EP1356106A2 - Procede pour la determination in vitro des agressions ou du vieillissement cutanes - Google Patents

Procede pour la determination in vitro des agressions ou du vieillissement cutanes

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Publication number
EP1356106A2
EP1356106A2 EP01986926A EP01986926A EP1356106A2 EP 1356106 A2 EP1356106 A2 EP 1356106A2 EP 01986926 A EP01986926 A EP 01986926A EP 01986926 A EP01986926 A EP 01986926A EP 1356106 A2 EP1356106 A2 EP 1356106A2
Authority
EP
European Patent Office
Prior art keywords
skin
proteins
mrna molecules
column
fragments
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01986926A
Other languages
German (de)
English (en)
Inventor
Dirk Petersohn
Marcus Conradt
Kay Hofmann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henkel AG and Co KGaA
Original Assignee
Henkel AG and Co KGaA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Henkel AG and Co KGaA filed Critical Henkel AG and Co KGaA
Publication of EP1356106A2 publication Critical patent/EP1356106A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6881—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/20—Dermatological disorders

Definitions

  • the present invention relates to a method for determining skin stress and / or skin aging in humans or animals in vitro, test kits and biochips for determining skin stress and / or skin suppuration, and the use of proteins, mRNA molecules or fragments of proteins or mRNA molecules as stress and / or aging markers of the skin; furthermore a test method for proving the effectiveness of cosmetic or pharmaceutical active substances against skin stress and / or skin aging as well as a screening method for the identification of cosmetic or pharmaceutical active substances against skin stress and / or skin aging and a method for the production of a cosmetic or pharmaceutical preparation against skin stress and / or or skin aging.
  • Every living cell is able to react to signals from its environment.
  • the reactions of the cells are realized by an orderly regulation of the gene expression, so that the metabolism of cells is not static but very dynamic.
  • the human genome comprises around 140,000 genes.
  • each cell uses only a small, specific part for the synthesis of proteins, which is reflected in the gene expression pattern.
  • Exogenous signals are received by cells and lead to changes in the gene expression pattern, partly via complex signal transduction cascades. In this way, each cell responds to signals from its environment by adapting its metabolism.
  • the cells of the skin notice the high-energy radiation from the sun and react to it by changing their RNA and protein synthesis performance.
  • Some molecules are increasingly synthesized after a stress stimulus (e.g. sunlight) (e.g. MMP-1), others are produced to a lesser extent (e.g. collagen ⁇ (I)).
  • a stress stimulus e.g. sunlight
  • MMP-1 e.g. MMP-1
  • others are produced to a lesser extent (e.g. collagen ⁇ (I)).
  • no significant change will take place in a large number of the synthesis processes (for example TIMP-1).
  • Human skin is the largest organ in the human body. It is a very complex organ, which consists of a variety of different cell types and forms the body's interface with the environment. This fact makes it clear that the cells of the skin are particularly exposed to exogenous signals of the environment, physical and chemical nature and therefore regulate their gene expression continuously. To understand skin reactions to exogenous stimuli, the analysis of gene expression in the skin is therefore of crucial importance.
  • the macroscopic phenomena of aging skin are based on the one hand on intrinsic or chronological aging (skin aging), and on the other hand on extrinsic aging due to environmental stress (skin stress).
  • skin aging intrinsic or chronological aging
  • extrinsic aging due to environmental stress (skin stress).
  • the ability of living skin cells to react to your environment changes over time - there are aging processes that lead to senescence and ultimately cell death.
  • the visible signs of aged skin are to be understood as an integral of intrinsic and extrinsic aging (e.g. due to sunlight), the events of extrinsic aging accumulating in the skin over a longer period of time.
  • the skin consists of several different cell types (fibroblasts, keratinocytes in different differentiation states, melanocytes, Merkel cells, Langerhans cells, hair follicle cells, sweat gland cells etc.), so that the complexity of genes expressed in the skin is very great. It has never been possible to describe this immense complexity. So far, it has not been possible to identify genes from this complexity that are related to skin aging and can serve as molecular markers of skin aging.
  • mRNA molecules are present in the cell in concentrations between a few and several hundred copies.
  • the weakly expressed genes have so far not been available or have been very difficult to access, but they can certainly play a decisive role in aging processes and for the skin's home ostasis.
  • transcriptome The entirety of all mRNA molecules that are synthesized by a cell or a tissue at a specific point in time is referred to as a "transcriptome”. To date, it has not been possible to describe the complete transcriptome, i.e. the entirety of all transcribed genes, in human skin.
  • the cosmetic antiage products on the market exert their effects on one of the few known markers of extrinsic skin aging, such as collagen synthesis, collagenase activity or collagenase inhibitors.
  • methods for determining skin stress and / or skin aging are to be provided by means of the identified genes.
  • This first object is achieved according to the invention by a method (1) for identifying the genes which are important for skin aging and / or skin stress in humans or animals in vitro, characterized in that a) a first mixture of expressed in human or animal skin, ie transcribed and possibly also translated genetically coded factors, ie proteins, mRNA molecules or fragments of proteins or mRNA molecules from young human or animal skin, b) a second mixture of expressed, ie transcribed and optionally in human or animal skin also translates translated genetically coded factors, ie of proteins, mRNA molecules or fragments of proteins or mRNA molecules from old human or animal skin and c) subjecting the mixtures obtained in a) and b) to a serial analysis of gene expression (SAGE), and thereby identifying the genes which are expressed differently (differentially) in old and young skin.
