Patent Application
20110262025
The present invention relates to gene panels and genomic transcriptional profiling-derived biomarkers employed methods to identify and evaluate agents which transcriptionally regulate genes implicated in maintaining or improving stratum corneum (SC) barrier functioning of human skin. More particularly, the invention provides cosmetic agents, compositions and methods for increasing lipid synthesis, increasing hydration status, and thickening/strengthening the barrier to prevent, reverse or reduce the visible signs of aging in skin.
Skin aging is a multifactorial process driven by both intrinsic (effects of natural course of chronological aging, e.g.) and extrinsic factors, including ultraviolet (UV) exposure, environmental toxins/pollutants and smoking.
The SC is the outermost layer of skin and provides the chemical and physical barrier between the body and the environment. Cells are generated at the basal layer of the epidermis and rise up to the outer surface. As other newer cells are generated, the cells at the outermost layer regularly slough off to complete an overall process that takes about a month in healthy human skin. As the cells migrate from the basal to outer layer they produce and accumulate keratin, which gradually replaces the cell's cytoplasm in a process referred to as “cornification.” At the point where no cytoplasm remains, the cell dies and sheds off the body.
The barrier forms from this “cornification” and the cornified barrier provides a number of functions. In healthy skin, dead skin cells remain at the outermost surface for a brief period of time, held together by a corneodesmosome bond between surface corneocytes. This delays desquamation of the skin cells and reinforces the barrier. Important functions of the barrier include attenuation of the penetration of free radicals and prevention of penetration of harmful radiation including UV radiation, into deeper layers. The SC also acts as a permeability barrier and functions to prevent loss of body water to the outside environment.
The SC is a densely packed structure comprising an intracellular fibrous matrix that is hydrophilic and able to trap and retain water. The intercellular space is filled with lipids formed and secreted by the keratinocytes and which provide a diffusion pathway to channel substances with low solubility in water. A commonly espoused skin metaphor portrays the stratum corneum as a brick wall wherein each brick is a corneocyte and the intercellular lipid matrix is the mortar.
It is well known in the art that the ability of the SC to cyclically generate new layers of skin diminishes with age so that the SC turnover rate is substantially reduced in aged skin, with the cornified layer becoming gradually thinner. This results in a reduction in the functioning capacity of the barrier so that harmful stimuli penetrate the SC more easily, leading to UV-damage, for example, of the underlying dermal layers, degradation of collagen and elastin, and eventually manifests in appearance as skin atrophy and wrinkling. Thinning of the SC by the sum of intrinsic and extrinsic aging factors increases the visible appearance of fine lines and wrinkles. Further, the barrier suffers from an age-related increase in permeability to free radicals and a reduction in the amount of lipid in the intercellular matrix, decreasing barrier capacity to diffuse toxins from deeper layers. Recovery capacity of the barrier to environmental insult is also substantially reduced with age.
The evolution of the 2.1 billion dollar per year cosmetics industry is driven by a desire on the part of consumers to reduce the appearance of age. The industry expends considerable effort and resources to develop and provide consumers with easy-to-use, non-invasive, topically applied cosmetic compositions and treatment regimens that provide the desired “anti-aging” effect. Traditionally the creams, lotions, ointments and other product formulations were limited in effect to providing aesthetic benefit without conferring any real benefit to the health of the skin. Transient mimicking of a youthful appearance substituted for a true restoration of youthful skin attributes.
More recently, a greater understanding of the biochemical processes responsible for aging has invigorated the cosmetics industry and resulted in the emergence of a new class of cosmetic actives sometimes referred to as “cosmeceuticals.” The purported effects, including but not limited to antioxidant, anti-inflammatory and free-radical-scavenging effects, derive from the underpinning science. A continuing problem with the new era of treatment agents, however, is that the actual physiological effect, when measured against objective standards, did not appear commensurate in scope with the effect predicted by the scientific theory allegedly being exploited. Generally, the SC barrier of the skin, sought to be enhanced by the topically applied products comprising the agents, provides an element of unpredictability based upon its barrier functioning. The degree to which a compound penetrates the barrier, the effect of pH, the stability of the compound in a skin environment, exposure to degrading forces upon application and/or penetration, and other factors interfere with effectuating the theoretical correlate when compounds are applied topically to the skin.
The present inventors recognized the deficiencies associated with evaluation of known cosmetic agents for cosmetically functional effects and the deficiencies associated with identification of agents that actually achieve a theoretically-based effect to provide actual, verifiable anti-aging benefit to skin.
Accordingly, it is an objective of the present invention to apply the relatively new technologies of genomics and proteomics in a rigorous and objective manner to identify and evaluate cosmetic agents for desired anti-aging benefit. It is another object to provide compositions comprising known and newly identified agents demonstrated by application of these technologies to confer real, measurable substantive anti-aging benefit to skin, in particular by parsing the components of the skin aging process and adapting gene and biomarker panels to identify agents and combinations of agents which specifically target those components. Particular pathways include those relating to lipid content, hydration status and the thickness and maturity of the corneocyte layer.
Global gene expression profiling provides a useful means to identify key aspects of the skin aging process and provides information permitting the development of present inventive skin technologies. An important aspect of skin aging that can be addressed by application of genomics and proteomics includes skin barrier functioning. The present inventors further developed and confirmed the validity of human skin equivalent cultures, which permit topical application of test compounds and combinations to a SC surface derived from specifically aged skin cells, and measure relevant biomarkers including transcriptional profiles. Using this transcriptional analysis approach it is possible to detect skin barrier enhancement in response to the compounds. Gene microarrays are new tools for understanding changes occurring at the transcriptional level during skin aging. The present inventors gathered gene expression data and applied advanced bioinformatics to identify particular pathways affected by aging, including lipid synthesis and the dermal matrix.
Generally the present invention results from the discovery by the inventors of a very specific subset of genes relating to lipid synthesis and processing which are regulated in skin cells as a function of aging.
