EP2242849A2 - Bei der behandlung von alopecia verwendete egr1-modulatoren - Google Patents

Bei der behandlung von alopecia verwendete egr1-modulatoren

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Publication number
EP2242849A2
EP2242849A2 EP08858303A EP08858303A EP2242849A2 EP 2242849 A2 EP2242849 A2 EP 2242849A2 EP 08858303 A EP08858303 A EP 08858303A EP 08858303 A EP08858303 A EP 08858303A EP 2242849 A2 EP2242849 A2 EP 2242849A2
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European Patent Office
Prior art keywords
expression
gene
anagen
egr1
transcription factor
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EP08858303A
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English (en)
French (fr)
Inventor
Sandrine Rethore
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Galderma Research and Development SNC
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Galderma Research and Development SNC
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Publication of EP2242849A2 publication Critical patent/EP2242849A2/de
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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • 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
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • 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
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the invention relates to the identification and use of Early Growth Response 1 (EGR1) transcription factor modulator compounds for the treatment of alopecia. It also relates to methods of in vitro diagnosis or prognosis in vitro of this pathology.
  • EGR1 Early Growth Response 1
  • hair growth is cyclic and comprises three successive phases: the anagen phase, the catagen phase and the telogen phase.
  • Each follicle of the hair thus renews continuously, cyclically and independently to the adjacent follicles (Kligman 1959, Montagna and Parakkal, 1974).
  • the anagen phase or growth phase during which the hair lengthens, lasts several years. This phase recapitulates hair morphogenesis and is divided into 7 different stages (anagen I to anagen VII) (Muller-Rover et al., 2001).
  • the anagen phase is generally reduced to three stages, each grouping several stages: early for stages I-III, anagen medium for stages IV to V and late anagen for stages VI and VII.
  • the catagen phase that follows the anagen phase is very short and lasts only a few weeks. This phase is divided into 8 different stages (catagene I to catagen VIII) (Muller-Rover et al., 2001). During this phase, the hair undergoes an involution, the follicle atrophies and its dermal implantation appears more and more. higher. The telogen phase, which lasts a few months, corresponds to a rest period of the follicle where the hair eventually falls. After this resting phase a new follicle is regenerated, on the spot, and a new cycle begins again. (Montagna and Parakkal, 1974).
  • the hair follicles In mice and other fur mammals, the hair follicles also have a renewal cycle comprising the three phases anagen, catagen and telogen, cut into different stages.
  • the hair cycles of young animals are often "synchronized", that is to say in the same phase of the cycle at the same time within the same anatomical region (Muller-Rover et al., 2001).
  • the fall or natural loss of hair is a physiological phenomenon that occurs constantly and can be estimated, on average, a few hundred hairs a day for a normal physiological state. However, it happens that the hair cycle can be disrupted and that hair loss accelerates and leads to a temporary or permanent loss of hair called alopecia. Different causes can cause alopecia.
  • Hereditary androgenetic alopecia is the most common: it is manifested by a decrease in hair volume, even baldness, and affects 70% of men;
  • the hair loss is directly related to the hair cycle, the follicle no longer entering the anagen phase, or the anagen phase is not maintained, which implies that the follicle no longer produces hair shaft so more hair.
  • the Applicant has now found that the gene coding for Early Growth Response 1 is expressed specifically in the keratocytes of the hair follicle, and that its expression was induced at the time of entry into anagen, in vivo, in a model of induction of Anagene entry by gonadectomy It therefore proposes to target this gene or its expression product, to prevent or improve the phenomena of alopecia.
  • Alopecia refers to all forms of alopecia, namely androgenetic alopecia, acute or areata
  • EGRl The Early Growth Response 1 gene (or "EGR1") encodes a zinc finger protein of the type
  • EGR1 gene or "EGR1 nucleic acid” means the gene or nucleic acid sequence that encodes the EGR1 protein. If the targeted target is preferably the human gene or its expression product, the invention may also use cells expressing the transcription factor Early Growth Response 1, by genomic integration or transient expression of an exogenous nucleic acid coding for the transcription factor.
  • EGR1 is a nuclear protein that functions as a transcription factor by modulating genes involved in differentiation and mitogenesis.
  • EGR1 is known to be expressed and play an important role during tooth morphogenesis (Karavanova, 1992).
  • a large number of genes and signaling pathways, present during tooth morphogenesis, are involved in the hair cycle and especially at the time of entry into anagen.
  • the BMP pathway controls the development of the teeth and the entry into the growth phase of the adult hair follicle (Botchkarev and Sharov, 2004).
  • the specific expression of the EGR1 transcription factor in hair keratinocytes and its induction during anagen entry suggests that it plays an important role in the homeostasis of the hair follicle.