  • SAGE serial analysis of gene expression
  • the method according to the invention can preferably be applied to human skin, but can also be applied to animal skin and to skin models based on human or animal skin.
  • SAGE TM serial analysis of gene expression
  • collagen (I) ⁇ 1 is overexpressed by a factor of 4-5 in young skin
  • collagen (I) ⁇ 2 by a factor of 4
  • collagen (III) ⁇ 1 by a factor of 8.
  • Other gene classes are those of young skin overexpressed correspond to marker proteins, for example of the cytoskeleton.
  • transgelin, desmin, actin, myosin, calponin and tropomyosin are overrepresented between 6 and 37 times.
  • some keratins and other keratinocyte-specific genes are overrepresented in the library from old skin.
  • epidermal keratins 5, 10 and 14 which are strongly expressed in both libraries are e.g. B. represented about 2 times more strongly in the old skin, while keratin 1, which is also strongly expressed, does not show this tendency.
  • Stratifin, Desmocollin, MMP2 (gelatinase) and CLSP (keratinocyte-specific calmodulin) are 5 to 8 times overrepresented.
  • Tables 1 to 4 contain a detailed list of the genes determined with the aid of the method according to the invention, which are differentially expressed in old and young skin, stating a serial number in column 1, the tag sequence used in column 2, and the determined relative expression frequency in young skin in column 3, the determined expression frequency in old skin in column 4, the quotient of the frequencies (from column 3 and column 4) in column 5, the significance in column 6, the UniGene accession number in column 7 and a brief description of the Gene or gene product in column 8.
  • the quotient in column 5 indicates the strength of the differential expression, i. i.e. by which factor the respective gene is expressed more strongly in young skin than in old skin, or vice versa.
  • the database is accessible on the Internet at the following address: http://www.ncbi.nlm.nih.gov/.
  • genes or gene products are also available at the Internet addresses httD.7 / www.ncbi.nlm.nih.gov / UniGene / Hs.Home.html or http://www.ncbi.nlm.nih.gov/genome/ quide directly accessible. Table 1 lists all genes that are at least 2-fold and less than 5-fold differentially expressed.
  • Table 2 lists all genes that are at least 5-fold and less than 7-fold differentially expressed.
  • Table 3 lists all genes that are at least 7-fold and less than 10-fold differentially expressed.
  • Table 4 lists all genes that are at least 10-fold differentially expressed.
  • Table 5 lists genes that are at least 2-fold differentially expressed; to which, however, no database entry could be assigned and which can therefore only be identified by the tag sequence in column 2.
  • Table 6 lists genes that are at least 2-fold differentially expressed and that in column 2 by their UniGene Accession Number, in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBUGenbank number To be defined.
  • Table 7 lists genes that are differentially expressed between 2.89 and 11.10-fold. The assignment of the tags to the genes, which are defined by your UniGene Accession Number in column 6, was done by manual annotation.
  • the databases were downloaded from the NCBI, formatted for a local version of the BLAST program (also NCBI) and compared for identical hits with the tags detected in the SAGE analysis.
  • the genes / clones found were checked for redundancy and reworked as follows:
  • ESTs are from dbEST through 19-Oct-2001
  • the second object underlying the present invention is achieved according to the invention by a method (2) for determining skin stress and / or skin aging in humans or animals, in particular in women, in vitro, which is characterized in that a) a mixture of proteins , mRNA molecules or fragments of proteins or mRNA molecules from human or animal skin, b) the mixture obtained is examined for the presence and, if appropriate, the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are identified as differentially expressed in old and young skin by means of serial analysis of gene expression (SAGE), c) compares the test results from b) with the expression patterns identified by means of serial analysis of gene expression (SAGE) and d) the mixture examined in b) is assigned to old or stressed skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more strongly in old or stressed skin than in young or undressed skin , or assigns the mixture examined in b) to young or undressed skin
  • step b) of the method for determining skin stress and / or skin aging it may be sufficient to examine the mixture obtained for the presence of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules by means of serial analysis gene expression (SAGE) can be identified as differentially expressed in old and young skin if these are only expressed in old or only in young skin. In all other cases, the amount of differentially expressed molecules must also be examined in step b). that is, expression must be quantified.
  • SAGE serial analysis gene expression
  • step d) of the method for determining skin stress and / or skin aging the mixture of old or stressed skin examined in b) is assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more in old or stressed skin than in young or stressed skin, d. that is, the mixture either contains more different compounds typically expressed in old skin than those typically expressed in young skin (qualitative differentiation), or contains more copies of compounds typically expressed in old skin than is typically present in young skin are (quantitative differentiation).
  • the assignment to young or undressed skin is carried out in a complementary manner.
