amber vial with 4 ml methanol and shaken overnight the absorbable dose : after eliminating sc from skin samples , the skin was chopped into small pieces and the test substances were extracted with 2 ml of methanol for 24 - hours under constant shaking . the receptor fluid samples were diluted before the analysis . the day after the extraction process , all the samples were filtered through 0 . 22 μm membrane filter into hplc vials . the samples with gibberellic acid and naringenin , together with known concentration standards were assayed by hplc - uv ( agilent , usa ). the samples with n - acetyl aspartic acid and known concentration standards were assayed by lc / electrospray ionization - mass spectrometry ( lc / esi - ms ) ( shimadzu lc - load , thermoscientific ). the results ( fig1 ) showed that topical application of gibberellic acid , naringenin and n - acetyl aspartic acid 0 . 5 % w / w in a basic formulation for 24 hours , resulted in the penetration of each active into the skin . fig1 shows the range of each ingredient in the non absorbed dose ( skin surface + strip ), the potentially absorbable dose ( sc acting as a reservoir ) and in the absorbed dose ( quantity in the skin and in the receptor fluid ). mmp inhibition activity for compound of the invention was tested on human skin explants . skin explants were sourced from 2 healthy caucasian female breast skin donor of 46 and 33 years of age . a cream containing all - trans retinoic acid ( tretinoin ) ( 0 . 05 %) was added topically using positive displacement pipette ( 5 ul / cm 2 ) to each explants . a similar cream base without tretinoin was used as placebo control . n - acetyl aspartic acid and gibberellic acid were prepared in propylene glycol ( 1 %). solutions were kept under nitrogen and used immediately after preparation . propylene glycol was used as vehicle control for the compounds of the invention in this study . mmp - inhibitory activity for compounds of the invention was also assessed on human dermal fibroblasts . dermal fibroblasts were cultured in dulbecco &# 39 ; s modified eagle medium ( dmem ) ( invitrogen ) with 10 % ebs ( ex . invitrogen ) and 1 % pencillin / streptavidin ( ex . invitrogen ). to induce inflammation in the cells , pma ( phorbol myristate acetate ) ( ex . sigma ) was used at a concentration of 10 nm . cells were pre - treated with gibberellic acid ( ga ), naringenin ( ng ) or n - acetyl aspartic acid ( triple a ) or benchmark controls ( retinol and retinoic acid ( ra )) or a positive control for inhibition of inflammation : dexamethasone ( dex ) ( ex . sigma ) for 1 h before pma was added to the culture . the cells were stimulated for 24 h and the culture supernatant was collected and stored at − 20 ° c . until further analysis . fluorokine multianalyte profiling ( xmap ) using the bioplex ( ex . biorad ) dual laser flow - based sorting and detection analyzers manufactured by luminex corporation ( luminex , austin , tex .) was employed to measure levels of mmp in conditioned media samples of stimulated dermal fibroblasts . the technology incorporated polystyrene microspheres dyed internally with differing ratios of two spectrally distinct fluorophores to create a family of different spectrally addressed bead sets . the bead utilized in this study was conjugated with a biotinylated capture antibody specific for a unique mmps , the assay was performed without antecedent serum depletion . results were compared with medium from cells treated with vehicle only ( dmso ). the assays utilized a 96 - well microplate format and were processed according to the manufacturer &# 39 ; s protocol , including generation of a standard curve for each target prepared in background assay diluents . values that fell within the linear range of the calibration assay were accepted . samples were analyzed using the bio - plex suspension array system and bio - plex manager software 4 . 0 ( bio - rad laboratories , hercules , calif .). quantities were determined by comparison to standard curves obtained for the different mmp . naringenin , gibberellic acid and n - acetyl aspartic acid show an ability to inhibit mmp production which results in less collagen breakdown in the skin ( fig2 , 3 and 4 ). collagen loss contributes to unwanted features of skin aging and photo - induced aging , and through inhibition of mmp production the compounds of the invention will alleviate skin wrinkle and line formation and prevent the breakdown of skin integrity proteins which result in reduced skin elasticity and flaccidness . procollagen - 1 induction activity of arachidonic acid , quercetin , n - acetyl aspartic acid , docosahexaenoic acid ( ethyl ester ), gibberellic acid , naringenin and β - aescin ( anti - wrinkle , anti - photoaging ) fibroblast cells were cultured as outlined in example 3 . 