In one embodiment of the invention, gene panels are provided which comprise genes relating to SC lipid synthesis and which are regulated in response to extrinsic and/or intrinsic aging conditions. The panels comprise at least two genes, and any of various combinations or subsets of the genes identified in Tables A-D as set forth in
The invention further provides methods for assessing the age status of human skin, comprising extracting nucleic acid from a sample of skin and contacting the nucleic acid with an inventive microarray, performing a transcriptional analysis to obtain a transcriptional profile; and comparing the transcriptional profile to a reference profile derived from a control. Other methods enable identification or evaluation of agents for efficacy specifically in improving SC barrier maintenance and/or repair properties in aged skin. According to this embodiment the method comprises contacting skin, skin cells or a skin equivalent with a proposed agent; generating a transcriptional profile based on an inventive gene panel, comparing the transcriptional profile to a reference transcriptional profile, and identifying the agent as effective if the test transcriptional profile exhibits directional regulation which increases an amount of at least one lipid in the SC compared to the reference.
Another embodiment of the invention provides cosmetic compositions effective for improving SC barrier maintenance and/or repair properties in aged skin. The compositions comprise at least one agent that transcriptionally regulates genes constituting a gene panel of the invention to ultimately increase an amount of at least one lipid in the SC, wherein the lipid is selected from the group consisting of cholesterol, fatty acid and ceramide, or precursors thereof.
The invention further provides a biomarker panel indicative of hydration status of skin and certain methods and compositions relating thereto. The biomarker panel includes aquaporin-3, CD44 antigen and Claudin 1.
In another embodiment, an improved method for assessing SC corneocyte maturity is provided, along with methods for evaluating compounds and compositions for efficacy in improving barrier functioning through promotion of corneocyte maturity.
Features and benefits of the various embodiments of the present invention will become apparent from the following description, which includes figures and examples of specific embodiments intended to give a broad representation of the invention. Various modifications will be apparent to those skilled in the art from this description and from practice of the invention. The scope is not intended to be limited to the particular forms disclosed and the invention covers all modifications, equivalents and alternatives falling within the spirit and scope of the invention as defined by the claims.
B. Table of transcriptional changes in cellular cholesterol influx genes.
C. Table of transcriptional changes in cellular cholesterol efflux genes.
D. Table of transcriptional changes in cellular mitochondrial cholesterol utilization genes.
B. Table of transcriptional changes in cellular fatty acid synthesis genes.
C. Table of transcriptional changes in cellular fatty acid influx genes.
D. Table of transcriptional changes in cellular mitochondrial fatty acid utilization genes.
B. Table of transcriptional changes in lamellar body secretion genes.
C. Table of transcriptional changes in ceramide precursor processing genes.
B. Effects of Dex and GFF on enzymatic activity of caspase-14.
B. Illustrates synergy between Hexamidine, Niacinamide and Pentanediol on C44 Expression in Keratinocytes.
The present invention is based in part on discovery by the inventors that the effects of aging on SC lipid pathways reflect regulation by a unique set of genes. Transcription of those genes may be impacted by particular conditions, treatments or agents. Aging may be either intrinsic aging resulting from factors relating to chronological aging, or extrinsic aging resulting from environmental factors, or combined intrinsic and extrinsic aging. The invention therefore provides transcriptional profiles for genes relating to lipid synthesis and metabolism as a function of age and provides the opportunity to attack/ameliorate the effects of aging by manipulating transcription of genes to produce downstream translation products which provide benefits to the skin in a natural way, in contrast to conventional methods which rely on direct application of “benefit agents” which suffer from the deficiencies in the art.
Since penetration and stability of agents in the skin context is multifactorial and difficult to assess, the genomic technology providing assessment via transcriptional profiling permits the investigator to leapfrog over those issues and test for effects that subsume those factors. One embodiment of the instant invention provides a gene panel comprising genes relating to SC lipid formation in human skin and which generate a transcriptional profile that is in part a function of aging. The genes, listed in
In particular a gene panel comprises at least two genes selected from Tables A-D as set forth in
Similarly, a gene panel according to the invention may consist of genes relating to an amount of sphingolipid in the SC of human skin which are regulated in response to extrinsic and/or intrinsic aging conditions. The panel comprises at least 2 genes selected from Table D set forth in
A microarray is a technology widely used in molecular biology and genomic studies. A microarray comprises an arrayed series of nucleic acid oligonucleotides, each containing a probe for a target gene. A probe is a short section of a target gene (or other target DNA) that is designed to hybridize to a target cDNA or cRNA sample. The hybridization is thereafter detected and quantified by methods well-known in the art, which may include fluorophore-labeled targets, to determine relative abundance of the specific sequence in the sample. An array may contain thousands of probes, and a “global” array is understood to contain a probe for an entire population of known genes in a species. Generally the probes are attached via a linking chemistry to a solid substrate such as a glass or silicon chip. Colloquially these are known as Affy chips when an Affymetrix™ brand chip is employed. Such microarrays are also known as gene chips. Many other microarray platforms exist however, and a person of ordinary skill in the art will understand that the microarray detection system employed for transcriptional assay may vary without departing from the spirit of the present invention.
Generally, microarray technology is sophisticated and a person of skill in the art, given a target gene, is capable of designing a suitable probe using methods routine in the art. Probes for genes having the sequences set forth in the instant Sequence Listing are well-known in the art and low density microarrays for any subset of target nucleic acid molecules are readily designed and constructed. A person of ordinary skill in the art can readily construct a microarray comprising immobilized oligonucleotides which hybridize specifically to nucleic acids corresponding to the genes constituting a gene panel according to the present invention. The genes are known in the art, and suitable probes for the genes are known in the art. One embodiment of the invention provides a unique assembly of hybridizing oligonucleotides targeting a unique panel of genes, however, which permits, inter alia, practice of the methods of the invention.
The instant inventors discovered profiles for the transcriptional regulation of genes relating to lipid formation and aging. Accordingly, one embodiment of the invention is directed to methods for assessing the age status of human skin. The method comprises extracting nucleic acid from a sample of skin from a human subject; contacting the nucleic acid with a microarray according to the invention, performing a transcriptional analysis to obtain a transcriptional profile; and comparing the transcriptional profile to a reference profile derived from a control population standardized as a function of age. In another related embodiment the extracted nucleic acid is quantified and quantities are compared to reference quantities as a function of age.