  • a preferred embodiment of the method according to the invention for determining skin stress and / or skin aging is characterized in that in step b) the mixture obtained is checked for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of protein NEN or mRNA molecules that are defined in Tables 1 to 4 in column 7 by their UniGene Accession Number, in step c) the test results from b) with those in Tables 1 to 4 in columns 3 and 4 compares the specified expression frequencies and the expression quotients specified in column 5 and in step d) assigns the mixture of old or stressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are in old age or stressed skin are expressed at least twice as strongly as in young or undressed skin, or assigns the mixture of young or undressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are expressed at least twice as strongly in young or undressed skin
  • a further preferred embodiment of the method according to the invention for determining skin stress and / or skin aging is characterized in that in step b) the mixture obtained is checked for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or Examined mRNA molecules, which are defined in Tables 2 to 4 in column 7 by their UniGene Accession Number, in step c) the test results from b) with the relative given in Tables 2 to 4 in columns 3 and 4 Expression frequencies and the expression quotients indicated in column 5 are compared and in step d) the mixture of old or stressed skin examined in b) is assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are in old or stressed skin are expressed at least 5 times as strongly as in young or undressed skin, or in b) the examined mixture of young or undressed skin is assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed in young or undressed skin at least
  • step b) the mixture obtained is examined for the presence and, if appropriate, the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules, which are listed in tables 3 and 4 in column 7 by their UniGene accession number in step c) the test results from b) are compared with the relative expression frequencies given in tables 3 and 4 in columns 3 and 4 as well as with the expression quotients given in column 5 and in step d) the mixture of old resp assigned stressed skin if it contains predominantly proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 7-fold as strongly in old or stressed skin as in young or undressed skin, or that in b ) examined mixture of young or undressed skin assigned if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 7-fold as strongly in young or undressed skin as in old
  • a further preferred embodiment of the method according to the invention for determining skin stress and / or skin aging is characterized in that in step b) the mixture obtained is checked for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or investigated mRNA molecules, which are defined in Table 4 in column 7 by their UniGene Accession Number, in step c) the test results from b) with the relative expression frequencies given in table 4 in columns 3 and 4 and the column 5 compares the given expression quotient and in step d) assigns the mixture of old or stressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules that are at least 10 times in old or stressed skin are expressed as strongly as in young or undressed skin, or the gemi examined in b) assigned to young or undressed skin if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 10 times as strongly in young or undressed skin as in old or stressed skin
  • a further preferred embodiment of the method according to the invention for determining skin stress and / or skin aging is characterized in that in step b) the mixture obtained is checked for the presence and optionally the amount of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules investigated, which are defined in Table 5 or in Table 7 in column 2 by their 11-base tag sequence, in step c) the test results from b) with those in Table 5 or in Table 7 in columns 3 and 4 specified relative expression frequencies and the expression quotient indicated in column 5 and in step d) assigns the mixture of old or stressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are contained in old or stressed skin at least twice, especially 5 times, preferably 7 times, especially b e are preferably expressed 10 times as strongly as in young or undressed skin, or assigns the mixture of young or undressed skin examined in b) if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA
  • the condition of the skin can also be described by quantifying several markers (expression products of the genes which are important for skin aging and / or skin stress), which must then be active in a characteristic relationship with one another in order to represent young skin, or in a characteristic ratio different from this must be active in order to represent old skin.
  • the present invention therefore furthermore relates to a method (3) for determining skin stress and / or skin aging in humans or animals, in particular in women, in vitro, which is characterized in that a) a mixture of proteins, mRNA Molecules or fragments of proteins or mRNA molecules from human or animal skin wins, b) in the mixture obtained at least two of the proteins, mRNA molecules or fragments of proteins or mRNA molecules quantified by means of method (1) as for skin aging and / or the skin stress are significantly identified, c) the expression ratios of the at least two proteins, mRNA molecules or fragments of proteins or mRNA molecules relative to one another are determined, d) the expression ratios from c) are compared with the expression ratios, which are typically young or old for the molecules quantified in b) Skin is present, in particular with the expression ratios that result from Tables 1 to 5, columns 3 and 4, and e) assigns the mixture of old or stressed skin obtained in a) if the expression ratios of the examined skin match the expression
  • the mixture is preferably obtained from a skin sample, in particular from a whole skin sample or from an epidermis sample.
  • a skin sample opens up more extensive comparison options with the SAGE libraries, which are also obtained from whole skin.
  • the epidermis sample on the other hand, is easier to obtain, for example by applying an adhesive tape to the skin and tearing it off, as described in WO 00/10579, to which reference is hereby made in full.
  • the mixture is obtained in step a) by means of microdialysis.
  • microdialysis A method for measurement of local tissue metabolism", Nielsen PS, Winge K, Petersen LM; Ugeskr Laeger 1999 Mar 22 161: 12 1735-8; and in "Cutaneous microdialysis for human in vivo dermal absorption studies ", Anderson, C. et al. ; Drugs Pharm. Sci., 1998, 91, 231-244; and also described on the Internet at http://www.microdialysis.se/techniqu.htm, to which reference is hereby made in full.
  • microdialysis When using microdialysis, a probe is typically inserted into the skin and the probe is slowly rinsed with a suitable carrier solution. After the acute reactions after the puncture have subsided, the microdialysis delivers proteins, mRNA molecules or fragments of proteins or mRNA molecules which occur in the extracellular space and which can then be isolated and analyzed in vitro, for example by fractionation of the carrier liquid. Microdialysis is less invasive than taking a whole skin sample; however, it is disadvantageously limited to the extraction of compounds occurring in the extracellular space.
