Patent Application
20060204992
This invention relates to a process for determining hair cycle markers in vitro, to test kits and biochips for determining hair cycle markers and to the use of proteins, mRNA molecules or fragments of proteins or mRNA molecules as hair cycle markers; to a test method for demonstrating the effectiveness of cosmetic or pharmaceutical active substances for influencing the hair cycle and to a screening process for identifying cosmetic or pharmaceutical active substances for influencing the hair cycle and to a process for the production of a cosmetic or pharmaceutical preparation for influencing the hair cycle.
Besides its actual biological function, the hair has a psychosocial function which is not to be underestimated. Unwanted hair loss or excessive hair growth can have a serious negative impact on the self-consciousness of the person affected (Paschier et al. (1988), Int. J. Dermatol. 27: 441-446). Except for rare congenital hair diseases caused by mutations in keratins or other structural proteins, excessive hair loss and excessive hair growth are caused by a disturbed hair cycle. Hair follicles pass through a cycle of three stages: anagen (growth phase), catagen (regression phase) and telogen (resting phase). Androgenic alopecia is characterized, for example, by an increasingly shorter anagen phase coupled with a reduction in size of the hair follicle (see, for example, Paus and Cotsarelis (1999), New Eng. J. Med., 341: 491-497).
Assigning the hair follicle to a stage of the hair cycle is essentially done on the basis of a microscopic-morphological analysis of the hair. Knowledge of the molecular mechanisms which play a role in the progression through the hair cycle is only fragmentary. Consequently, molecular markers characteristic of a certain stage of the hair follicle are lacking as are molecular targets through which the state of the hair follicle can be influenced. Although a number of different markers of hair-covered human skin were identified in DE 102 60931 to Applicants, those markers are characteristic of the anagenic hair follicles which make up most of the hair-covered skin.
The inadequate number of markers characteristic of other stages of the hair cycle leads to deficiencies in the general description of the growth phases of the hair in vivo, in cultivated hair follicles in vitro (Philpott Model; Philpott M. et al. (1990). Human Hair Growth in vitro; J. Cell Sci. 97: 463-471, 1990) and in reconstructed hair follicle models. In the latter systems in particular, morphological classification in stages of the hair cycle is no longer readily possible. Hair follicles cultivated in vitro are evaluated by microscopic measurement of the growth in length with a measuring ocular, including photographic documentation, and by histological evaluation of complicated vertical sections. This form of analysis is very time-consuming and requires a large number of hair follicles to cover the individual variations. For evaluating reconstructed hair follicle models, characterization via molecular markers of the corresponding stage is crucially important.
Besides the ratio of proliferation to apoptosis in the follicles, the DNA/protein and keratin synthesis and the ATP content, markers for the growth phase of hair follicles have hitherto been purely individual markers, for example matrix proteins, such as collagen type IV, fibronectin and laminin (Couchman, J. R. et al. (1985), Dev. Biol. 108: 290-298), growth factors, such as Transforming Growth Factor TGF-β1 and TGF-β2 (Foitzik et al. (2000), FSEB, J. 14: 752-760; Tsutomu, S. et al. (2002), J. Invest. Dermatol. 118: 993-997) and Fibroblast Growth Factor FGF-7 (Herbert, J. M. et al. (1994), Cell 78: 1017-1025). However, problems have arisen from the fact that many of these markers resulted from studies of the synchronized hair cycle of mice and cannot readily be applied to the human hair cycle.
In addition, the fragmentary knowledge of the molecular mechanisms playing a role in the progression through the hair cycle leads to an inadequate number of targets which are available for cosmetically or pharmacologically influencing the hair follicles. Thus, the enzyme 5α-reductase (type II) is the only validated target for androgenic alopecia. Inhibition of this enzyme, for example by the active principle finasteride, results in a reduced concentration of dihydrotestosterone in the skin and in the serum and hence in inhibition of the androgen-dependent miniaturization of the hair follicles. The disadvantage of finasteride undoubtedly lies in the side effects associated with its use: pregnant women in particular should not use finasteride. In addition, finasteride may not be used in cosmetic formulations.
The analysis of molecular markers in hair follicles is complicated as only relatively small quantities of mRNA can be obtained from the follicles and the concentration of such mRNA molecules is quite low, e.g., only a few to several hundred copies per cell in the hair follicles. Weakly expressed genes have only been accessible to existing analysis techniques with great difficulty, if at all, but can play a crucial role in the hair follicle.
There has never been a description of the transcriptome, i.e. the totality of all transcribed genes, of the hair follicles in various stages of the cell cycle.
Transcriptome analyses of the skin by various processes, including SAGE™ analysis, are already known. However, they are conducted with isolated keratinocytes (in vitro) or epidermis explantates which, as explained above, are not models representative of the complex events in the skin.
It is known from applicants' DE-A-101 00 127.4-41 that skin cells can be subjected to SAGE™ analysis in order to characterize the overall transcriptome of the skin. Applicants' DE-A-101 00 121.5-41 discloses the identification of markers of stressed or aged skin on the basis of a comparative SAGE™ analysis between stressed or aged skin and unstressed or young skin. However, there is no information on specific hair cycle markers in either of these documents.
It is known from J. Invest. Dermatol. 2002 July; 119(1): 3-13; “A serial analysis of gene expression in sun-damaged human skin”; Urschitz, J. et al., that markers of sun-damaged skin can be determined by a comparative SAGE™ analysis of whole skin explantates taken from in front of the auricle (sun-damaged) and behind the auricle (protected from the sun). Knowledge of specific hair cycle markers cannot be acquired from this publication either.
Accordingly, a need exists for the identification of genes which are markers important to the hair cycle.
In accordance with the present invention, a large number of the genes important to the hair cycle have been identified thereby enabling further genetic characterization of hair cycle regulation and screening processes for identifying active substances for influencing the hair cycle.