1 and treated with compounds of the invention the secretion of pro - collagen in the cell culture supernatants were analyzed using pro - collagen 1 specific elisa (# 8003 ; ex . quidel ). results were compared with cell culture supernatants from dmso vehicle treated cells . the assays utilized a 96 - well microplate format and were processed according to the manufacturer &# 39 ; s protocol , including generation of a standard curve prepared in background assay diluents . absorbance of the assay dye was read at a synergy ht plate reader . values that fell within the linear range of the calibration assay were accepted . quantities were determined by comparison to standard curve obtained for pro - collagen 1 . fig5 shows procollagen - 1 stimulation in dermal fibroblasts after 24 hour stimulation with compounds of the invention . fig5 shows the average of three separate experiments for three different donors . n - acetyl aspartic acid and gibberellic acid significantly increase the production of pro - collagen type 1 , the precursor to collagen type 1 . docosahexaenoic acid ( ethyl ester ), naringenin and β - aescin display a convincing trend towards increasing the production of pro - collagen type 1 . positive stimulatory effect on fibronectin in human epidermal keratinocytes , arachidonic acid , β - aescin and docosahexaenoic acid ( ethyl ester ), ( anti - wrinkle ) keratinocytes were cultured in commercially available keratinocyte medium m154 ( cascade biologics / invitrogen ) # m154500 with a final calcium 0 . 05 um . supplements were added meeting following final concentrations of growth factors ; bovine pituitary extract ( bpe ), 0 . 2 % v / v · bovine insulin , 5 μg / ml · hydrocortisone , 0 . 18 μg / ml · bovine transferrin , 5 μg / ml · human epidermal growth factor , 0 . 2 ng / ml . medium was changed every other day . cells were grown to 80 % confluence prior stimulation with compounds . experiments were performed in 24 - well plates . the cells were treated with individual compounds of the invention for 6 days . after 3 days the medium was refreshed . a source of calcium ions was used as a positive control . after 6 days tissue culture medium was stored in − 20 ° c . until analysis . experimental method as outlined in example 3 . 1 was modified in order to analyze fibronectin levels extracted from keratinocytes stimulated for 6 days with all - trans retinoic acid , retinol , arachidonic acid , β - aescin , and dodecahexaenoic acid ( ethyl ester ) at a range of concentrations . experiments were performed in duplicates . fig6 shows stimulation of fibronectin in human keratinocytes stimulated with all - trans retinoic acid , retinol , arachidonic acid , β - aescin , docosahexaenoic acid ( ethyl ester ), and the positive calcium control . the compounds of the invention had superior effect on fibronectin release in human keratinocytes after 6 days stimulation compared to all - trans retinoic acid and retinol . retinoic acid is a known activator of fibronectin in skin ( schwartz and kligman 1995a ). decreased fibroblast contractile activity and reduced fibronectin expression are involved in skin photo - aging ( knott et al . 2010 ). interleukin inhibitory activity of gibberellic acid , naringenin and n - acetyl aspartic acid ( anti - inflammation , anti - redness , anti spider veins , combats puffiness around eyes .) human dermal fibroblast cell cultures were prepared according to example 3 . 1 . to induce inflammation in the cells pma ( phorbol myristate acetate ) ( ex . sigma ) was used at a concentration of 10 nm . cells were pre - treated with gibberellic acid ( ga ), naringenin ( ng ) or n - acetyl aspartic acid ( triple a ) or benchmark controls ( retinol and retinoic acid ( ra )) or a positive control for inhibition of inflammation : dexamethasone ( dex ) ( ex . sigma ) for 1 h before pma was added to the culture . the cells were stimulated for 24 h and the culture supernatant was collected and stored at − 20 ° c . until further analysis experimental method as outlined in example 3 . 1 was modified in order to analyze il - 6 levels extracted from dermal fibroblasts stimulated for 6 days with all - trans retinoic acid , retinol , arachidonic acid , β - aescin , and dodecahexaenoic acid ( ethyl ester ) at a range of concentrations . experiments were performed in duplicates . fig7 shows inhibition of interleukin - 6 [ il - 6 ] production in dermal fibroblasts by gibberellic acid , naringenin and n - acetyl aspartic acid . gibberellic acid , naringenin and n - acetyl aspartic acid inhibit interleukin - 6 production and therefore demonstrate an anti - inflammatory effect . the prevention of inflammation can help combat skin redness . anti - inflammatory action can have a positive effect on the reduction of vegf - induced anti - aging signs , such as spider veins and puffiness around eye region . gm - csf inhibition ( skin lightening activity of gibberellic acid , naringenin and n - acetylaspartic acid , prevention of inflammation - induced redness ) to induce inflammation in the cells pma ( phorbol myristate acetate ) ( ex . sigma ) was used at a concentration of 10 nm . cells were pre - treated with gibberellic acid ( ga ), naringenin ( ng ) or n - acetyl aspartic acid ( triple a ) or benchmark controls ( retinol and retinoic acid ( ra )) or a positive control for inhibition of inflammation : dexamethasone ( dex ) ( ex . sigma ) for 1 h before pma was added to the culture . the cells were stimulated for 24 h and the culture supernatant was collected and stored at − 20 ° c . until further analysis . experimental method as outlined in example 3 . 