  • a further preferred embodiment of the method according to the invention for determining skin stress and / or skin aging is characterized in that in step b) in method (2) the examination for the presence and optionally the amount of at least one of the proteins or protein fragments; or in method (3) the quantification of at least two proteins or protein fragments is carried out by means of a method which is selected from
  • 2D gel electrophoresis is described, for example, in LD Adams, Two-dimensional Gel Electrophoresis using the Isodalt System or in LD Adams & SR Gallagher, Two-dimensional Gel Electrophoresis using the O'Farrell System; both in Current Protocols in Molecular Biology (1997, Eds. FM Ausubel et al.), Unit 10.3.1-10.4.13; or in 2-D E-lectrophoresis manual; T. Berkelman, T. Senstedt; Amersham Pharmacia Biotech, 1998 (Order No. 80-6429-60).
  • the mass spectrometric characterization of the proteins or protein fragments is carried out in a manner known in the art, for example as described in the following references:
  • a further preferred embodiment of the method according to the invention for determining skin stress and / or skin aging is characterized in that in step b) in method (2) the examination for the presence and, if appropriate, the amount of at least one of the mRNA molecules or mRNA molecules molecular fragments; or in method (3) performs the quantification of at least two mRNA molecules or mRNA molecule fragments by means of a method which is selected from Northern blots,
  • RT-PCR Reverse transcriptase polymerase chain reaction
  • RNase protection experiments iv. Dot blots
  • v. cDNA sequencing vi. Clone hybridization
  • vii. Differential display viii. Subtractive hybridization
  • TOGA Total Gene Expression Analysis
  • SAGE Serial analysis of gene expression (SAGE) and especially xii. Use of nucleic acid chips, or by means of suitable combinations of these methods.
  • a further preferred embodiment of the method according to the invention for determining skin stress and / or skin aging is characterized in that in step b) the presence and optionally the amount of 1 to about 5000, preferably 1 to about 1000, in particular about 10 to about 500, preferably about 10 to about 250, particularly preferably about 10 to about 100 and very particularly preferably about 10 to about 50 of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which i. in tables 1 to 4 in column 7 by their UniGene Accession Number, or ii.
  • Another object of the present invention is a test kit for determining skin stress and / or skin aging in humans or animals in vitro, comprising means for carrying out the method according to the invention for determining skin stress and / or skin aging.
  • Another object of the present invention is a biochip for determining skin stress and / or skin aging in humans or animals in vitro, comprising i. a solid, ie rigid or flexible support and ii. on this immobilized probes which are capable of specific binding to at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are defined in Tables 1 to 4 in column 7 by their UniGene accession number, or which are defined in Table 5 or in Table 7 in column 2 by their 11-base tag sequence.
  • a BioChip is a miniaturized functional element with molecules immobilized on a surface, in particular biomolecules, which can serve as specific interaction partners.
  • DNA DNA
  • RNA in the case of nucleic acids and their chemical derivatives there may be single strands, triplex structures or combinations thereof
  • saccharides e.g. Antibodies, antigens, receptors
  • peptides proteins
  • proteins e.g. Antibodies, antigens, receptors
  • derivatives of combinatorial chemistry e.g. organic molecules
  • BioChips have a 2D base area for coating with biologically or biochemically functional materials.
  • the base surfaces can, for example, also be formed by walls of one or more capillaries or by channels.
  • the prior art can e.g. For example, reference is made to the following publications: Nature Genetics, Vol. 21, Supplement (total), Jan. 1999 (BioChips); Nature Biotechnology, Vol. 16, pp. 981-983, Oct. 1998 (BioChips); Trends in Biotechnology, Vol. 16, pp. 301-306, Jul.
  • the DNA chip technology which is particularly preferred in the context of the present invention is based on the ability of nucleic acids to enter into complementary base pairings. This technical principle, known as hybridization, has been used for years in Southern blot and Northern blot analysis. Compared to these conventional methods, in which only a few genes are analyzed, DNA chip technology allows a few hundred to several tens of thousands of genes to be examined in parallel.
  • a DNA chip essentially consists of a carrier material (e.g. glass or plastic) on which single-stranded, gene-specific probes are immobilized in a high density at a defined location (spot).
  • a carrier material e.g. glass or plastic
  • probe immobilization is considered problematic. According to the current state of the art, several ways of immobilizing probes are realized:
  • E. M. Southern (EM Southern et al. (1992), Nucleic Acid Research 20, 1679-1684 and EM Southern et al. (1997), Nucleic Acid Research 25, 1155-1161) describes the production of oligonucleotide arrangements by direct synthesis on a glass surface, which was derivatized with 3-glycidoxypropyltrimethoxysilane and then with a glycol.
  • a similar method realizes the in situ synthesis of oligonucleotides using a photosensitive, combinatorial chemistry that can be compared with photolithographic techniques (Pease, AC et al. (1994), Proc. Natl Acad Sei USA 91, 5022- 5026).
  • photolithographic techniques Pease, AC et al. (1994), Proc. Natl Acad Sei USA 91, 5022- 5026.
  • existing DNA molecules can also be bound to surfaces of support material.
  • P.O. Brown (DeRisi et al. (1997), Science 278, 680-686) describes the immobilization of DNA on glass surfaces coated with polylysine.