Transcriptional profiling provides a superior method for evaluating a cosmetic agent for efficacy in improving SC barrier maintenance and/or repair properties in aged skin that does not rely on self-report of the consumer or tests which cannot reliably factor penetration, metabolic state and other conditions that might affect cosmetic efficacy. According to one embodiment, the method comprises contacting skin, skin cells or a skin equivalent with a proposed agent; generating a transcriptional profile based on a gene panel according to the invention, comparing the transcriptional profile to a reference transcriptional profile, and identifying the agent as effective if the test transcriptional profile exhibits directional regulation which increases an amount of at least one lipid in the SC compared to the reference.
The barrier function of the epidermis of skin is primarily localized to the SC. The SC is a two-compartment system of corneocytes embedded in a lipid-enriched extracellular matrix. Within the SC, several defensive barrier functions can be localized to either the lipid matrix or corneocytes. Primary strategies for improving barrier function therefore include (1) increasing the number of fully differentiated (mature) SC corneocytes and (2) increasing integrity of lipid matrix.
The present inventors applied the genomic and transcriptional profiling technologies set forth above to investigate molecular regulation of skin barrier formation and function. Major lipids of the human SC are ceramides, cholesterol, and fatty acids (FAs), comprising approximately 50%, 25% and 10% of the total lipid mass, respectively. These lipids are produced by granular keratinocytes and delivered to the SC, along with processing enzyme, in lamellar bodies (LBs) which fuse with the keratinocyte plasma membrane and extrude their contents locally in the SC. Once released into the SC these lipids are enzymatically processed to form the highly structured lipid lamellae matrix responsible for several barrier functions. An overall strategy for building a better lipid matrix, thus improved barrier function, would be to increase the de novo synthesis, cellular uptake, and processing of these SC lipids.
Ceramides are a family of lipid molecules comprising a sphingosine and a FA. Ceramides are found primarily in cell membranes as one of the components of sphingomyelin. Ceramide is understood to have many functions aside from providing structure, including regulation of cell differentiation, proliferation and apoptosis. Synthesis of ceramide occurs in the endoplasmic reticulum and begins with condensation of palmitate and serine to form 3-keto-dihydrosphingosine, catalyzed by the enzyme serine palmitoyl transferase, which is the rate limiting enzyme. 3-keto-dihydrosphingosine is reduced to dihydrosphingosine, which is then followed by acylation by the enzyme (dihydro) ceramide to produce dihydroceramide with the final reaction catalyzed by dihydroceramide desaturase. Ceramide is transported to the Golgi. Ceramide may be metabolized into sphingomyelin and glycosphingolipids. These end products provide another source of ceramide via degradation of the sphingolipids.
Cholesterol is a waxy steroid metabolite that is an important structural component of cell membranes. It is known that cholesterol regulates membrane fluidity and in its structural capacity it reduces the permeability of the plasma membrane to protons and sodium ions. Cholesterol has important functions relating to intracellular transport, cell signaling and nerve conduction. Certain disease states, such as atopic dermatitis, which reflect impaired stratum corneum barrier function to cause increased transepidermal water loss (TEWL), are associated with deficiencies in both ceramide and cholesterol, as well as abnormal ratios wherein a decrease in the amount of ceramide relative to cholesterol causes an increase in TEWL and dry, flaky skin.
The cholesterol metabolic pathways pertinent to SC lipid matrix production include those relating to biosynthesis and cellular uptake/efflux. Mitochondrial utilization of cholesterol or cholesterol precursors may also affect overall equilibrium. The present inventors noted that with aging there is transcriptional down-regulation of genes which encode many of the enzymes necessary for biosynthesis and cellular cholesterol uptake and processing, while transcriptional up-regulation of genes which encode for enzymes involved in cholesterol efflux were observed. Agents evaluated as effective in barrier maintenance may act to reduce or reverse the regulatory effect of aging shift the transcriptional profile in a directional manner to restore a profile generated from young skin.
Another group of lipids important to skin barrier functioning is FAs. The metabolites of certain essential FAs are important for structural integrity, while others are precursors for the production of regulatory prostaglandins. Metabolites of FAs are further known to confer a water proofing effect to the corneocyte layer and to guide transport and metabolism of cholesterol. Increased TEWL is also a symptom of a deficiency in FAs. The synthesis, uptake and utilization of FAs are critical for proper SC lipid matrix formation. In an evaluation for agents which are effective in restoring/promoting increased FA content, a transcriptional profile reflecting up-regulation of genes which encode for enzymes involved in both biosynthesis and uptake of FAs is desired, while a profile reflecting down-regulation of those genes and/or up-regulation of genes which encode enzymes involved in cellular efflux or proteins involved in transporting FAs into the mitochondria for energy production is not desired.
It is known in the art of skin barrier health that lipid content decreases as a function of age, resulting in a thinning of the barrier, greater water loss, dryness, and increased permeability to toxins and free radicals. By utilizing the instantly inventive technologies, a cosmetic composition may more reliably and validly be designed and tested for efficacy in improving SC barrier maintenance and/or repair properties in aged skin by restoring lipid content. The instant invention provides compositions comprising one or more agents that transcriptionally regulate genes constituting a gene panel according to the invention to increase an amount of at least one lipid in the SC, the lipid selected from the group consisting of cholesterol, FA and sphingolipid/ceramide.
As noted above, a second broad strategic approach to affecting and improving barrier functioning involves increasing the number and quality of stratum corneum corneocytes. The ratio of mature to immature corneocytes at the stratum corneum surface is known to decrease with both chronological age and with exposure to extrinsic aging factors such as environmental insult.
As keratinocytes migrate from the epidermal basal layer to the SC, they undergo a differentiation or “maturation” process resulting in the formation of the corneocyte envelope (CE). The CE is made up of several different proteins, one of which is involucrin, that are highly cross-linked to produce the insoluble (hydrophobic) structure of the SC corneocytes. A method for assessing CE maturity was developed by Hirao et al. who described a tape stripping method for simultaneously identifying “mature” and “immature” corneocytes (Exp Dermatol 2001: 10: 35-44). The method is based upon staining with nile red (red fluorescence), which characterizes corneocyte hydrophobicity, and involucrin (green fluorescence) staining, which characterizes the degree of cross-linking. The premise for this identification is that fully “mature” CE would stain only with nile red because the epitope for the involucrin antibody would not be recognized due to the high degree of cross-linking. In contrast, “immature” CE would stain solely with involucrin because of the lack of cross-linking. Successive tape stripping, demonstrates that the ratio of mature (red) and immature (green) corneocytes changes at different levels of the SC.