In one aspect, an in vitro method for determining hair cycle phase in humans is provided. An exemplary method entails providing a plurality of genetically encoded markers isolated from hair covered human skin or from human hair follicles which are differentially expressed at the anagenic phase of the hair cycle when compared to expression in cells in the catagenic phase of the hair cycle. A sample of hair covered skin or human hair follicles is obtained and analyzed for the presence and optionally the quantity of at least one genetically encoded molecule which is differentially expressed in anagenic and catagenic hair follicles. The sample is then designated as comprising healthy cells in the anagenic phase of the cycle if it contains markers which are expressed at higher levels in anagenic hair follicles or cells in regression in the catagenic phase of the hair cycle if it contains molecules which are expressed at higher levels in catagenic hair follicles. The genetically encoded markers encompassed by the foregoing method comprise at least one mRNA molecule, at least one protein or polypeptide or fragments thereof.
Tables 2 to 9 provide a plurality of markers that are differentially expressed in anagenic phase of the hair cycle when compared to the catagenic phase of the hair cycle. Such markers can be used to advantage in the methods of the present invention.
In another embodiment of the invention, the expression levels of at least two molecules in the sample which are differentially expressed in cells from the anagenic phase of the hair cycle when compared to expression levels in the catagenic phase of the hair cycle are quantified and the expression ratios of the at least two molecules determined thereby forming an expression quotient. The expression ratios obtained are compared with those in column 5 of Tables 2 to 6 and the sample designated as healthy cells in the anagenic phase of the hair cycle if the expression ratios observed in the follicles correspond to the ratios observed in anagenic hair follicles or cells in regression in the catagenic phase of the hair cycle if the expression ratios correspond to those observed in catagenic hair follicles.
Also encompassed by the present invention is a test kit for determining hair cycle phase in a human subject. An exemplary test kit comprises reagents suitable for performing the method described above. Thus, a kit of the invention comprises a plurality of probes corresponding to those provided in Tables 2-9 which are optionally detectably labelled, a solid support such as a biochip and physiological buffers for assessing gene expression levels. The kit may also comprise means for obtaining genetically encoded molecules or markers from hairy skin or hair follicles.
Thus, in yet another aspect of the invention, a biochip for determining hair cycle phase in human beings in vitro is provided comprising a solid, i.e. rigid or flexible, carrier and a plurality of probes immobilized thereon which are capable of specifically binding to at least one molecule selected from the group consisting of SEQ ID NO:1 to SEQ ID NO: 570 or the corresponding gene product. SEQ ID NOS:1-570 represent markers for determining hair cycle phase in human beings in vitro Exemplary markers are selected from the group consisting of at least one molecule having a Swissprot Accession Number provided in column 8 of Table 8, a Swissprot Accession Number provided in column 9 of Table 7, a Swissprot Accession Number provided in column 9 of Table 9, a UniGene Accession Number provided in column 7 of Tables 2 to 6, and a Swissprot Accession Number in column 8 of Tables 2 to 6.
Also provided in the present invention is an in vitro method for identifying a pharmaceutically active agent which modulates the hair cycle. An exemplary method entails providing hair covered human skin or human follicles comprising cells; determining the phase of the hair cycle of said cells as described above; contacting the cells with the agent at least once; and repeating the determination of the phase of the hair cycle to determine whether said agent alters the phase of the hair cycle. In a preferred embodiment, the method is performed on a biochip. A test kit for performing the method described above is also provided herein. Finally, a pharmaceutical preparation comprising the agent identified in the foregoing screening method having efficacy against diseases or impairment of hair and its growth in a pharmaceutically acceptable carrier is also disclosed.
Diseases or disorders of the hair cycle include, for example pili torti (corkscrew hair, twisted hair), monilethrix (spindle hair), woolly hair (kinked hair), hair shaft defects with breakages [Trichorrhexis nodosa, Trichorrhexis invaginata, Trichoschisis, trichoptilosis (split hair shafts)], hair shaft defects through metabolic disorders, pili recurvati, rolled hair, changes in hair color [heterochromy, albinism, poliosis (acquired patch-like absence of pigment in the hair), canitis (physiological graying)], hypertrichoses, hirsutism, alopecias (irreversible alopecia: for example, androgenetic alopecia in men and women); reversible alopecia: for example symptomatic diffuse alopecias through infections, chem. noxas and medicaments, hormonal disorders, diseases, etc.) and alopecia greata.
The totality of all the mRNA molecules synthesized at a certain time by a cell or a tissue is known as a transcriptome. The technique of serial analysis of gene expression (SAGE™) (Velculescu, V. E. et al., 1995, Science 270, 484-487) is used for understanding the transcriptome of human hair follicles. This technique facilitates the simultaneous identification and quantitation of the genes expressed in hair follicles. Comparison of the transcriptome of anagenic hair follicles with the transcriptome of catagenic hair follicles identifies those genes which are important for these stages of the hair cycle. These may be genes which are highly expressed in anagenic hair follicles or conversely, genes which are only weakly expressed when compared to expression levels observed in catagenic hair follicles.
Although gene expression can also be analyzed by the quantitation of specific mRNA molecules (for example Northern Blot, and/or RNase protection experiments), only a limited number of genes can be measured by these techniques. Theoretically, SAGE™ analysis could be replaced by MPSS (massive parallel signature sequencing) or by techniques based on differential display. In practice, however, the SAGE™ technique is faster and more reliable than alternative methods and is therefore preferred.
The SAGE method is based on two principles. First, only a short nucleotide sequence from the 3′ region of the mRNA is required for identification of the gene. A sequence of nine base pairs allows the differentiation of 262,144 (49) transcripts. This is more than the number of all the genes present in the genome. Second, concatenation of the short sequences allows efficient automated analysis by sequencing. An advantage of this technique not to be underestimated is the ability to determine the reading direction of the genes. If two opposite transcripts of a gene in the reading direction are started, this can only detected by the SAGE technique.
Typically, double-stranded cDNA is synthesized with biotinylated primers from polyA-RNA. The cDNA is digested with a restriction enzyme (anchoring enzyme) recognizing 4 bp which statistically cuts all 256 bp. The 3′ end of the cDNA is isolated by binding to Streptavidin beads. The sample is divided into two halves and the cDNA end is ligated with a linker (1 or 2) which has a recognition site for a type IIS restriction enzyme (tagging enzyme). This cuts up to 20 bp staggered from the asymmetric recognition site. This results in the formation of a short sequence (tag) tied to the linker which is unique to each gene. In order to obtain relatively large quantities of material, the linker1 tags are ligated with the linker2 tags after the projecting ends have been filled (linker ditag). The ligation products are amplified with linker-specific primers (1 or 2). The linker no longer in use is then released by another enzymatic digestion with the anchoring enzyme. The isolated ditags are concatenated by ligation (concatemers), cloned in a vector and transfixed in cells. From the cells, the concatemers are amplified via PCR and, finally, sequenced.