  • the DNA probes can be applied to a carrier using a so-called “pin spotter”.
  • a pin spotter thin metal needles, for example with a diameter of 250 ⁇ m, are immersed in probe solutions and then transfer the attached sample material with defined volumes to the carrier material of the DNA Crisps.
  • the probe is preferably applied by means of a piezo-controlled nanodispenser, which, similar to an inkjet printer, applies probe solutions with a volume of 100 picoliters to the surface of the carrier material in a contact-free manner.
  • the probes are immobilized, for example, as described in EP-A-0 965 647:
  • the generation of DNA probes is carried out by means of PCR using a sequence-specific primer pair, a primer at the 5 'end being modified and a linker with a free one Amino group carries. This ensures that a defined strand of the PCR products can be bound to a glass surface which has been treated with 3-aminopropyltrimethoxysilane and then with 1, 4-phenyldiisothiocyanate.
  • the gene-specific PCR products should ideally have a defined nucleic acid sequence with a length of 200-400 bp and contain non-redundant sequences.
  • mRNA is isolated from two cell populations to be compared.
  • the isolated mRNAs are converted into cDNA by means of reverse transcription using, for example, fluorescence-labeled nucleotides.
  • the samples to be compared are marked with, for example, red or green fluorescent nucleotides.
  • the cDNAs are then hybridized with the gene probes immobilized on the DNA chip and the bound fluorescence is then quantified.
  • the biochip according to the invention preferably comprises 1 to approximately 5000, preferably 1 to approximately 1000, in particular approximately 10 to approximately 500, preferably approximately 10 to approximately 250, particularly preferably approximately 10 to approximately 100 and very particularly preferably approximately 10 to approximately 50 different probes.
  • the different probes can each be present in duplicate on the chip.
  • the biochip according to the invention preferably comprises nucleic acid probes, in particular RNA or PNA probes, particularly preferably DNA probes.
  • the nucleic acid probes preferably have a length of approximately 10 to approximately 1000, in particular approximately 10 to approximately 800, preferably approximately 100 to approximately 600, particularly preferably approximately 200 to approximately 400 nucleotides.
  • the biochip according to the invention comprises peptide or protein probes, in particular antibodies.
  • the biochip according to the invention comprises probes which are capable of specific binding to at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are listed in Table 6 or 8 in column 2 by their UniGene accession. Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene.
  • Another object of the present invention is the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which i. in tables 1 to 4 in column 7 by their UniGene Accession Number, or the ii. in Table 6 or 8 in column 2 by their UniGene Accession Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene , or the iii. in Table 5 or in Table 7 in column 2 are defined by their 11-base tag sequence as stress and / or aging markers of the skin in humans or animals.
  • Another object of the present invention is a test method for demonstrating the effectiveness of cosmetic or pharmaceutical active substances against skin stress and / or skin aging in vitro, characterized in that a) the skin status is determined by a method according to the invention for determining skin stress and / or skin aging, or using a test kit according to the invention for determining skin stress and / or skin aging, or using a biochip according to the invention, b) applying an active ingredient against skin stress and / or skin aging to the skin one or more times, c) renewing the skin status by means of an inventive method Method for determining skin stress and / or skin aging, or using a test kit according to the invention for determining skin stress and / or skin aging, or using a biochip according to the invention, and d) the effectiveness of the active ingredient by comparing the results from a) and c) determined.
  • Another object of the present invention is a test kit for demonstrating the effectiveness of cosmetic or pharmaceutical active substances against skin stress and / or skin aging in vitro, comprising means for carrying out the test method according to the invention.
  • Another object of the present invention is the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which i. in tables 1 to 4 in column 7 by their UniGene Accession Number, or ii. in Table 6 or 8 in column 2 by their UniGene Accession Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene , or the iii. in Table 5 or in Table 7 in column 2 are defined by their 11-base tag sequence to demonstrate the effectiveness of cosmetic or pharmaceutical active ingredients against skin stress and / or skin aging.
  • Another object of the present invention is a screening method for the identification of cosmetic or pharmaceutical active ingredients against skin stress and / or skin aging in vitro, characterized in that a) the skin status by an inventive method for determining the skin stress and / or Skin aging, or by means of a test kit according to the invention for determining skin stress and / or skin aging, or by means of a biochip according to the invention, b) applying a potential active substance against skin stress and / or skin aging to the skin one or more times, c) the skin status again determined by a method according to the invention for determining skin stress and / or skin aging, or by means of a test kit according to the invention for determining skin stress and / or skin aging, or by means of a biochip according to the invention, and d) active ingredients by comparing the results from a ) and c) determined.
  • Another object of the present invention is the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which i. in tables 1 to 4 in column 7 by their UniGene Accession Number, or the ii. in Table 6 or 8 in column 2 by their UniGene Accession Number, or in column 3 by their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene , or the iii. in Table 5 or in Table 7 in column 2 are defined by their 11-base tag sequence for the identification of cosmetic or pharmaceutical active substances against skin stress and / or skin aging.
  • Another object of the present invention is a method for producing a cosmetic or pharmaceutical preparation for skin stress and / or skin aging, characterized in that a) active ingredients with the aid of the screening method according to the invention, or the use for the identification of cosmetic or pharmaceutical active ingredients against skin stress and / or skin aging and b) active ingredients found to be effective mixed with cosmetically and pharmacologically suitable and compatible carriers.