1 was modified in order to analyze gm - csgf levels in human dermals fibroblasts stimulated with all - trans retinoic acid , retinol , arachidonic acid , β - aescin , and dodecahexaenoic acid ( ethyl ester ) at a range of concentrations . experiments were performed in duplicates . fig8 shows inhibition of gm - csf production in human dermal fibroblasts by gibberellic acid , naringenin and n - acetyl aspartic acid ( and demonstrating anti - pigmentation activity ). full thickness skin from surgical waste material from breast reductions was collected . subcutaneous fat was removed with a scalpel . 8 mm punch biopsies was taken from the skin and referred to as skin explants . thereafter , the explants were put on millipore cell culture inserts ( 12 mm in diameter ). inserts containing skin explants were put in 6 - well plate ( 1 insert / well ) and 1 ml of supplemented keratinocyte medium ( cascade biologics ) was added to each well to allow survival and nutrition of the explants . the skin explants were treated with 1 % w / w solutions of gibberellic acid , n - acetyl aspartic acid and naringenin in propyleneglycol for 24 h , or with the benchmark control : tretinoin ( aberela ® ex . janssen ) and the corresponding placebo . after pre - treatment the explants were uv - irradiated with 120 mj / cm 2 uva and uvb and thereafter re - stimulated topically with gibberellic acid , naringenin and n - acetyl aspartic acid for 24 hours . supernatants were thereafter collected and analyzed for gm - csf production . fig9 shows the effect on topical treatment of skin explants with 1 % w / w solution of gibberellic acid , naringenin and n - acetyl aspartic acid in propylene glycol on the suppression of uv - induced expression of gm - csf . gibberellic acid , naringenin and n - acetylaspartic acid inhibit the paracrine production of the melanogenic factor gm - csf stimulated by either pma or uv in fibroblasts and skin explants . these results implicate the efficacy of the described ingredients in uv or inflammation - induced hyperpigmentation . inhibition of gm - csf production can reduce in reduction in skin pigmentation and uv - induced skin pigmentation , can reduce redness in the skin and provide a more even skin complexion . vegf - inhibition activity of naringenin , n - acetyl aspartic acid , gibberellic acid and β - aescin ( combating redness , broken veins , puffy eyes ) cell culture and cytokine analysis was carried out on human dermal fibroblasts as described in example 3 . 1 . the methodology was adapted in order analyze vegf production . na , ga and triplea all reduce inflammatory induced vegf synthesis in cultured dermal fibroblasts . in accordance with the known pro - inflammatory effect of ra an additional increase in vegf could be detected in samples treated with ra together with the inflammatory inducer pma . treatment with retinol did not have that effect , is resulted a small but insignificant decrease in vegf . treatment with be also resulted in a decreased vegf expression , although insignificant . fig1 — shows the inhibition of inflammation induced vegf by naringenin , n - acetyl aspartic acid , gibberellic acid and β - aescin . naringenin , n - acetyl aspartic acid , gibberellic acid and β - aescin inhibit vegf activity and are therefore useful as angiogenesis inhibitors for prevention of associated unwanted features of skin aging such as anti - redness , broken veins and puffiness around the eyes . naringenin and docosahexaenoic acid ( ethyl ester ) stimulate keratin - 6 production in normal human keratinocytes [ wound healing ] cells were cultured as described in example 5 . 1 . and the methodology described in example 3 . 1 was adapted in order analyze keratin - 6 production . fig1 shows stimulation of keratin 6 in human keratinocytes stimulated with compounds of the invention , retinoic acid ( ra ) and retinol ( rol ) and the positive control calcium . compounds naringenin and docosahexaenoic acid ( ethyl ester ) stimulated keratin - 6 production in normal human keratinocytes and can assist the process of wound healing and skin repair . n - acetylaspartic acid , docosahexaenoic acid ( ethyl ester ) and β - aescin . stimulate fibronectin production in normal human keratinocytes [ wound healing ] cells were cultured as described in example 5 . 1 . and the methodology described in example 3 . 