Another promising method is the microarray or chip technique. Here, entire gene libraries are placed on a chip. The genes on the chip are hybridized with fluorescence-marked cDNA generated from the mRNA of the tissue sample to be analyzed. By comparing anagenic with catagenic follicle material, all interesting genes can be detected in a single test on the basis of the differences in fluorescence. However, this does presuppose a knowledge of the clones in the gene library.
A very advantageous analysis method is the combination of SAGE analysis with the microarray technique. The SAGE method provides new or known genes which can be meaningful to the hair cycle. These are projected onto a chip with which samples of individual candidates can be measured.
Human hair follicles from healthy female donors were used for the SAGE™ analysis. The follicles were isolated from pieces of tissue taken from above the ear of the donor and were divided on the basis of their morphology into catagenic and anagenic hair follicles. In order to minimize the detection of donor-specific variances, the catagenic and anagenic hair follicles of a total of five donors were combined. The same number of catagenic and anagenic follicles of a donor were used and the total number of follicles of the individual donors were assimilated to one another.
The SAGE™ analysis was carried out as described in Velculescu, V. E. et al., 1995 Science 270, 484-487. A SAGE™ bank for catagenic hair follicles and one for anagenic hair follicles were analyzed. For further analysis, the two SAGE™ banks were standardized to the mean tag count. The two banks were compared with one another in order to identify genes demonstrating hair-cycle-specific regulation. As expected for two banks of the same tissue type, the tag repertoire of the two follicle banks is largely similar. Despite the similarity of the tissue and the relatively small number of tags, 197 tags show a differential expression with a significance of p>0.05. The significance was determined as described in Audic, S., Clayerie, J. M. (1997): “The significance of digital gene expression profiles”, Genome Res. 7: 986-95.
Table 1 lists markers for which a differential expression as a function of the stage of the hair cycle has already been described. They serve as positive controls for the experiment. Table 1 shows
The quotient in column 3 indicates the strength of the differential expression, i.e. the factor by which the particular gene is expressed more strongly in anagenic hair follicles than in catagenic hair follicles or vice versa.
The particular genes or gene products for Tables 1-6 are disclosed under their UniGene Accession Number in the data bank of the National Center for Biotechnology Information (NCBI). This data bank is accessible on the world wide web at ncbi/nim.nih.gov. In addition, the genes or gene products are directly accessible at the following world wide web addresses ncbi.nlm.nih.gov/UniGene/Hs.Home.html or ncbl.nlm.nih.gov/genome/guide.
Mice comprising inactivated vitamin D receptor demonstrate hair loss. It was shown that, after stimulation of the anagen stage by shaving, mice with an inactive vitamin D receptor are unable to initiate the hair cycle (Kong et al. (1002), J. Invest. Dermatol., 118: 631-8).
Thrombospondin-1 was shown to play a role in the induction of hair follicle involution and in vascular degradation during the catagen phase (Yano et al. (2003), J. Invest. Dermatol., 120: 14-9). Whereas no expression of the thrombospondin can be detected in the early to middle anagen phase, high expression levels can be detected during the catagenic phase in accordance with the expression data found there.
Although the role of neurotrophin-5 for human hair follicles has never been described, studies of the family member neurotrophin-3 in murine hair follicles have been conducted. Maximal expression of neutrotrophin was observed in the catagenic stage (Botchkarev et al. (1998), Am. J. Pathol., 153: 785-99). A corresponding expression pattern was found there for neurotrophin-5.
In the course of SAGE™, the number of individual tags was determined in a first step and, where possible, assigned to genes or inputs in the UniGene data bank. By comparison of the tags in the various SAGE™ banks, differentially expressed genes can be identified. Accordingly, a first classification was made based on the significance of the differential expression of the identified genes as genes which are significantly differentially expressed are considered marker genes for particular stages of the hair cycle.
The genes for which a significant differential expression was found are listed in Tables 2 to 6.
Tables 2 to 6 contain a detailed list of the genes differentially expressed in anagenic hair follicles and in catagenic hair follicles, as determined by the process according to the invention, with an indication of
The quotient in column 5 indicates the strength of the differential expression, i.e. the factor by which the particular gene is expressed more strongly in anagenic hair follicles than in catagenic hair follicles or vice versa.
Table 2 lists all the genes which exhibit at least five-fold differential expression levels in anagenic hair follicles when compared to levels observed in catagenic hair follicles with a p value of p<0.01 (significance>2.0).
Table 3 lists all the genes which exhibit at least two-fold differential expression levels in anagenic hair follicles when compared to those observed in catagenic hair follicles with a p value of p<0.01 (significance>2.0).
Table 4 lists all the genes which exhibit at least 1.3 fold differential expression levels in anagenic hair follicles when compared to those observed in catagenic hair follicles with a p value of p<0.01 (significance>2.0).
Table 5 lists all the genes which exhibit at least five-fold differential expression levels in anagenic hair follicles when compared to levels observed in catagenic hair follicles with a p value of p<0.05 (significance>1.3).
Table 6 lists all the genes which exhibit at least two-fold differential expression levels in anagenic hair follicles when compared to those observed in catagenic hair follicles with a p value of p<0.05 (significance>1.3).
The clear expression difference in the ribosomal RNAs is particularly noticeable. Slight expression differences in ribosomal RNAs have hitherto been described as typical artefacts of SAGE™. In the present case, however, the expression differences are strikingly high and uniform. There is a much stronger expression of rRNA in anagenic hair follicles than in catagenic hair follicles. Accordingly, the strength of expression of ribosomal RNA is itself a marker criterion for anagenic hair follicles.
In addition, there are some other biologically interesting expression differences. First, the expression of attractin in catagenic hair follicles is increased. Attractin is a protein from the agouti/melanocortin signal transduction pathway. The gene product plays a role in determining the hair color of mice (Gunn et al. (1999), Nature, 398: 152-6; Barsh et al. (2002), J. Recept. Signal Transduct. Res., 22: 63-77).