  • the present invention further provides a cosmetic or pharmaceutical preparation for skin stress and / or skin aging, comprising at least one nucleic acid construct which is suitable for suppressing or reducing the activity of at least one of the proteins which are more strongly expressed in old or stressed skin than in young or undressed skin, or to induce or to increase the activity of at least one of the proteins which are expressed more strongly in young or undressed skin than in old or stressed skin.
  • the proteins are preferably selected from those in Tables 1 to 4 in column 7 by their UniGene accession number, or in Table 6 or 8 in column 2 by their UniGene accession number, or in column 3 their Swissprot or TREMBL number, or in column 4 by their EMBL / Genbank number, or in Table 9 by the name of the gene, or the tag sequence in Table 5 or in Table 7 in column 2 by their 11 bases
  • the nucleic acid construct is preferably selected from DNA, RNA or PNA. However, linear combinations of these nucleic acids or hybrid molecules, for example RNA / DNA hybrids, are also possible.
  • the nucleic acid construct is also preferably selected from protein-coding sequences, ribozymes, antisense nucleic acids, triple helix formers and rRNA.
  • the preparation according to the invention can contain approximately 1000, in particular approximately 10 to approximately 500, preferably approximately 10 to approximately 250, particularly preferably approximately 10 to approximately 100 and very particularly preferably approximately 10 to approximately 50 nucleic acid constructs which differ from one another.
  • the nucleic acid construct is present in the preparation according to the invention in particular enclosed in lipid vesicles, for example in liposomes, niosomes or transfersomes, preferably in liposomes.
  • Suitable nucleic acid constructs for the purposes of the invention are DNA and RNA sequences which code for one of the age markers and constructs of these polynucleotides.
  • the invention also includes partial sequences of age genes and genes whose sequence has been changed in comparison to the age genes mentioned by directed or other types of mutagenesis.
  • polynucleotides for insertion into the skin are DNA and RNA sequences which comprise part of the sequence of one or more of the genes mentioned and themselves have a desired biological activity (for example anti-sense RNA, rRNA or short double-stranded DNA molecules) ,
  • Any gene which can be used according to the invention, but preferably those which were found to be weakly expressed in the aging skin, can be introduced into the cells of the skin with the aim of its increased expression.
  • Any construct from one of the genes which can be used according to the invention, functionally linked to a eukaryotic promoter, can be used.
  • the constructs with one of the age genes can be any eukaryotic expression constructs.
  • bacterial plasmids, viral constructs, or other DNA constructs can be genetically modified to produce a recombinant DNA (or RNA) molecule that contains a sequence that encodes one of the age genes and expresses the desired gene product in skin cells ,
  • the preparation according to the invention is preferably applied to the skin.
  • the DNA contained in it can be either linear or circular, preferably circular DNA molecules.
  • the polynucleotide or the polynucleotide-containing construct can be reproduced and purified by known methods and is used as a pure molecule or in one of the u.g. Wording used.
  • Constructs which carry a promoter which enables expression of the DNA of interest are preferred. Depending on the nature of the gene and the purpose of its use, different promoters can be used. Strong, constitutive promoters can be used, such as the immediate early promoter gene "of cytomegalovirus (CMV) or the promoter of the" long terminal repeat "of Rous Sarcoma virus (RSV).
  • CMV cytomegalovirus
  • RSV Rous Sarcoma virus
  • tissue-specific promoters or cell type-specific promoters can be used.
  • the promoter can be selected so that it effects the specific expression in skin cells or certain skin cell types.
  • tissue-specific or cell-type-specific promoters include the keratin promoters (e.g. human keratin 14 promoter (Wang et al. 1997 Proc. Natl. Acad. Be US 94: 219-226)) or tyrosinase promoters (specific for melanocytes).
  • inducible promoters can be used.
  • the constructs can also contain other elements which enhance the transcriptional or translational expression of the gene product.
  • the construct may include an "internal ribosomal entry site" (IRES) to enhance translation of the downstream sequence (see Murakami et al. 1997, Gene 202: 23-29).
  • IRS internal ribosomal entry site
  • Other components that are present on the construct may include markers (e.g., antibiotic resistance genes such as the ampicillin resistance gene) for selecting cells containing the construct, an origin of replication that causes the construct to replicate stably in prokaryotic cells, a nuclear localization signal, or other elements that inhibit the production of the construct Support DNA construct and / or the encoded protein.
  • the constructs can also contain a polyadenylation signal.
  • the sequence of such a signal can be selected from various known polyadenylation signals.
  • a preferred example is the SV40 early polyadenylation signal.
  • the constructs can also include one or more introns that can enhance expression of the DNA.
  • the invention also includes constructs that code for fusion proteins of one of the age markers with a second protein or for a fusion protein of two age markers.
  • Preferred nucleic acid constructs are those with a plurality of expression cassettes, on which two or more age markers are coded, or which code one of the age genes and a gene for a further protein.
  • Co-transfection with one or more further vectors which can support the expression of the gene of interest is also possible.
  • Vectors which carry one or more of the features mentioned and, in addition, can also contain further functional units have been described many times in the literature and are commercially available. Such vectors are e.g. pCI and pSI (Promega GmbH) or pDEST (Gibco BRL).