The invention further provides an improved method for assessing CE maturity which is adapted for more convenient clinical applications and which provides greater resolution to enable more refined classification, and use of imaging software to fine-tune the classification and analysis. Details of the differences between the instant techniques and those known in the art are set forth in Example 7, below. According to one embodiment, a method is provided for determining a ratio of mature to immature corneocytes. The method comprises (1) extracting a sample of corneocytes from the skin surface by tape-stripping wherein a tape is adapted to permit uniform sampling of a fixed area of the skin surface; (2) differentially staining the extracted corneocytes according to degree of cross-linking and hydrophobicity; (3) imaging the stained corneocytes; and (4) importing images into an analyzer that permits at least five levels of resolution for classification of the differentially stained corneocytes; and (5) conducting a statistical analysis of the classification to determine the ratio of mature to immature corneocytes. The invention also provides a cosmetic composition formulated for topical administration, comprising one or more compounds effective for increasing a ratio of mature to immature corneocytes at a skin surface, wherein the ratio is determined by the improved methods. In specific embodiments the composition comprises one or more of Hexamidine, Niacinamide and Pentanediol. Methods for inhibiting and/or reversing skin damage due to environmental stress are also provided wherein the skin is treated with compositions which are effective for increasing the ratio of mature to immature corneocytes. The inventive methods provide a technique for evaluating agents proposed for efficacy in enhancing a barrier function of the SC. The method comprises inspection of skin treated with the proposed agent wherein skin in tangential proximity to the treated skin, that is, skin from the same subject or sample, provides the control. By “tangential” it is understood merely that the control and treatment skin derive from substantially the same skin area of the subject or from the same skin equivalent since it is known that skin from different regions of the body comprise different optimal ratios of mature to immature corneocytes. One embodiment of the method comprises selecting a first and second skin surface in substantially tangential proximity to one another, wherein the first surface is a control surface and the second surface is a treatment surface; contacting the second surface with a proposed agent in a vehicle for a period of time while simultaneously contacting the first surface with vehicle or nothing (no treat control) for the period of time; conducting a corneocyte ratio assessment as provided herein on both the first and second surfaces; and evaluating the agent as effective for enhancing a barrier function of the SC if a ratio of mature to immature corneocytes is significantly greater in the second surface than in the first surface.
The corneocytes of the SC are essentially “dead” anucleated cells derived from outer stratum granulosum keratinocytes during terminal differentiation, embedded in a lipid-enriched extracellular matrix, secreted from epidermal LB s. Permeability barrier insults stimulate rapid secretion of preformed LBs from the outer stratum granulosum, regulated through modulations in ionic gradients and serine protease (SP)/PAR-2 signaling. Because corneocytes are also required for barrier function, it is hypothesized that corneocyte formation may be regulated by barrier function, or vice versa. Barrier abrogation by two unrelated methods initiates a wave of cornification, assessed as TdT-mediated dUTP nick end-labeling-positive cells in stratum granulosum and newly cornified cells by electron microscopy. Because cornification is blocked by occlusion, corneocytes formed specifically in response to barrier requirements, rather than injury or cell replacement needs. SP inhibitors and hyperacidification (which decreases SP activity) block cornification after barrier disruption. Similarly, cornification is delayed in PAR-2−/− mice. Although classical markers of apoptosis [poly(ADP-ribose)polymerase and caspase (Casp)-3] remain unchanged, barrier disruption activates Casp-14. Moreover, the pan-Casp inhibitor Z-VAD-FMK delays cornification, and corneocytes are structurally aberrant in Casp14−/− mice. Thus, permeability barrier requirements coordinately drive both the generation of the stratum corneum lipid-enriched extracellular matrix and the transformation of granular cells into corneocytes, in an SP- and Casp-14-dependent manner, signaled by PAR-2 (Demerjian et al. American Journal of Pathology: 127(1); (2008)).
Protease inhibitors are known to prevent breakage of the corneodesmosome bonds between corneocytes at the skin surface. Exfoliation and other methods of intentional enhancement of the sloughing off process (desquamation) are considered beneficial for smoothing the skin surface and improving appearance.
Abnormally high SP activity has been observed in several skin diseases associated with decreased barrier function, such as atopic dermatitis, contact dermatitis, and psoriasis. A sustained increase in SC SP activity leads to decreased barrier function and increased inflammation, which has been attributed to 1) SP degradation of SC lipid processing enzymes, and 2) loss of SC cohesion as a result of SP-mediated degradation of corneocyte cell-cell connections (corneodesmosomes). Additionally, acute disruption of the SC barrier by tape striping, solvent treatment, or detergent treatment elicits an increase in epidermal SP activity which negatively affects barrier recovery kinetics. However, the addition of SP inhibitors accelerates barrier recovery.
Additional embodiments of the invention relate to methods and compositions for improving barrier functioning by regulation of hydration status of skin. In particular, a novel biomarker panel comprising at least 2 biomarkers selected from the group consisting of aquaporin 3, CD44 antigen and Claudin 1 are provided.
Aquaporins are a class of membrane proteins within mammalian skin that regulate transport of water, glycerol and other solutes across the plasma membrane. Without being limited by theory, it is thought that two major aquaporin membrane proteins encoded by AQP-3 and AQP-9 are expressed in skin cells. Aquaporin 3 is a transporter protein in the plasma membrane of keratinocytes, which transports water and glycerol into the vascular-free epidermis from the dermis. When the AQP-3 gene is inactivated, multiple symptoms of damaged skin, such as lower water content, leaky skin barrier, delayed wound healing, and impaired skin elasticity are observed. Investigators have previously shown that an increase in transcription of AQP-3 and expression of aquaporin 3 improves skin hydration, thus minimizing the visual signs of dry or photo-damaged skin.
CD44 protein is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. It is known as a receptor for hyaluronic acid and also interacts with other ligands including osteopontin, various collagens and matrix metalloproteinases (MMPs). Claudins are a family of proteins that are the most important components of the tight junctions, where they establish the paracellular barrier that controls the flow of molecules in the intercellular space between the cells of an epithelium. They have four transmembrane domains, with the N-terminus and the C-terminus in the cytoplasm.
