US 2014O163118A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0163118A1 Giuliani et al. (43) Pub. Date: Jun. 12, 2014 (54) EXPRESSION SIGNATURES OF GENES AND Publication Classification GENE NETWORKS ASSOCATED WITH SKN AGING (51) Int. C. (75) Inventors: Giammaria Giuliani, Milan (IT): Raymond Rodriguez, Davis, CA (US); GOIN33/50 (2006.01) Somen Nandi, Davis, CA (US) (52) U.S. C. (73) Assignee: DERMACHIP INC., Davis, CA (US) CPC .................................. G0IN33/5023 (2013.01) USPC .......................... 514/789; 435/6.13:435/6.11 (21) Appl. No.: 14/115,352 (22) PCT Fled: May 3, 2012 (57) ABSTRACT (86) PCT NO.: PCT/US12A36226 S371 (c)(1), Described herein are expression signatures of genes and coor (2), (4) Date: Jan. 27, 2014 dinately expressed gene networks associated with skin aging, Related U.S. Application Data methods used to determine the physiological age of skin, and (60) Provisional application No. 61/482,071, filed on May methods used to screen for compounds used to reduce the 3, 2011. visible signs of aging of the skin. Patent Application Publication Jun. 12, 2014 Sheet 1 of 2 US 2014/O163118A1 Figure 1. A genetic regulatory module responsible for a single gene signature in One aging related network. White = up-regulated, grey = down-regulated). This network which created using only top 100 up & down regulated genes illustrates the complexity of the aging process. This network is connected to other networks involved in skin aging as well as to aging in other tissues. It is the gene signatures in different but connected and interacting networks that determine the aging process. It is the genetic regulatory module (e.g., IL-8, IFNG) that will serve as targets for drugs and natural Compounds to reverse skin aging. It is believed that the loss of Connections between networks reduces the ability of the cell to make coordinated responses to external Stimuli and thus the cause of disease and aging. Patent Application Publication Jun. 12, 2014 Sheet 2 of 2 US 2014/O163118A1 s sy i dows 3. i down r 83. ti aa. dows s: viks. t; 3. SS s tags s low Expression high Expression Figure 2. Master regulators correlate with the differential gene expression signatures between old and young cohorts. Low expression level in old cohorts is at the left of the bottom row which shows expression levels gradually increased to the right. The regulator gene symbols are listed at left side of the graph and are inside boxes. Each regulator has two rows of up and down regulated genes that are positioned according to their gene expression levels in the data sets. The P-value of the correlations between the gene expression signature of overall datasets (bottom row) and individual regulation patterns are listed right below each gene Symbol. The lower the P-value, the high possibility the regulator has to be responsible for the signature changes. US 2014/0163118 A1 Jun. 12, 2014 EXPRESSION SIGNATURES OF GENES AND regulate body temperature through Sweat glands and helps GENE NETWORKS ASSOCATED WITH SKN regulate metabolism. The skin consists of three functional AGNG layers: Epidermis, the Dermis (or corium) and the Subcutis (or hypodermis). RELATION TO OTHERAPPLICATIONS 0006 Various cell types are present in the skin. Kerati 0001. This application claims the benefit of and priority to nocytes is the most abundant cell type in the epidermis. These U.S. Provisional application No. 61/482,071 filed 3 May cells produce keratin proteins. Fibroblasts differentiate into 2011. cells that form the dermis and produce collagen and elastin. Melanocytes produce the pigment melanin that accumulates GOVERNMENT SPONSORSHIP around the nuclei of the keratinocytes absorbing harmful 0002. None ultraviolet (UV) light. Langerhans cells (macrophages) reside in the dermis mediating humoral and cellular immune FIELD OF THE INVENTION functions. Merkel’s cells, which are present in small numbers but are more numerous in the skin of the palms and soles of 0003. The invention relates to newly discovered expres the feet, are sensory mechanical receptors that respond to sion signatures of coordinately expressed genes and gene certain stimuli such as pressure or touch. The epidermis is the networks associated with the physiological and pathological outermost skin layer. As skin cells migrate to the Surface, processes of skin aging and to methods for identifying and farther away from their source of nourishment, they flatten using compounds that regulate and in some cases recalibrate and shrink. They lose their nuclei, move out of the basal layer gene sets and gene pathways associated with skin ageing to the horny layer (stratum corneum), and die. This process, wherein such regulation and recalibration effectively reverses called keratinization, takes about 4 weeks. About 10 percent the effects of ageing of the skin to produce more youthful of epidermal cells are melanocytes that pigment the skin. The looking skin so that the skin is more Supple, Smooth and epidermis is differentiated into five layers: horny layer (stra hydrated and has less wrinkles and fine lines typically asso