  • sequences of the age genes and partial sequences of the genes which can be determined according to the invention and in particular those listed in Tables 1 to 9 can be used for the selective inhibition of the expression of individual genes.
  • Oligonucleotides and polynucleotides suitable for this purpose include antisense nucleotides, ribozyme nucleotides and double-stranded RNAs.
  • Antisense nucleotides are well known in their ability to hybridize with sense strands of the mRNA and thereby interfere with the expression of the mRNA (see, for example, Wingers et al., Laboratory Investigation 79, 1415-1424 (1999). An overview of the use of antisense nucleotides in the skin gives Wraight et al., Pharmacol & Ther 90, 89-104 (2001).
  • Ribozyme nucleic acids are also known as single-stranded RNA molecules that have the ability to selectively cleave ssRNA and ssDNA, and thereby selectively inhibit the expression of the target molecules.
  • Post-transcriptional gene repression can also be caused by double-stranded RNAs. These dsRNAs will soon interfere with the target RNA after cleavage. right segments by ribonuclease III. Duplexes from short RNA sequences can also be used for gene repression (SM Elbashir et al., Nature 411, 494-498 (2001). These double-stranded RNA molecules can also be used in the sense of the invention for the repression of the found age genes.
  • Unmodified DNA or RNA molecules are rapidly degraded in tissues or cells.
  • modifications have been developed which affect either the backbone or the pyridine or pyrimidine bases of an oligonucleotide.
  • modified DNA or RNA sequences can also be used for the purposes of the invention. Suitable modifications are e.g. Phosphorothioates, methylphosphonates or peptide linkages (PNAs) for the sugar backbone as well as C5-propynyl-dU, dC for the nucleoside bases.
  • PNAs Phosphorothioates, methylphosphonates or peptide linkages
  • the DNA or RNA molecules of the invention can e.g. can be applied topically.
  • the molecules can either be used without further substances influencing the penetration, or in a mixture or associated with molecules which influence the penetration through the stratum corneum and the transfection of the cells of the skin.
  • a preferred embodiment of the topical application of the age genes is that in a formulation with lipids, optionally in a mixture with surfactants, preferably in the form of liposomes.
  • the formulation contains about 0.1 ⁇ g - 5 mg DNA or RNA per mg liposome.
  • the components of the liposomes can be neutral or charged and in the form of e.g. multilamellar vesicles or unilamelar vesicles are present.
  • Suitable lipids for the production of liposomes are, for example, natural phosphatidylcholine, which can be obtained, for example, from egg, soybean, olives, coconut, whale fat, saffron, linseed, evening primrose or primrose.
  • Other suitable lipids are, for example, natural or synthetic phosphatidylethanolamine, synthetic phosphatidylcholine, phosphatidic acids or their esters, phosphatidylserine and phosphatid dyl (poly) alcohols, such as phosphatidylinisitol or phosphatidylglycerol.
  • lipids mentioned are DPPC (dipalmitoylphosphatidylcholine), DOPE (dioleylphosphatidylethanolamine), DOTMA (N [1- (2,3, dioleoyloxy) propyl] -N, N, N trimethylammonium chloride), DOTAB (N-1- (2,3-Dioleoyloxy) propyl N, N, N-trimethylammonium chloride), DPPA (dipalmitoylphosphatidic acid), DPPG (dipalmitoylphosphatidylglycerol).
  • DPPC dipalmitoylphosphatidylcholine
  • DOPE dioleylphosphatidylethanolamine
  • DOTMA N [1- (2,3, dioleoyloxy) propyl] -N, N, N trimethylammonium chloride
  • DOTAB N-1- (2,3-Dioleoyloxy) propyl N, N, N-trimethylammoni
  • liposomes Individual surface-active compounds, which are used, for example, as detergents or emulsifiers, can also be used to form liposomes. Examples include DODAC (dioctadecylammonium chloride) and CTAC (cetyltrimethylammonium chloride). Suitable constituents of liposomes and production processes are described in the literature (see, for example, “Liposome Drug Delivery Systems”, G. Betageri (ed.), Lancaster Techonomic Publishing Company (1993) or “Liposome Technology”, Gregoriadis (ed.), CRC Press ). In addition to liposomes, other lipid vesicles such as niosomes and transfersomes (see e.g. WO9817255) can also be used.
  • DODAC dioctadecylammonium chloride
  • CTAC cetyltrimethylammonium chloride
  • Suitable constituents of liposomes and production processes are described in the literature (see, for example, “L
  • Suitable penetration enhancers include organic solvents (eg ethanol), pyrrolidones, sulfoxides (eg DMSO), fatty acids (saturated or unsaturated, branched or unbranched with a preferred chain length of 8-18), terpenes (eg L-menthol or 1,8-cineol) surfactants (e.g. polysorbates (Tween), polyethylene alkylphenols (Brij), alkyl ether sulfates as well as betaines and amphoteric glycinates).
  • organic solvents eg ethanol
  • pyrrolidones eg pyrrolidones
  • sulfoxides eg DMSO
  • fatty acids saturated or unsaturated, branched or unbranched with a preferred chain length of 8-18
  • terpenes eg L-menthol or 1,8-cineol
  • surfactants e.g. polysorbates (Tween), polyethylene alkylphenols
  • invasive or minimally invasive methods are also possible for improving the penetration of oligo- or polynucleotides in the sense of the invention.