In addition, cobalamin adenosyl transferase, an enzyme in the vitamin B12 metabolism pathway, is induced in catagenic hair follicles. In human beings, a vitamin B12 deficiency leads to depigmentation of the hair (Mori et al. (2001), J. Dermatotol. 28: 282-5). Dopachrome tautomerase, an enzyme involved in the biosynthesis of melanin, is also induced in catagenic hair follicles. All the genes mentioned above are relevant to hair follicle biology, particularly to pigmentation, but have not hitherto been described in connection with regulation of the hair cycle.
It is also noticeable that the transcription factors Fos-B and Egr1 are induced in catagenic hair follicles. These two transcription factors belong to the group of so-called immediate-early genes and have wide-reaching regulatory functions.
On the other hand, the angiopoietin-like protein CDT6 is repressed in catagenic hair follicles. This protein is assumed to have a regulatory function in angiogenesis (Peek et al. (2002), J. Biol. Chem., 277: 686-93). Control of angiogenesis and hence the supply of blood to the hair follicle is coupled to the hair cycle (see above, thrombospondin-1).
Also noteworthy is the induction of the 14-3-3 sigma protein, stratifin, and the simultaneous repression of the 14-3-3 tau/theta protein. The family of 14-3-3 proteins regulate a number of enzymes, including those involved in primary metabolism and the cell cycle. They also have a chaperone function. They can activate the transcription of inducible genes and regulate signal transduction and apoptosis processes. A role in the differentiation of keratinocytes was described in particular for the 14-3-3 sigma protein, stratifin (Dellambra et al. (1995), J. Cell Sci. 108:3569-79). A specific regulation of the members of this protein family in the various hair follicle stages is therefore extremely likely. Finally, keratin 6A and acidic hair keratin are also repressed in catagenic hair follicles.
Any evaluation of whether or not the differential expression of various genes is significant is critically determined by the number of sequenced tags. Non-significant expression differences can become statistically significant through an increase in the number of sequenced tags.
The relevance of subsignificant expression differences can be evaluated using various data analysis methods through which expert biological knowledge flows into the evaluation of the expression differences. One method is the clustering of the identified genes according to their GO annotation. The GO annotation derives from the inputs in the data bank of the Gene Ontology (GO) Consortium, in which individual genes/proteins are classified according to their (primary) function. See world wide website geneontology.org/. By using these relationship features, expression differences which are statistically not outside the confidence interval can also assume a significance.
Table 7 contains a detailed list of the genes differentially expressed in anagenic hair follicles and in catagenic hair follicles, as determined by the process according to the invention, with an indication of
The quotient in column 5 indicates the strength of the differential expression, i.e. the factor by which the particular gene is expressed more strongly in anagenic hair follicles than in catagenic hair follicles or vice versa.
The particular genes or gene products are accessible on the internet under their GO number at the following world wide web address geneontology.org.
For example genes of the DPP-IV cluster, a family of dipeptidyl peptidases (attractin [anagen 8 tags: catagen 23 tags], DPP-9 [0:9], DPP-4 [0:2], DPP-8 [0:1]), are clearly induced in catagenic hair follicles. The dipeptidyl peptidases of the DPP-IV family are proline-specific proteases which function to regulate various pathological and physiological processes (Aleski and Malik (2001), Biochim. Biophys. Act, 1550: 107-116). In addition, there is a weak, but consistent induction of various DNA repair helicases, for example RecQ-like 5 [3:8], RecQ-like 4 [1:2], RuvB-like [0:3], etc. This induction can be found in all annotated helicases of this set of data. In addition, the melanin biosynthesis cluster, which includes inter alia dopachrome tautomerase [0:7] and silver/pMEL [7:17], is also clearly induced.
By contrast, various subunits of type IV collagen (α1 [5:1], α2 [1:0], α6 [4:0]) are induced in anagenic hair follicles. Type IV collagen is a typical constituent of the follicle matrix and the expression of this protein can be expected to be increased in the growth phase of the follicle. The synaptosome cluster is also induced in anagenic hair follicles. This cluster includes the SNARE proteins VAMP-2 [5:0] and VAMP-3 [4:0] which have a general role in secretion. This observation is supported by the general induction of genes which play a role in exocytosis. This induction of exocytosis genes is likely associated with the process of pigmentation of the hair. Pigmentation involves the transfer of melanin-synthesizing organelles, so-called melanosomes, from melanocytes to keratinocytes of the hair follicle. Melanosomes bear a large microscopic similarity to the synaptosomes of the nerve cells, secretory vesicles which enable neurotransmitters to be released. The role of SNARE proteins for the synaptosomes is sufficiently documented; the role of these proteins in melanosomes is under discussion at the present time (Scott et al. (2002); J. Cell. Sci., 115: 1441-51). Finally, genes belonging to the group with N-acetyl lactosamine synthase activity (chain 1 [3:0], chain 2 [8:2], chain 3 [1:0]) are induced in anagenic hair follicles. Poly-N-acetyl lactosamine structures are found both in N- and in O-linked glycans of the glycoproteins from mammals. These glycans presumably interact with selectins and other glycan-binding proteins (Zhou (2003), Curr. Protein Pept. Sci., 4:1-9).
Another method of increasing the relevance of subsignificantly differentially expressed genes is clustering according to sequence patterns. Such clustering is possible by co-ordinating the SAGE data with the data from available domain and pattern data banks, for example PROSITE and Pfam at world wide web site sanger.ac.uk/Software/Pfam/index.shtml and espasy.ch/prosite/.
Table 8 contains a detailed list of the genes differentially expressed in anagenic hair follicles and in catagenic hair follicles, as determined by the process according to the invention, with an indication of
The quotient in column 5 indicates the strength of the differential expression, i.e. the factor by which the particular gene is expressed more strongly in anagenic hair follicles than in catagenic hair follicles or vice versa.
Through this co-ordination, the significance of some already described genes is further increased. Thus, the GO cluster with dipeptidyl peptidase activity is extended by other members of the PF:PEPTIDASE_S9 family. In addition, proteins with a GRAM domain are clearly induced in the catagenic hair follicles. The function of the domain is not known at present (Doerks et al. (2000) Trends Biochem. Sci., 25: 483-485).