1 was adapted in order analyze fibronectin production . fig1 shows the stimulation of fibronectin in human keratinocytes stimulated with all - trans retinoic acid , retinol , compounds of the invention and the positive control calcium . compounds of the invention had superior effect on fibronectin release in human keratinocytes after 6 days stimulation compared to all - trans retinoic acid and retinol . retinoic acid is a known activator of fibronectin in skin ( schwartz and kligman 1995b ) compounds gibberellic acid , naringenin and n - acetyl aspartic acid stimulate fibroblast growth factor ( fgf ) production in human skin explants [ wound healing ] human skin explants were obtained and treated as described in example 7 . 3 . the methodology described in example 3 . 1 was adapted in order analyze fgf production in skin explants . fig1 shows the stimulation of fgf production in human skin explants by compounds of the invention stimulation of fgf production by naringenin , gibberellic acid and n - acetylaspartic acid in human skin explants suggests wound healing benefits from topical application of these compounds to the skin . stimulation of growth factors ctgf and hbegf on the gene level in affymetrix gene array by compounds of the invention skin explants were obtained as detailed in example 3 . 1 . compounds naringenin and n - acetyl aspartic acid were topically applied on skin explants for 24 hours and investigated with affymetrix gene array . table 3 shows the stimulatory action of naringenin and n - acetyl aspartic acid on ctgf expression and also naringenin &# 39 ; s stimulatory effect on hbegf ( log 2 − ratio |& gt ; 1 ). using a cut - off fold change | log 2 − ratio |& gt ; 1 the analysis which was suggested by ( quackenbush j et al 2002 ) the affymetrix gene array on skin explants showed that naringenin and n - acetyl aspartic acid induced ctgf . ng also stimulated the expression of hbegf . it is noteworthy that all - trans retinoic acid did not have any effect on ctgf . gibberellic acid , naringenin , n - acetyl aspartic acid , arachidonic acid and docosahexaenoic acid ( ethyl ester ) stimulate kallikrein 8 production in human normal keratinocytes ( desquamation / complexion brightening / depigmentation ) kallikrein 8 is involved in the late differentiation of keratinocytes : the desquamation process . kallikrein 8 is a biomarker for epidermal turnover and exfoliation . keratinocytes used for kallikrein 8 experiments were cultured as described in example 2 . 1 . experiments were performed in monolayers with 80 % confluence . the cells were stimulated for 4 days prior to kallikrein 8 analysis with the different actives and then culture supernatant were collected and stored in − 20 ° c . until further analysis . kallikrein 8 production was analysed in cell culture supernatants from stimulated keratinocytes using a elisa assay specific for kallikrein 8 ( uscn life science inc , e90690hu ). results were compared with cell culture supernatants from dmso vehicle treated cells . the assays utilized a 96 - well microplate format and were processed according to the manufacturer &# 39 ; s protocol , including generation of a standard curve prepared in background assay diluents . absorbance of the assay dye was read at a synergy ht plate reader . values that fell within the linear range of the calibration assay were accepted . quantities were determined by comparison to standard curves obtained for klk8 . fig1 shows significant stimulation of kallikrein 8 by gibberellic acid , naringenin , n - acetyl aspartic acid , arachidonic acid and docosahexaenoic acid ( ethyl ester ), as well as positive controls retinol and retinoic acid . gibberellic acid , naringenin , n - acetyl aspartic acid , arachidonic acid and docosahexaenoic acid ( ethyl ester ) induced a significant increase in the synthesis of kallikrein 8 in human normal keratinocytes . the process of desquamation is essential in maintaining an intact skin bather and for healthy looking skin . enhanced epidermal turnover and desquamation can result in exfoliation of excessive pigmentation and therefore contribute to combating skin darkening due to the presence of melanin in the skin and also result in improving skin luster and / or brightness of the complexion . compounds of the invention stimulate keratin 1 / 10 production in human normal keratinocytes cells were cultured as described in example 5 . 1 . and the methodology described in example 3 . 1 was adapted in order analyze keratin 1 / 10 production . fig1 shows synthesis of keratin 1 / 10 expressed at % of vehicle control synthesis . naringenin ( 150 microm ) and gibberellic acid ( 1 . 2 microm ) induced a significant increase in the synthesis of keratin 1 / 10 in human normal keratinocytes . the process of keratinocyte differentiation is essential in maintaining an intact skin bather and for healthy looking skin . compounds gibberellic acid , naringenin and n - acetyl aspartic acid inhibit genes involved in acne exacerbation androgen stimulation is an important factor in adolescent and adult acne . androgen causes an increased production of sebum ( thiboutot 2004 ). the cmap ingredients showed anti - steroid 5 - α - reductase expression and anti - hydroxysteroid ( 17 - beta ) dehydrogenase 2 ( hsd17b2 ) activities in affymetrix gene arrays which implicates control of estrogen and androgen levels in the human skin . the therapeutic potential of this enzyme family in the treatment of acne has extensively been described . ( poirier 2003 ) skin explants were obtained as detailed in example 3 . 