tum corneum); clear layer (stratum lucidum); granular layer ciated with ageing. (stratum granulosum); prickle-cell layer (stratum spinosum); and the basal layer (stratum basale). The dermis is the layer BACKGROUND just below the outer keratinized epidermal layer. The dermis 0004 Skin aging is natural process associated with a num contains cells, water, collagen fibers, glycosaminoglycans ber of pathophysiologies that can reduce quality of life and and fibronectins that form a hydrated gel and are responsible longevity. The goal of this study is to gain new insights into for the high elasticity and tensile strength of the dermis. how skin ages as a function of time. The working hypothesis Embedded in this layer are lymph channels, blood vessels, for this study is that skin aging results from the dysregulation nerve fibers, muscle cells, hair follicles, sebaceous glands, of key regulatory genes (i.e., gene signatures) that control and Sweat glands. thousands of genes in multiple, interacting and overlapping networks and pathways. Dysregulation of gene functions 0007 Glycosaminoglycans are mucopolysaccharides leads to physiological aging of tissues, cells and Subcellular present in the dermis that can bind large amounts of water. As components. This dysregulation may be the result of damage the skin ages, the interweaving of the collagen fibers to the products of key regulatory genes (i.e., oxidative dam increases and the water-binding capacity diminishes and the age to regulatory proteins) or to transcriptional silencing of skin tends to wrinkle. Glycosaminoglycans bind with the these genes by mutagenic and/or epigenetic processes. This proteins in the connective tissue matrix to form proteogly study is the first of its kind to examine, in a comprehensive cans. These proteoglycans form a gel-like material that can fashion, the gene expression profiles in human skin (specifi absorb and expel water like a sponge. Glycosaminoglycans cally adult females who self-identified themselves as white) are subject to a continuous turnover. In contrast, the collagen of various age and to identify gene networks and gene signa fibers are only renewed when necessary, such as when injury tures associated with the aging process. This information is sustained. The ability of the skin to store water and thereby should yield a small number of robust and predictive genes/ remain soft and Supple depends in part on the presence of gene-network whose expression can be used to: (a) under lipids, arginine, and other “natural moisturizing factors' stand the relationship between chronological age and physi (NMF) that originate from the cornification (differentiation) ological age and (b) assess the ability of compounds (e.g., of the keratinocytes, for example, pyrrolidine carboxylic drugs, natural products and dietary factors) to regulate and/or acid, and secretions from the Sweat and sebaceous glands dysregulate genes and gene networks. The present study pro including urea, salts, and organic acids. vides information that will guide future research and Suggest 0008. The dermis also contains collagens. Type I collagen new therapies for preventing, mitigating or treating skin is the most abundant protein in skin connective tissue, which pathologies related to aging. also contains other types of collagen (III, V, VII), elastin, 0005. The skin is the largest human organ comprising proteoglycans, fibronectin, and other extracellular matrix about one sixth of total body weight. The skin performs a proteins. Newly synthesized type I procollagen is secreted complex role in human physiology: serves as a barrier to the into the dermal extracellular space where it undergoes enzy environment, and the sebum produced by Some of its glands matic-processing, arranging itself into a triple helix configu (sebaceous) have anti-infective properties. The skin acts as a ration. The triple helix complexes associate with other extra channel for communication to the outside world, protects us cellular matrix proteins such as leucine-rich Small from water loss, friction wounds, and impact wounds and proteoglycans, to form regularly arranged fibrillar structures. uses specialized pigment cells to protect us from ultraviolet This process, called fibrillogenesis, results in formation of rays of the sun. Skin produces vitamin D in the epidermal collagen bundles that are responsible for the strength and layer, when it is exposed to the Sun's rays. The skin helps resiliency of the skin. US 2014/0163118 A1 Jun. 12, 2014 0009 Skin aging is influenced by several factors, includ 0014 Increased intracellular ceramide content may also ing genetics, environmental exposure (ultraviolet (UV) irra contribute to activation of the MAP kinase pathways by UV diation, Xenobiotics, and mechanical stress), hormonal irradiation. UV-induced ceramide generation seems to be changes, and metabolic processes (generation of reactive dependent on increased ROS production, since ceramide and chemical compounds such as activated oxygen species, Sug ROS levels rise in parallel, and UV-induced ceramide pro ars, and aldehydes).