As already described for GO clusters, type IV collagen subunits (C4 domain) are repressed in catagenic hair follicles in this arrangement also. The induction of proteins with a Gla domain in the anagenic hair follicles is noteworthy. These proteins are matrix-Gla and osteocalcin proteins. The matrix-Gla protein was described as an BMP-2 antagonist in hair follicle development and in the cycle (Nakamura et al. (2003), FASEB J., 17: 497-9).
In addition, the significance of differential gene expression can be increased by lexical analysis. In this case, a search is made for corresponding keywords in the descriptive texts of the various genes, as found for example in the data bank annotations.
Table 9 contains a detailed list of the genes differentially expressed in anagenic hair follicles and in catagenic hair follicles, as determined by the process according to the invention, with an indication of
As a result of this analysis, catagenic hair follicles show a significant induction of the cluster with the keyword “autophagy” (Apg4 [2:7], Apg3 [0:2], Apg10 [0:2], Apg5 [0:1]. Autophagy is a process in which cells envelop macroscopic cell constituents, such as organelles for example, in autophagosomes and then digest them in the lysosome. Autophagy occurs primarily during cell supply deficiencies; excessive autophagy is regarded as a mechanism of non-apoptotic programmed cell death. In addition, clusters formed on the basis of the keywords “dsc2” and “desmocollin” are repressed in catagenic hair follicles. Localization in the hair follicle has been reported in particular for desmocollin-3 (Kurzen et al. (1998), Differentiation, 63: 295-304; Nuber et al. (1996), Eur. J. Cell Biol., 71: 1-13).
Previously, it had been demonstrated that ribosomal RNA expression was repressed in catagenic hair follicles. These data are confirmed by the analytic methods described herein.
Finally, the repression of selenoproteins in catagenic hair follicles is also striking.
In yet another aspect of the invention a process (2) for determining the hair cycle in human beings, more particularly in women, in vitro, is provided. An exemplary method entails
a) obtaining a mixture of proteins, mRNA molecules or fragments of either from hair-covered human skin or from human hair follicles,
b) analyzing the mixture of a) for the presence and optionally the quantity of at least one of the proteins, mRNA molecules or fragments of either which are differentially expressed in anagenic and catagenic human hair follicles as shown by (SAGE),
c) comparing the analysis results from b) with the expression patterns identified by serial analysis of gene expression (SAGE) and
d) assigning the mixture to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of either which demonstrate elevated expression levels in anagenic hair follicles when compared to those observed in catagenic hair follicles or to hair in regression or unhealthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which demonstrate elevated expression in catagenic hair follicles than in anagenic hair follicles.
Diseases or disorders of the hair cycle include, for example pili torti (corkscrew hair, twisted hair), monilethrix (spindle hair), woolly hair (kinked hair), hair shaft defects with breakages [Trichorrhexis nodosa, Trichorrhexis invaginata, Trichoschisis, trichoptilosis (split hair shafts)], hair shaft defects through metabolic disorders, pili recurvati, rolled hair, changes in hair color [heterochromy, albinism, poliosis (acquired patch-like absence of pigment in the hair), canitis (physiological graying)], hypertrichoses, hirsutism, alopecias (irreversible alopecia: for example, androgenetic alopecia in men and women); reversible alopecia: (for example symptomatic diffuse alopecias through infections, chem. noxas and medicaments, hormonal disorders, diseases, etc.) and alopecia greata.
The mixture obtained in step a) above may be obtained from whole skin samples, hair-covered skin equivalents, isolated hair follicles, hair follicle equivalents or cells of hair-covered skin.
It may be sufficient in step b) to analyze the mixture obtained for the presence of at least one of the proteins, mRNA molecules or fragments of either which are identified by serial analysis of gene expression (SAGE) as differentially expressed in anagenic and catagenic hair follicles where they are expressed solely in anagenic hair follicles or solely in catagenic hair follicles. In other cases, the quantity of the differentially expressed molecules must also be determined in step b), i.e. the expression must be quantitated.
In step d), the mixture analyzed in step b) is assigned to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which demonstrate elevated expression levels in anagenic hair follicles when compared to those observed in catagenic hair follicles, i.e. the mixture contains either more different compounds typically expressed in anagenic hair follicles than those which are typically expressed in catagenic hair follicles (qualitative differentiation) or more copies of compounds typically expressed in anagenic hair follicles than are typically present in catagenic hair follicles (quantitative differentiation). For assignment to hair in regression or unhealthy hair, the complementary procedure is followed.
A preferred embodiment of the method of the invention for determining the hair cycle is characterized in that, in step b), the mixture obtained is analyzed for the presence and optionally the quantity of at least one of the proteins, mRNA molecules or fragments of either which are identified by their Swissprot Accession Number in column 9 of Table 9 and, in step d), the mixture analyzed in b) is assigned to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more strongly in anagenic hair follicles than in catagenic hair follicles or the mixture analyzed in b) is assigned to hair in regression or unhealthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more strongly in catagenic hair follicles than in anagenic hair follicles.
Another preferred embodiment of the method of the invention for determining the hair cycle is characterized in that, in step b), the mixture obtained is analyzed for the presence and optionally the quantity of at least one of the proteins, mRNA molecules or fragments of either which are identified by their Swissprot Accession Number in column 8 of Table 8 and, in step d), the mixture analyzed in b) is assigned to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of either which are expressed more strongly in anagenic hair follicles than in catagenic hair follicles or the mixture analyzed in b) is assigned to hair in regression or unhealthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more strongly in catagenic hair follicles than in anagenic hair follicles.
Another preferred embodiment of the process according to the invention for determining the hair cycle is characterized in that, in step b), the mixture obtained is analyzed for the presence and optionally the quantity of at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are identified by their Swissprot Accession Number in column 9 of Table 7 and, in step d), the mixture analyzed in b) is assigned to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed more strongly in anagenic hair follicles than in catagenic hair follicles or the mixture analyzed in b) is assigned to hair in regression or unhealthy hair if it predominantly contains proteins, mRNA molecules or fragments of either which are expressed more strongly in catagenic hair follicles than in anagenic hair follicles.