The invention further provides cosmetic compositions effective for increasing hydration status of the skin as indicated by measurement of the biomarkers constituting the biomarkers of the inventive panel. In specific embodiments the composition comprise Hexamidine, Niacinamide and Pentanediol in particularly effective ratios. The present inventors surprisingly discovered that synergy among the proposed ingredients with respect to the target endpoint is a function of the ratio of the ingredients. Certain ratios do not provide synergy. In specific embodiments the effective ratio of Hexamidine:Niacinamide:Pentanediol is in a range of about 1-5:40-60:5-50. In very specific embodiments the effective ratio of Hexamidine:Niacinamide:Pentanediol is 1:50:30 and in other very specific embodiments the ratio may be 1:50:5. The compositions may therefore effectively employed in methods for moisturizing skin comprising contacting the skin with an effective amount of the composition.
Efficacy of the methods may also be a function of treatment frequency, duration and/or regimen. A consumer may apply single or multiple daily doses for a number of consecutive days contemplated as between about 4 and about 21 for initial benefit with sustained benefit in accordance with continued treatment. In very specific embodiments an initial hydrating benefit is realized after 14 consecutive treatments.
Hexamidine in accordance with the instant invention is understood to include isomers, tautomers, salts and derivatives including but not limited to organic acids such as carboxylic acids and mineral acids such sulfonic acid. A technical name for Hexamidine is 4,4′-(hexamethylenedioxy)dibenzenecarboximidamide. Dermatologically acceptable salts include alkali metal salts such as potassium and sodium; alkaline earth metal salts, such as calcium and magnesium; non-toxic heavy metal salts, and ammonium and trialkylammonium salts. Alternatively, Hexamidine is hexamidine diisethionate, which is commercially available under the tradename ELESTAB HP100 from Laboratoires Serobiologiques of Pulnoy, France.
The compositions according to the instant invention may comprise Hexamidine from about 0.0001% to about 25%, alternatively from about 0.001% to about 10%, alternatively from about 0.01% to about 5%, and alternatively from about 0.02% to about 2.5% by weight of the composition.
Many of the methods of the instant invention may be practiced in vitro using skin equivalent technologies. In several experiments designed to test the validity and reliability of skin equivalent cultures, both validity and reliability were confirmed. Skin equivalent models provide a high through put means to test cosmetic agents. In vitro human skin cultures have been tissue-engineered to reproduce key structural and functional aspects of natural skin. Of particular value for skin aging research are cultures containing a stratified cornified epidermis either alone or in combination with a basement membrane and a fibroblast-containing dermal matrix. Such cultures can be constructed of skin cells from donors of varying ages and conditions. The presence of an air-interface allows for topical application of test materials, including undiluted or diluted test compounds, combinations of compounds, or product formulations.
In vitro skin models, however, do not exhibit the macroscopic features analogous to signs of facial skin aging such as fine lines and wrinkles. Hence, it is necessary to identify and evaluate biomarkers of skin and barrier functioning, such as proteins, mRNA or other macromolecules that may be detected or measured as a function of aging. Epidermal-dermal constructs can express major structural collagens in the dermal matrix, such as collagens I and III and procollagen I, and basement membrane zone-specific markers such as collagen IV, in addition to epidermal hyaluronic acid. These types of structural elements of skin are known to be decreased in expression or functionality as skin ages.
Biomarker endpoints and certain clinical skin instrumental measurements, such as those used for skin barrier determinations, can be detected in skin equivalent cultures. As noted above, the stratum corneum barrier consists of flattened, terminally differentiated corneocytes surrounded by specialized lamellar lipids, which act to retard loss of water from the skin's surface. Water loss can be measured functionally by use of an evaporimeter instrument wherein the endpoint measured is TEWL, and is expressed in units of grams of water per skin per hour. In one validity test the present inventors took epidermal cultures from a commercial supplier (Epiderm 201 cultures from MatTek Corp., Ashland Mass.) grew them in cultures for four days whereby the number of differentiated cell layers and stratum corneum thickness was increased during this period. In parallel, evaporimeter measurements indicated a decrease in TEWL values to levels comparable to those measured in human back skin in vivo. TEWL levels could be reduced further by 24 hour treatment with 1% Niacinamide. These findings indicate that in vitro epidermal constructs form a functional water barrier that can be enhanced by compounds such as Niacinamide.
Methods for manufacturing cosmetic compositions according to certain embodiments of the invention are also contemplated. Specifically, compositions are formulated to comprise agents selected to one or more of the inventive methods of screening, evaluating and identifying as effective as set forth herein. Techniques for formulating compositions comprising particular selected cosmetic actives as suitable for topical application are well known by practitioners of the cosmetic arts. As non-limiting examples, compositions may be formulated as lotions, creams, gels, balms, oils, ointments and the like and effective non-invasive application may be, for example, via direct consumer application to the skin or by dermal patch or cosmetic mask intended to provide sustained contact over a period of time.
The following examples are intended to illustrate particular aspects of the invention and should not be construed as limiting the scope of the invention as defined by the claims.
Hexamidine is analyzed as a single variable in an aged-equivalent skin culture system (Dermaquant model (Epistem Manchester, UK)-keratinocytes and fibroblasts taken from 54 year old female donors.) This example illustrates the validity of a skin-equivalent substrate in methods relating to transcriptional profiling and further illustrates the efficacy of Hexamidine in enhancing barrier functioning through genomic regulation.
The Experimental design is set forth in Table I. Cultures were treated and harvested at indicated time points. Total RNA extraction, labeling and hybridization to Affymetrix™ U133A plus 2 gene chips were conducted. Data were subjected to normalization and statistical analysis.
Results: Genomic data reveals that Hexamidine beneficially affects pathways involved in keratinocyte stratum corneum lipid metabolism. Transcription data are set forth in
The increased uptake and synthesis of stratum corneum lipid precursors, accompanied by their decreased mitochondrial utilization suggests that these precursors are being packed into LB s and delivered to the stratum corneum. Increased expression of genes involved in LB formation (ABCAl2) and secretion (ABHD5) was observed, suggesting increased LB delivery to the SC.