  • Common minimally invasive methods include electroporation and iontophoresis. In both methods, a voltage is applied to the surface of the skin with the help of electrodes. Electroporation uses a short pulse of high voltage to permeate the skin. For this purpose, a small voltage with constant current is used in iontophoresis. Low frequency ultrasound can also be used to increase skin permeability to DNA or RNA.
  • 6.1 ⁇ l of the resulting liposome dispersion are diluted with 1.5 ml PBS and mixed with 20 ⁇ g plasmid dissolved in 1.5 ml HBS (150 mM NaCl, 20 mM Hepes, pH 7.4).
  • 1.5 ml HBS 150 mM NaCl, 20 mM Hepes, pH 7.4
  • 6.7 ⁇ l of the resulting liposome dispersion are diluted with 1.5 ml PBS and mixed with 20 ⁇ g plasmid dissolved in 1.5 ml HBS (150 mM NaCl, 20 mM Hepes, pH 7.4).
  • Figure 1 TEM image of the liposome-DNA complexes from Example 2 in cryomode. Magnification: 10000 times
  • TRANSPORTER 1. (4F2 LC) 4F2 LIGHT CHAIN. (SLC7A5).
  • MSP58 NUCLEOLAR PROTEIN CELL CYCLE-REGULATED FACTOR P78.
  • NFATX4 (NFATC3 OR NFAT4) NUCLEAR FACTOR OF ACTIVATED T-CELLS, CYTOPLASMIC 3 (T CELL TRANSCRIPTION FACTOR NFAT4) (NF-ATC3) (NF-AT4) (NFATX).
  • NMT1 (NMT1 OR NMT) GLYCYLPEPTIDE N- TETRADECANOYLTRANSFERASE 1 (EC 2.3.1.97) (PEPTIDE N-MYRISTOYLTRANSFERASE 1) (MYRISTOYL-COA.PROTEIN N- MYFERISTASE) (NM
  • NR4A1 Hs.1119 P22736 NR4A1: (NR4A1 OR HMR OR NAK1 OR GFRP1) ORPHAN NUCLEAR RECEPTOR HMR (EARLY RESPONSE PROTEIN NAK1) (TR3 ORPHAN RECEPTOR)
  • NRF1 (NFE2L1 OR NRF1 OR TCF11 OR HBZ17) NUCLEAR FACTOR ERYTHROID 2 RELATED FACTOR 1 (NF-E2 RELATED FACTOR 1) (NFE2-RELATED FACTOR 1) (NUCLEAR FACTOR, ERYTHROID DERIVED 2, LIKE TRANSCRIPTION FACTOR 11) (TRANSCRIPTION FACTOR HBZ17)
  • ODC1 ORNITHINE DECARBOXYLASE (EC 4.1.1.17) (ODC).
  • PGS2 BONE PROTEOGLYCAN II PRECURSOR (PG-S2) (DECORIN) (PG40).
  • PPP2R1A SERINE / THREONINE PROTEIN PHOSPHATASE 2A, 65 KDA REGULATORY SUBUNIT A, ALPHA ISOFORM (PP2A, SUBUNIT A, PR65-ALPHA ISOFORM) (PP2A, SUBUNIT A, R1-ALPHA TEMPERAMENT) ANTIGEN-ASSOCIATED 61 KDA PROTEIN)
  • PSMA6 PROTEASOME IOTA CHAIN (EC 3.4.99.46)
  • MACROPAIN IOTA CHAIN MULTICATALYTIC ENDOPEPTIDASE COMPLEX IOTA CHAIN
  • 27 KDA PROSOMAL PROTEIN PROS-27

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Abstract

L'invention concerne un procédé servant à déterminer in vitro les agressions ou le vieillissement cutanés chez l'homme ou chez les animaux, des nécessaires d'essai et des biopuces servant à déterminer les agressions ou le vieillissement cutanés, ainsi que l'utilisation de protéines, de molécules d'ARNm ou de fragments de protéines ou de molécules d'ARNm en tant que marqueurs d'agressions ou de vieillissement cutanés. L'invention concerne également un procédé de test servant à mettre en évidence l'efficacité de principes actifs cosmétiques ou pharmaceutiques contre les agressions ou le vieillissement cutanés, ainsi qu'une méthode de sélection servant à identifier les principes actifs cosmétiques ou pharmaceutiques contre les agressions ou le vieillissement cutanés et un procédé pour la production d'une préparation cosmétique ou pharmaceutique contre les agressions ou le vieillissement cutanés. L'invention concerne en outre une préparation cosmétique ou pharmaceutique contre les agressions ou le vieillissement cutanés.
EP01986926A 2001-01-03 2001-12-20 Procede pour la determination in vitro des agressions ou du vieillissement cutanes Withdrawn EP1356106A2 (fr)

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DE10100121A DE10100121A1 (de) 2001-01-03 2001-01-03 Verfahren zur Bestimmung des Hautstreß oder der Hautalterung in vitro
DE10100121 2001-01-03
PCT/EP2001/015178 WO2002053773A2 (fr) 2001-01-03 2001-12-20 Procede pour la determination in vitro des agressions ou du vieillissement cutanes

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