Another preferred embodiment of the process according to the invention for determining the hair cycle is characterized in that, in step b), the mixture obtained is analyzed for the presence and optionally the quantity of at least one of the proteins, mRNA molecules or fragments of either which are identified by their Unigene Accession Number in column 7 of Table 6, by their Swissprot Accession Number in column 8 or by the brief description of the gene or gene product in column 9 and, in step d), the mixture analyzed in b) is assigned to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of either which are expressed at least twice as strongly in anagenic hair follicles as in catagenic hair follicles or the mixture analyzed in b) is assigned to hair in regression or unhealthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least twice as strongly in catagenic hair follicles as in anagenic hair follicles.
Another preferred embodiment of the method according to the invention for determining the hair cycle is characterized in that, in step b), the mixture obtained is analyzed for the presence and optionally the quantity of at least one of the proteins, mRNA molecules or fragments of either which are identified by their Unigene Accession Number in column 7 of Table 5, by their Swissprot Accession Number in column 8 or by the brief description of the gene or gene product in column 9 and, in step d), the mixture analyzed in b) is assigned to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least five times as strongly in anagenic hair follicles as in catagenic hair follicles or the mixture analyzed in b) is assigned to hair in regression or unhealthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least five times as strongly in catagenic hair follicles as in anagenic hair follicles.
Another particularly preferred embodiment of the method according to the invention for determining the hair cycle is characterized in that, in step b), the mixture obtained is analyzed for the presence and optionally the quantity of at least one of the proteins, mRNA molecules or fragments of either which are identified by their Unigene Accession Number in column 7 of Table 4, by their Swissprot Accession Number in column 8 or by the brief description of the gene or gene product in column 9 and, in step d), the mixture analyzed in b) is assigned to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments thereof which are expressed at least 1.3 times as strongly in anagenic hair follicles as in catagenic hair follicles or the mixture analyzed in b) is assigned to hair in regression or unhealthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least 1.3 times as strongly in catagenic hair follicles as in anagenic hair follicles.
Another particularly preferred embodiment of the method according to the invention for determining the hair cycle is characterized in that, in step b), the mixture obtained is analyzed for the presence and optionally the quantity of at least one of the proteins, mRNA molecules or fragments of either which are identified by their Unigene Accession Number in column 7 of Table 3, by their Swissprot Accession Number in column 8 or by the brief description of the gene or gene product in column 9 and, in step d), the mixture analyzed in b) is assigned to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least twice as strongly in anagenic hair follicles as in catagenic hair follicles or the mixture analyzed in b) is assigned to hair in regression or unhealthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least twice as strongly in catagenic hair follicles as in anagenic hair follicles.
Another most particularly preferred embodiment of the method according to the invention for determining the hair cycle is characterized in that, in step b), the mixture obtained is analyzed for the presence and optionally the quantity of at least one of the proteins, mRNA molecules or fragments of either which are identified by their Unigene Accession Number in column 7 of Table 2, by their Swissprot Accession Number in column 8 or by the brief description of the gene or gene product in column 9 and, in step d), the mixture analyzed in b) is assigned to growing or healthy hair if it predominantly contains proteins, mRNA molecules or fragments of either which are expressed at least five times as strongly in anagenic hair follicles as in catagenic hair follicles or the mixture analyzed in b) is assigned to hair in regression or unhealthy hair if it predominantly contains proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at least five times as strongly in catagenic hair follicles as in anagenic hair follicles.
The hair cycle can also be described by quantitating several markers (expression products of the genes of importance to anagenic or catagenic hair follicles) which then have to be active in a characteristic ratio to one another in order to represent healthy or growing hair or in a different characteristic ratio to one another in order to represent hair in regression or unhealthy hair.
Accordingly, the present invention also relates to a method (3) for determining the hair cycle in human beings, more particularly in women, in vitro. An exemplary method entails
The mixture obtained in step a) of the method according to the invention is preferably obtained from a skin sample, more particularly from a whole skin sample.
In another embodiment of the method according to the invention, the mixture obtained in step a) is obtained by microdialysis. The technique of microdialysis is described, for example, in “Microdialysis: A method for measurement of local tissue metabolism”, Nielsen, P. S., Winge, K., Petersen, L. M.; 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 on the internet at world wide web address microdialysis.se/technique.htm, which is incorporated by reference herein.
In the technique of microdialysis, a probe is typically inserted into the skin and then slowly rinsed with a suitable carrier solution. After the acute reactions have abated following the insertion, the microdialysis yields proteins, mRNA molecules or fragments thereof which are present in the extracellular space and which can then be isolated in vitro, for example by fractionation of the carrier liquid, and analyzed. Microdialysis is less invasive than removing a whole skin sample, but has the disadvantage that it is limited to obtaining molecules occurring in the extracellular space.
Another preferred embodiment of the process according to the invention is characterized in that, in step b) of process (2), the analysis for the presence and optionally the quantity of at least one of the proteins or protein fragments or, in process (3), the quantitation of at least two proteins or protein fragments is carried out by a method selected from
Suitable analytical methods for use in the invention are described in the overview article by Akhilesh Pandey and Matthias Mann: “Proteomics to study genes and genomes”, Nature, Volume 405, Number 6788, 837-846 (2000), and the references cited therein, which is incorporated herein by reference.
2D gel electrophoresis is described, for example, in L. D. Adams, “Two-dimensional gel electrophoresis using the Isodalt System” or in L. D. Adams and S. R. Gallagher, Two-dimensional Gel Electrophoresis using the O'Farrell System”; both in Current Protocols in Molecular Biology (1997, Eds. F. M. Ausubel et al.), Unit 10.3.1-10.4.13; or in 2D Electrophoresis 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 methods known to those of skill in the art, for example as described in the following literature references:
Another preferred embodiment of the process according to the invention is characterized in that, in step b) of process (2), the analysis for the presence and optionally the quantity of at least one of the mRNA molecules or mRNA molecule fragments or, in process (3), the quantitation of at least two mRNA molecules or mRNA molecule fragments is carried out by a method selected from
These methods are suitable for use in the invention and are described in the overview articles by Akhilesh Pandey and Matthias Mann: “Proteomics to study genes and genomes”, Nature, Volume 405, Number 6788, 837-846 (2000), and “Genomics, gene expression and DNA arrays”, Nature, Volume 405, Number 6788, 827-836 (2000) and the references cited therein, which are incorporated by reference herein. The TOGA process is described in J. Gregor Sutcliffe et al. “TOGA: An automated parsing technology for analyzing expression of nearly all genes, Proceedings of the National Academy of Sciences of the United States of America (PNAS), Vol. 97, No. 5, pp. 1976-1981 (2000)”, which is also incorporated herein by reference. The MPSS® process is described in U.S. Pat. No. 6,013,445, which is also incorporated herein by reference.