Hexamidine is a known SP inhibitor. Proteases are known to degrade the corneodesmosomes, increasing the rate of desquamation leading to loss or thinning of the outer-most protective cornified layer with resulting diminishment in barrier functioning. This experiment demonstrates that Hexamidine likely exerts an effect on lipid formation independent of its SP inhibition activity, and the effect is exerted at the level of transcriptional regulation. Hence, the present inventors have determined that the positive effects of Hexamidine on barrier functioning arise from at least two mechanisms. It was previously postulated that through protease inhibition Hexamidine increases thickness and integrity of the cornified layer, thereby discouraging loss of water. In parallel, this experiment suggests that Hexamidine acts to increase synthesis and transport of lipid to the SC also resulting in a thickening and strengthening of the stratum corneum. Deficiency in lipid content of the SC is known to be associated with an increase in keratinocyte proliferation, an increase in cellular turn-over and presence of disease states characterized by dry, flaky skin.
This Example investigates the effects of application of a composition containing agents which operate via multiple mechanisms to enhance barrier functioning. Specifically, a skin equivalent culture treated with a composition comprising a Hexamidine, Niacinamide and Pentanediol is subjected to a barrier lipid genomic analysis.
Experimental: Epidermal cultures (Epi 200 MatTek Corp., Ashland, Mass.) were treated and harvested at 12, 24 and 48 hour time points. Total RNA extraction, labeling and hybridization to Affymetrix™ U133A plus 2 gene chips were conducted. Transcriptional data were subjected to normalization and statistical analysis.
Significantly, two genes associated with lamellar body (LB) secretion, annexin II and abhydorlase containing member 5, were up-regulated in the treated cultures.
This Example illustrates the transcriptional and expressional changes in keratinocyte cholesterol metabolism pathway components in the SC upon treatment with a composition comprising Hexamidine, Niacinamide and Pentanediol. A Summary of the observed transcriptional changes is set forth in
Experimental: Experimental design is as set forth in Example 2.
The cholesterol synthetic pathway is localized in the endoplasmic reticulum (ER). The rate limiting biosynthetic step is conversion of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonate, catalyzed by 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase (HMGCR). The activity of HMGCR is under transcriptional control mediated through sterol-regulatory element binding transcription factor proteins (SREBFs). SREBFs along with other proteins activate the transcription of HMGCR and other genes involved in lipid metabolism. Increased expression of these transcriptional activators suggests expressional regulation of HMGCR and other cholesterol synthetic genes. It is understood that in SREBF control of lipid synthesis, SCAP complexes with SREBFs on the ER and translocates to the Golgi where membrane-bound transcription factor peptidase site 1 (MBTPS1) and site 2 (MBTPS2) proteases cleave SREBFs, ultimately releasing the active beta helix-loop-helix fragment which trans-locates to the nucleus and activates transcription of genes involved in lipid synthesis. Expressional regulation of these genes by Hexamidine, Niacinamide and Pentanediol are set forth in
In the present experiment, expressional changes in HMGCR and other cholesterol synthetic genes were observed and are given in the tables set forth in
The amount of cholesterol available for cellular needs is influenced by cholesterol influx and efflux processing. The major mechanism by which exogenous cholesterol is taken up into a cell is through the low-density lipoprotein receptor (LDLr) the expression of which was up-regulated in this experiment. Transcriptional up-regulation of several genes for proteins involved in processing and transport of cholesterol taken up via LDLr were also observed (
Mitochondrial cholesterol utilization also influences cholesterol availability for cellular needs. An important cholesterol-metabolizing enzyme resides on the matrix side of the inner mitochondrial membrane, Cyp 27, which converts cholesterol to 27-hydroxycholesterol used for bile acid production, a more soluble transport form of cholesterol, and a potent repressor SREBF processing. In order for cholesterol to be utilized by Cyp27 it must be transported through the outer mitochondrial membrane by the peripheral benzodiazepine receptor (BZRP), which showed down-regulation at the 24-hr time point. Transcription of Cyp27 and other bile acid synthetic genes also were observed to be down-regulated in the experiment (
Summarily, changes in transcription of genes regulating cholesterol metabolism by treatment with a composition comprising Hexamidine, Niacinamide and Pentanediol were observed to occur in the following manner:
The increased uptake and synthesis of cholesterol, accompanied by its decreased efflux and mitochondrial utilization, suggests that cholesterol is being packed into LB and delivered to the SC.
This example illustrates transcriptional changes involved in Fatty Acid (FA) metabolism in the SC as a result of treatment with a composition comprising Hexamidine, Niacinamide and Pentanediol.
Experimental: experimental design is as set forth in Example 2.
The FA synthetic pathway is located in the cytoplasm of skin cells. The synthesis of malonyl-CoA is the rate limiting step and is catalyzed by acetyl-Coenzyme A carboxylase alpha (ACACA), which may therefore be considered the rate-limiting enzyme. Once malonyl-CoA is formed, fatty acid synthase (FASN) catalyzes the reductive synthesis of palmitate using one acetyl-CoA, seven amlonyl-coAs, and seven NADH. Palmitate is then transported to either the endoplasmic reticulum or mitochondria for chain elongation. Regulation of ACACA transcription is mediated in part through SREBFs, which, along with other proteins, activates the transcription of genes involve in lipid metabolism. In this experiment, upon treatment with a composition comprising Hexamidine, Niacinamide and Pentanediol, increased expression of these transcriptional activators was observed suggesting transcriptional regulation of the FA metabolism genes. Transcriptional changes in ACACA were also observed and are given in
In the process of generating acetyl CoA for FA synthesis, glucose is first degraded to pyruvate by aerobic glycolysis in the cytoplasm. The key enzyme is pyruvate kinase (PMK2). Pyruvate is transported into the mitochondria, where it is decarboxylated, forming Actyl CoA and other products. The key enzyme is pyruvate dehydrogenase. Acetyl CoA then serves as a substrate for citrate synthesis wherein the key enzyme is citrate synthase (CS). Citrate is transported out of the mitochondria to the cytoplasm where FA synthesis occurs, and there is split to generate cytoplasmic acetyl CoA for FA synthesis. The key enzyme is ATP citrate lysase (ACLY). The process of FA transport across mitochondrial membranes also appears to involve proteins transcriptionally regulated by the experimental actives. According to the current understanding of the process, first cytoplasmic long chain fatty acids (LCFAs) are converted to long chain Acyl-CoAs. Long Chain Acyl-CoAs are impermeable to the inner mitochondrial membrane, so they are converted to long chain acyl-carnitine by carnitine palmitoyltransferase 1A (CPT1A). Long chain Acyl-carnitine is transported across the membrane in exchange for carnitine by carnitine/Acyl-carnitine translocase (SLC25A20). Within the mitochondrial matrix acyl-carnitine reacts with CoA in a reaction catalyzed by carnitine acyltransferase II(CPT2), yielding long chain Acyl-CoA ready to undergo beta-oxidation.