1 . compounds gibberellic acid , naringenin and n - acetyl aspartic acid were topically applied on skin explants for 24 hours and investigated with affymetrix gene array . using a cut - off fold change | log 2 − ratio |& gt ; 1 the analysis which was suggested by quackenbush et al ( quackenbush 2002 ), the affymetrix gene array on skin explants showed that several genes involved inflammation , sebum , androgen and neuropeptine stimulation were downregulated by the compounds of the invention . n - acetyl aspartic acid shows anti - steroid 5 - α - reductase expression in affymetrix gene arrays and anti - hydroxysteroid ( 17 - beta ) dehydrogenase 2 ( hsd17b2 ) activity . gibberellic acid , naringenin and tretinoin all show strong anti - cyclooxygenase i ( cox i ) effect a strong downregulation in tac1 which encodes neurokinin a ( substance p ) was seen by naringenin and n - acteyl aspartic acid as well as tretinoin . gibberellic acid showed anti - arachidonate 5 - lipoxygenase activity . androgen stimulation is an important factor in adolescent and adult acne . androgen causes an increased production of sebum ( thiboutot 2004 ). the cmap ingredients showed anti - steroid 5 - α - reductase expression and anti - hydroxysteroid ( 17 - beta ) dehydrogenase 2 ( hsd17b2 ) activities in affymetrix gene arrays which implicates control of estrogen and androgen levels in the human skin . the therapeutic potential of this enzyme family in the treatment of acne has extensively been described ( poirier 2003 ). regulation of neuropeptides plays an important role in the exacethation of acne . specifically , substance p play a role in the exacethation of acne from a neurological point of view , where in vitro studies revealed that substance p promotes both the proliferation and the differentiation of sebaceous glands ( toyoda et al . 2002 ). a strong downregulation in tac1 which encodes neurokinin a ( substance p ) was seen by ng / n - triple a acid as well as tretinoin . the cmap ingredients showed gene regulation behavior linked to reduction of expression of enzymes involved in leukotriene and prostaglandin signalling pathways , i . e . arachidonate 5 - lipoxygenase , cyclooxygenase which both have been shown to be evaluated in acne - involved facial skin ( alestas et al . 2006 ). it is noteworthy that cox inhibitors have been shown to be effective in the management of premenstrual acne . we were also able to show that the cmap ingredients reduce il - 6 production in dermal fibroblasts which indicates dermal anti - inflammatory action which is an important target to combat acne . for example , long - term treatment with zileuton which directly targets reduced the content of neutral lipids and interleukin - 6 release ( zouboulis et al . 2009 ). gibberellic acid , naringenin and n - acetyl aspartic acid are non - irritating to the skin in vitro skin irritation studies carried out on pure gibberellic acid , naringenin and n - acetyl aspartic acid on human reconstructed epidermis ( skinethic model ) in accordance with the oecd guideline 439 , found all compounds to be non - irritating to skin . the studies , aimed at evaluating the capability of the test compound to induce skin irritation effects by a cytotoxicity assay on in vitro human reconstructed epidermis ( skinethic ), were carried out according to the ecvam protocol of apr . 27 , 2007 , the b46 method of the official journal of the european union , dated aug . 14 , 2009 and the oecd guideline 439 . it should be readily apparent to one of ordinary skill in the art that the examples disclosed herein below represent generalised examples only , and that other arrangements and methods capable of reproducing the invention are possible and are embraced by the present invention . the words “ comprises / comprising ” and the words “ having / including ” when used herein with reference to the present invention are used to specify the presence of stated features , integers , steps or components but do not preclude the presence or addition of one or more other features , integers , steps , components or groups thereof . it is appreciated that certain features of the invention , which are , for clarity , described in the context of separate embodiments , may also be provided in combination in a single embodiment . conversely , various features of the invention which are , for brevity , described in the context of a single embodiment , may also be provided separately or in any suitable sub - combination . abe , k ., butcher , r . w ., nicholson , w . e ., baird , c . e ., liddle , r . a ., and liddle , g . w . 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