According to the invention, however, other methods known to the skilled person for analyzing for the presence and optionally the quantity of at least one of the proteins, mRNA molecules or fragments thereof may also be used.
Another preferred embodiment of the process according to the invention is characterized in that step b) comprises analyzing for the presence and optionally the quantity of 1 to ca. 5,000, preferably 1 to ca, 1,000, more particularly ca. 10 to ca. 500, preferably ca. 10 to ca. 250, more preferably ca. 10 to ca. 100 and most preferably ca. 10 to ca. 50 of the proteins, mRNA molecules or fragments thereof which are defined by their Swissprot Accession Number in column 8 of Table 8, by their Swissprot Accession Number in column 9 of Tables 7 and 9 and by their UniGene Accession Number in column 7 of Tables 2 to 6, by their Swissprot Accession Number in column 8 or by the brief description of the gene or gene product in column 9.
The present invention also relates to a test kit for determining the hair cycle in human beings in vitro comprising means for carrying out the process according to the invention for determining the hair cycle in human beings.
The present invention also relates to a biochip for determining the hair cycle in human beings in vitro comprising
A biochip is a miniaturized functional element with molecules, more particularly biomolecules, which can act as specific interaction partners immobilized on one surface. The structure of these functional elements often comprises rows and columns which are known as chip arrays. Since thousands of biological or biochemical functional elements can be accommodated on one chip, they generally have to be made by microtechnical methods.
Biological and biochemical functional elements include, in particular, DNA, RNA, PNA (in the case of nucleic acids and their chemical derivatives, single strands, triplex structures or combinations thereof, for example, may be present), saccharides, peptides, proteins (for example antibodies, antigens, receptors) and derivatives of combinatorial chemistry (for example organic molecules).
Biochips generally have a 2D base surface for coating with biologically or biochemically functional materials. The base surfaces may also be formed, for example, by walls of one or more capillaries or by channels.
The prior art is represented, for example, by the following publications: Nature Genetics, Vol. 21, Supplement (whole), January 1999 (biochips); Nature Biotechnology, Vol. 16, pp. 981-983, October 1998 (biochips); Trends in Biotechnology, Vol. 16, pp. 301-306, July 1998 (biochips) and the above-cited overview articles by Akhilesh Pandey and Matthias Mann: Proteomics to study genes and genomes”, Nature, Volume 405, Number 6788, 837-846 (2000), and “Genomics, gene expression and DNA arrays”, Nature, Volume 405, Number 6788, 827,836 (2000), and the literature cited therein, which are all incorporated herein by reference.
A clear account of processes for the practical application of DNA chip technology is presented in the books “DNA Microarrays: A Practical Approach” (Editor: Mark Schena, 1999, Oxford University Press) and “Microarray Biochip Technology” (Editor: Mark Schena, 2000, Eaton Publishing), to the whole of which reference is hereby made.
DNA chip technology which is based on the ability of nucleic acid to enter into complementary base pairing is particularly preferred for the purposes of the present invention. This technical principle, known as hybridization, has already been used for years in Southern blot and Northern blot analysis. By comparison with these conventional methods, in which only a few genes are analyzed, DNA chip technology enables a few hundred to several thousand genes to be analyzed simultaneously.
A DNA chip consists essentially of a carrier material (for example glass or plastic) on which single-stranded, gene-specific probes are immobilized in high densities in a particular place (spot). The technique of probe application and the chemistry of probe immobilization are regarded as problematic. At present, there are several ways of achieving probe immobilization. E. M. Southern (E. M. Southern et al. (1992), Nucleic Acid Research 20, 1679-1684 and E. M. Southern et al. (1997), Nucleic Acid Research 25, 1155-1161) describes the production of oligonucleotide arrangements by direct synthesis on a glass surface which had been treated with 3-glycidoxypropyl trimethoxysilane and then with a glycol. A similar process achieves the in situ synthesis of oligonucleotides by a photosensitive combinatorial chemistry which can be compared with photolithographic techniques (Pease, A. C. et al. (1994), Proc. Natl. Acad Sci USA 91, 5022-5026).
Besides these techniques based on the in situ synthesis of oligonucleotides, already existing DNA molecules can also be immobilized on surfaces of carrier material. P.O. Brown (DeRisi et al. (1997), Science 278, 680-686) describes the immobilization of DNA on glass surfaces coated with polylysine. An article by L. M. Smith (Guo, Z. et al. (1994), Nucleic Acid Research 22, 5456-5465) discloses a similar process: oligonucleotides bearing a 5′-terminal amino group can be immobilized on a glass surface which had been treated with 3-aminopropyl trimethoxysilane and then with 1,4-phenyl diisothiocyanate.
DNA probes can be applied to a carrier with a so-called pin spotter. To this end, thin metal needles, for example 250 μm in diameter, dip into probe solutions and then transfer the adhering sample material in defined volumes to the carrier material of the DNA chip.
However, the probes are preferably applied by a piezo-controlled nanodispenser which, similarly to an ink jet printer, applies probe solutions contactlessly to the surface of the carrier material in a volume of 100 picoliters.
The probes are immobilized, for example, as described in EP-A-0 965 647. DNA probes are generated by PCR using a sequence-specific primer pair, one primer being modified at the 5′-end and carrying a linker with a free amino group. This ensures that a defined strand of the PCR products can be immobilized on a glass surface which had been treated with 3-aminopropyl trimethoxysilane and then with 1,4-phenyl diisothiocyanate. The gene-specific PCR products should ideally have a defined nucleic acid sequence in a length of 200 to 400 bp and comprise non-redundant sequences. After the immobilization of the PCR products via the derivatized primer, the counter-strand of the PCR product is removed by incubation for 10 minutes at 96° C.
In one application typical of DNA chips, mRNA is isolated from two cell populations to be compared. The isolated mRNAs are converted into cDNA by reverse transcription using fluorescence-marked nucleotides for example. The samples to be compared are marked, for example, with red or green fluorescing nucleotides. The cDNAs are then hybridized with the gene probes immobilized on the DNA chip and the immobilized fluorescent signals are then quantitated.