The observed transcriptional regulation of genes involved in producing the Acetyl CoA used for FA synthesis provides additional evidence that FA synthesis is up-regulated by the combination of Hexamidine, Niacinamide and Pentanediol. The Acetyl-CoA used for FA synthesis is derived from pyruvate produced during glycolysis. Expressional changes in the key enzymes discussed above were observed and are set forth in
It was initially believed that Long LCFAs enter eukaryotic cells merely by diffusion through the phospholipid bilayer. Subsequently investigators have discovered that in addition to simple diffusion, many cell types possess a saturable and competitive process occurring at low concentrations, indicative of protein-mediated transport. Proteins which have been identified as participating in LCFA transport include fatty acid transport proteins (FATPs), long chain acyl-CoA synthetases (LACs) and CD36. FATPs, LACs and CD36 are hypothesized to cooperate to facilitate efficient LCFA uptake.
Mitochondrial utilization also impacts the level of FA in the stratum corneum, rendering it unavailable for other cellular needs. Newly synthesized or up-taken FA can be utilized by cellular mitochondria for energy production in the process of beta oxidation. However, FA must be transported across mitochondrial membranes.
Summarily this experiment demonstrates that a composition comprising Hexamidine, Pentanediol, and Niacinamide regulates pathways involved in keratinocyte fatty acid metabolism in the following manner:
The increased uptake and synthesis of fatty acids, accompanied by its decreased mitochondrial utilization, suggests that fatty acids are being packed into LB and delivered to the SC.
This example illustrates how treatment of skin with a composition comprising Hexamidine, Niacinamide and Pentanediol transcriptionally regulates genes involved in Sphingolipid/ceramide metabolism.
Experimental: Experimental design is the same as described in Example 2, above.
Ceramides are the major lipid component of the stratum corneum, accounting for 30-40% of stratum corneum lipid content by weight. Human SC contains a heterogeneous family of 7 different ceramides (called ceramides 1-7), which are produced through specific modifications of two sphingolipid precursor molecules; glucosylceramide and sphingomyelin. The cellular biosynthetic pathway by which glucosylceramide and sphingomyelin are produced begins in the ER and ends at the Golgi. Serine palmitoyl transferase (SPTLC1/2) is the rate-determining enzyme in sphingolipid biosynthesis. It condenses serine with palmitoyl-CoA to produce 3-dehydrosphinganine, which undergoes further enzymatic modifications to produce ceramide. Once produced, the ceramide is transported from the ER to the Golgi by two different mechanisms dependant upon whether it is utilized for glucosylceramide or sphingomyelin production. Ceramide destined for sphingomyelin production is transported by a cytosolic protein, Ceramide transport protein (CERT) in an ATP-dependant manner. In contrast, ceramide utilized for glucosylceramide production is transported in a non-ATP/CERT dependant manner. Once translocated to the Golgi, ceramide is enzymatically modified to form the SC ceramide precursors glucosylceramide and sphingomyelin. Expressional regulation of these genes by Hexamidine, Niacinamide and Pentanediol are set forth in
Once synthesized, glycosylceramide and sphingomyelin are packed into LBs, and then released into the SC extracellular space following LB fusion with the plasma membrane of granular keratinocytes. Upon treatment with the test composition, the up-regulation of two genes involved in LB secretion; Annexin A2 and Abhydrolase domain containing 5 (
Processing of glycosylceramide is of particular importance, as it results in the formation of ω-OH Ceramide, which serves as the attachment point between the ceramide and specific amino acids of corneocyte envelop proteins. β-Glucosidase (GBA) is responsible for converting ω-OH glycosylceramide to ω-OH Ceramide. The activity of β-Glucosidase is dependent upon the presence of an “activator protein”, Saposin C, which properly positions the enzyme for glucose moiety cleavage. Four different saposins are found within the human epidermis (SAP A-D), each derived by proteolytic processing of a common precursor protein, prosaposin. Following this conversion, a portion of the ω-OH Ceramide undergoes further modification to produce ω-OH fatty acid, which may facilitate later desquamation, perhaps by reducing the interaction between CLE and unbound ceramide in the SC lamellar lipid matrix.
The processing of Sphingomyelin to SC ceramides 1 and 5 is carried out by sphingomyelin phosphodiesterase 1 (SMPD1). However, given that only two of the seven known human SC ceramide species are generated in part by sphingomyelin hydrolysis, and that all seven major SC ceramides are derived from glycosylceramide precursors, the glycosylceramide-to ceramide pathway appears to have a more critical role for generating the full spectrum of SC ceramide species.
Summarily, treatment of skin with a composition comprising Hexamidine, Niacinamide and Pentanediol is observed to transcriptionally modify pathways involved in keratinocyte sphingolipid metabolism in the following manner:
The ultimate metabolic result is an increased synthesis and transport of sphingolipids, accompanied by up-regulation of genes involved in LB secretion, suggesting increased delivery of ceramide “precursors” to the SC. This increased delivery is also associated with an up-regulation of glycosylceramide processing pathways, suggesting subsequent conversion to mature SC ceramide species.
This Example illustrates an improved method for assessing the corneocyte maturity level in human skin. The novel methods derive from a known method described by Hirao et al (Exp Dermatol 2001: 10: 35-44). Corneocyte envelopes (CE) are made up of several different proteins, one of which is involucrin, that are highly cross-linked to produce the insoluble (hydrophobic) structure of the SC corneocytes. Hirao described a tape stripping method for simultaneously identifying “mature” and “immature” corneocytes. The method is based upon staining with nile red (red fluorescence), which characterizes corneocyte hydrophobicity, and involucrin (green fluorescence) staining, which characterizes the degree of cross-linking. The premise for this identification is that fully “mature” CEs would stain only with nile red because the epitope for the involucrin antibody would not be recognized due to the high degree of cross-linking. In contrast, “immature” CE would stain solely with involucrin because of the lack of cross-linking. CE in between these two extremes stain for both, giving differential levels of yellow. Successive tape stripping, demonstrates that the ratio of fully mature (red) and fully immature (green) corneocytes changes at different levels of the SC. Generally, the methods of Hirao and the presently adapted method may be compared as follows:
Illustrates treatment with a cosmetic composition according to the invention effective for increasing/promoting corneocyte maturity.