A factor critical to the success of using DNA chip technology for analyzing the gene expression of the hair follicles is the composition of the gene-specific probes on the DNA chip. The relevant genes of the hair cycle as identified in SAGE™ analysis are particularly useful in this regard. Since extremely small quantities of mRNA occasionally have to be analyzed where a DNA chip is used for analyzing the relevant hair cycle genes, it may be necessary to enrich the mRNA before the analysis by means of so-called linear amplification (Zhao et al. (2002), BMC Genomics, 3:31). To this end, the mRNA of a sample is first transcribed into cDNA. The amplified RNA is obtained from this double-stranded cDNA by in vitro transcription.
The analysis chips mentioned in DE-A-100 28 257.1-52 and in DE-A-101 02 063.5-52 are most particularly preferred for the production of small biochips (containing up to ca. 500 probes). These analysis chips have an electrically addressable structure which enables the samples to be electrofocused. In this way, samples can advantageously be focused and immobilized irrespective of their viscosity at particular points of an array by means of electrodes. The focusing ability simultaneously provides for an increase in the local concentration of the samples and thus for higher specificity. During the analysis itself, the test material can be addressed at the individual positions of the array. Thus, each item of information analyzed can potentially be tracked with the highest possible sensitivity. Cross-contamination by adjacent spots is virtually impossible.
The biochip according to the invention comprises 1 to ca. 5,000, preferably 1 to ca. 1,000, more particularly ca. 10 to ca. 500, preferably ca. 10 to ca. 250, more preferably ca. 10 to ca. 100 and most preferably ca. 10 to ca. 50 different probes. The different probes can each be present on the chip in multiple copies.
The biochip according to the invention comprises nucleic acid probes, more particularly RNA or PNA probes and preferably DNA probes. The nucleic acid probes have a length of ca. 10 to ca. 1,000 nucleotides, preferably ca. 10 to ca. 800 nucleotides, more preferably ca. 100 to ca. 600 nucleotides and most preferably ca. 200 to ca. 400 nucleotides.
A particularly preferred biochip according to the invention is a DNA chip carrying probes selected from those listed in Tables 2 and 5 and in Table 3 (without mitochondrial and ribosomal tags) and the over-represented groups “DNA helicase activity”, “DPPIV activity” and “melanine biosynthesis from tyrosine” from Table 7.
In another preferred form, the biochip according to the invention comprises peptide or protein probes, more particularly antibodies.
The present invention also relates to the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are defined by their Swissprot Accession Number in column 8 of Table 8, by their Swissprot Accession Number in column 9 of Tables 7 and 9 and by their UniGene Accession Number in column 7 of Tables 2 to 6, by their Swissprot Accession Number in column 8 and by the brief description of the gene or gene product in column 9 as hair cycle markers in human beings.
The present invention also relates to a test method for demonstrating the effectiveness of cosmetic or pharmaceutical active principles for influencing the hair cycle, more particularly against diseases or impairment of the hair and its growth, in vitro, characterized in that
a) the hair status is determined by a process according to the invention for determining the hair cycle or by means of a test kit according to the invention for determining the hair cycle or by means of a biochip according to the invention,
b) an active principle against diseases or impairment of the hair and its growth is applied one or more times to the hair-covered skin,
c) the hair status is re-determined by a process according to the invention for determining the hair cycle or by means of a test kit according to the invention for determining the hair cycle or by means of a biochip according to the invention and
d) the effectiveness of the active principle is determined by comparison of the results from a) and c).
The test method according to the invention can be carried out with whole skin samples, hair-covered skin equivalents, isolated hair follicles, hair follicle equivalents or cells of hair-covered skin.
The present invention also relates to a test kit for demonstrating the effectiveness of cosmetic or pharmaceutical active principles against diseases or impairment of the hair and its growth, comprising means for carrying out the test method according to the invention.
The present invention also relates to the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are defined by their Swissprot Accession Number in column 8 of Table 8, by their Swissprot Accession Number in column 9 of Tables 7 and 9 and by their UniGene Accession Number in column 7 of Tables 2 to 6, by their Swissprot Accession Number in column 8 or by the brief description of the gene or gene product in column 9 for demonstrating the effectiveness of cosmetic or pharmaceutical active principles against diseases or impairment of the hair and its growth.
The present invention also relates to a screening process for identifying cosmetic or pharmaceutical active principles against diseases or impairment of the hair and its growth in vitro, characterized in that
a) the hair status is determined by a process according to the invention for determining the hair cycle or by means of a test kit according to the invention for determining the hair cycle or by means of a biochip according to the invention,
b) a potential active principle against diseases or impairment of the hair and its growth is applied one or more times to the hair-covered skin,
c) the hair status is re-determined by a process according to the invention for determining the hair cycle or by means of a test kit according to the invention for determining the hair cycle or by means of a biochip according to the invention and
d) effective active principles are determined by comparison of the results from a) and c).
The present invention also relates to the use of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are defined by their Swissprot Accession Number in column 8 of Table 8, by their Swissprot Accession Number in column 9 of Tables 7 and 9 and by their UniGene Accession Number in column 7 of Tables 2 to 6, by their Swissprot Accession Number in column 8 or by the brief description of the gene or gene product in column 9 for identifying cosmetic or pharmaceutical active principles against diseases or impartment of the hair and its growth.
The present invention also relates to a process for the production of a cosmetic or pharmaceutical preparation against diseases or impairment of the hair and its growth, characterized in that
effective active principles are determined by the screening process according to the invention or by its use for identifying cosmetic or pharmaceutical active principles against diseases or impairment of the hair and its growth and
active principles found to be effective are mixed with cosmetically and pharmacologically suitable and compatible carriers.
Pombe)
coli)
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S. cerevisiae)
cerevisiae)
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cerevisiae)
| Number | Date | Country | Kind |
|---|---|---|---|
| 103 40 373.6 | Aug 2003 | DE | national |
This application is a §365 (c) continuation application of PCT/EP2004/009435 filed 24 Jul. 2004, which in turn claims priority to DE application 103 40 373.6 filed 30 Aug. 2003. Each of the foregoing applications is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP04/09435 | Jul 2004 | US |
| Child | 11364118 | Feb 2006 | US |