Corneocytes consist of a stabilized array of keratin filaments contained within a covalently cross-linked cornified envelope. Fully mature corneocytes located in the outer SC layers, are characterized by being relatively large, very hydrophobic and highly cross-linked when compared to immature corneocytes of deeper SC layers. Immature corneocytes in the outer SC are known to be associated with poor barrier function. In this experiment skin is treated with a cosmetic formulation comprising Hexamidine, Niacinamide, Pentanediol and Pal-KT and biomarkers of corneocyte maturity such as size, hydrophobicity and degree of cross-linking are assessed as indicative of barrier function.
The cosmetic formulation exemplified herein comprises 5% Niacinamide, 0.1% Hexamidine, 3% Pentanediol, and 5.5 ppm Pal-KT.
20 female subjects (40-60 yrs old) applied the test cosmetic formulation to one side of their face twice daily for 4 wks, while the other side served as a non-treatment control. D-Squame® tapes were collected at baseline and 4 wks from an area just below the outside corner of the eye on both the treated and non-treated sides. Corneocytes were extracted from the 1st tape and double stained with nile red and an anti-involucrin antibody to evaluate the degree of hydrophobicity and envelope cross-linking. Corneocyte size (μm2) and staining intensity (red:green signal) for each corneocyte were measured microscopically using Image-Pro Plus®. Based upon the red:green signal ratio, the corneocytes were classified as red, mostly red, 50/50, mostly green, or green.
A visual increase in fully mature (red) corneocytes with 4 wks of treatment compared to non-treatment samples was observed. Figure X sets forth the results of two-tailed paired t-tests comparing corneocyte staining classifications and size (area) of non-treated samples to those treated with the cosmetic formulation.
Compared to control, there was a significant 18% (p=0.002) increase in the percentage of nile red staining fully mature corneocytes as a proportion of the total population of corneocytes. Additionally there was a significant decrease in fully immature corneocytes (p=0.067), as well as “less mature” corneocytes (p=0.038).
Concurrent with the higher number of mature corneocytes, there was also a significant 20% (p=0.01) increase in the size of mature corneocytes compared to control samples. A significant increase in size was also observed in “less mature” corneocytes (p=0.035).
The observed improvement of corneocyte maturity biomarkers suggests that use of the cosmetic compositions comprising Hexamidine, Niacinamde, Pentanedio and Pal-KT promotes a more mature SC. This SC contains larger, more hydrophobic corneocytes at the surface, providing greater tortuosity and better covalent bonding of SC intercellular lipids to protect the skin and prevent water loss.
Illustrates upregulation of biomarkers for skin hydration status by treatment with cosmetic compositions comprising Hexamidine, Niacinamide, Pentanediol.
A multiplex mRNA analysis was conducted. RNA was isolated from neonatal human keratinocytes. Neonatal human keratinocytes (Cascade Biologics # C-001-5C) were grown to 40% confluence in 6-well tissue culture plates then treated with compounds for 24 hours (3 replicates per treatment). The treatments included several concentrations of Hexamidine, Niacinamide, and /or Pentanediol. Additionally, 0.00194% (100 μM) caffeine and no-treatment controls were performed.
Several targets were measured with Panomics QuantiGene Plex Assay kit, a multi-analyte quantitative bead-based assay. The fluorescence intensity values, measured by a Bio-Rad Bio-Plex 100 instrument, for those samples yielding sufficient RNA are shown for Aquaporin 3, CDD44 antigen, and Claudin 1. Also measured were Glyceraldehyde-3-phosphate dehydrogenase, Collagen 1α2, Filaggrin, Serine palmitoyltransferase, Interleukin 1,3-hydroxy-3methylglutaryl-coenzyme A reductase, and Interferon gamma.
Charts depicting results are set forth as
Illustrates effects of Galactomyces Ferment Filtrate (GFF) on epidermal barrier marker Caspase-14 in human skin cells as measured via expression of late differentiation biomarkers.
As noted above, the SC of human epidermis is composed of terminally differentiated keratinocytes serving as an essential barrier to environmental stresses, such as UV induced photodamage, and water loss. The regulatory and proteolytic events that coordinate barrier formation are tightly controlled. Caspase-14 belongs to a conserved family of aspartate-specific proteases. Its epidermal expression is restricted almost exclusively to the suprabasal layers, implicating this protease in keratinocyte terminal differentiation and cornification. Thus, caspase-14 is a useful biomarker to monitor formation and homeostasis of the SC barrier.
Galactomyces Ferment Filtrate (GFF) is a known moisturizing agent in cosmetics. In previous studies the present inventors demonstrated that it has several beneficial effects on human skin, such as antioxidant effects through activation of ARE-related genes in human skin cells and induction of hyaluronan production in epidermal cells. The focus of this experiment is caspase-14 in order to elucidate further the effects of GFF on human epidermis.
In vitro human skin models, including skin keratinocytes and skin equivalents (SE) with partially (EPI-201) or completely formed (EPI-200) stratified, cornified epidermis were treated topically with GFF or Dexamethasone (Dex). The SE models were cultured in EPI-100MM medium without hydrocortisone. GFF was diluted with distilled water and added to stratum corneum surface of the skin cultures. Dex and RU-486 (a glucocorticoid receptor antagonist) were added to culture medium of the models. For western blot and caspase-14 (cas-14) analysis, protein was extracted from SE models in PBS by sonication. The data were compared using the Tukey's test. Differences were considered significant at p<0.05.
These results indicate that GFF increases caspase-14 expression by epidermal cells, specifically during late stage differentiation.
This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Ser. No. 61/301,870 filed Feb. 5, 2010.
| Number | Date | Country | |
|---|---|---|---|
| 61301870 | Feb 2010 | US |