  • An object of the invention is an in vitro method for diagnosing or monitoring alopecia evolution in a subject, comprising comparing the expression or activity of the Early Growth Response 1 (EGR1) protein. , the expression of its gene or the activity of at least one of its promoters, in a biological sample of a subject relative to a control subject.
  • EGR1 Early Growth Response 1
  • the expression of the protein can be determined by an assay of this EGR1 protein by an immunohistochemical or immunoassay test, for example by ELISA assay. Another method, especially for measuring the expression of the gene, is to measure the amount of corresponding mRNA, by any method as described above. An assay of the activity of the transcription factor EGR1 can also be envisaged. In the context of a diagnosis, the subject "control” is a "healthy" subject.
  • control subject refers to the same subject at a different time, which preferably corresponds to the beginning of treatment (To).
  • This measurement of the difference in the expression or activity of the EGR1 protein, the expression of its gene or the activity of at least one of its promoters makes it possible in particular to monitor the efficacy of a treatment. , including treatment with a modulator of the transcription factor EGR1, as envisaged above or by another treatment against alopecia
  • Such monitoring may support the patient as to the merits, or the need, to continue this treatment.
  • Another aspect of the present invention relates to an in vitro method for determining susceptibility of a subject to develop alopecia, comprising comparing the expression or activity of the Early Growth Response 1 (EGR1) protein, the expression of its gene or the activity of at least one of its promoters, in a biological sample of a subject in relation to a control subject
  • EGR1 Early Growth Response 1
  • the expression of the protein may be determined by an assay of the EGR1 protein, by an immunohistochemical or immunoassay test, for example by ELISA assay.
  • Another method, especially for measuring the expression of the gene is to measure the amount of mRNA corresponding by any method as described above.
  • An assay of the activity of the transcription factor EGR1 can also be envisaged
  • the tested subject is here an asymptomatic subject, having no hair disorder related to alopecia.
  • the subject "control” in this method means a "healthy" reference subject or population. The detection of this susceptibility allows the establishment of a preventive treatment and / or increased monitoring of the signs related to alopecia.
  • the biological sample tested may be any sample of biological fluid or a sample of a biopsy.
  • the sample may nevertheless be a preparation of skin cells, obtained by example by hair removal or biopsy
  • Another subject of the invention is an in vitro method for screening candidate compounds for the preventive and / or curative treatment of alopecia, comprising determining the ability of a compound to modulate the expression or activity of the alopecia.
  • transcription factor Early Growth Response 1 (EGR1) or the expression of its gene or the activity of at least one of its promoters said modulation indicating the utility of the compound for the preventive or curative treatment of alopecia
  • EGR1 Early Growth Response 1
  • the method thus makes it possible to select the compounds capable of modulating the expression or the activity of the transcription factor EGR1, or the expression of its gene, or the activity of at least one of its promoters
  • the invention relates to an in vitro method for screening candidate compounds for the preventive and / or curative treatment of alopecia, comprising the following steps: a. preparation of at least two biological samples or reaction mixtures; b. contacting one of the samples or reaction mixtures with one or more of the test compounds; vs. measuring the expression or the activity of the EGR1 protein, the expression of its gene or the activity of at least one of its promoters, in the biological samples or reaction mixtures; d. selection of compounds for which a modulation of the expression or activity of the EGR1 protein, the expression of its gene or the activity of at least one of its promoters, is measured in the sample or the mixture treated in b), relative to the untreated sample or mixture.
  • Modulation means any effect on the level of expression or activity of the EGR1 transcription factor, the expression of its gene or the activity of at least one of its promoters, namely possibly an inhibition, but preferably a stimulation, partial or complete.
  • the compounds tested in step d) above preferably induce the expression or the activity of the EGR1 protein, the expression of its gene or the activity of at least one of its promoters.
  • expression of a protein means the amount of that protein
  • protein activity is meant its biological activity
  • promoter activity is meant the ability of this promoter to trigger the transcription of the coded DNA sequence downstream of this promoter (and thus indirectly the synthesis of the corresponding protein).
  • the compounds tested can be of any type. They can be of natural origin or have been produced by chemical synthesis. It can be a library of structurally defined chemical compounds, compounds or uncharacterized substances, or a mixture of compounds
  • the biological samples are cells transfected with a reporter gene that is operatively all or part of the promoter of the EGR1 gene, and step c) described above consists in measuring the expression of said gene. reporter.
  • the reporter gene may in particular code for an enzyme which, in the presence of a given substrate, leads to the formation of colored products, such as CAT (chloramphenicol acetyltransferase), GAL (beta galactosidase), or GUS (beta glucuronidase).
  • the assay of the protein encoded by the reporter gene, or its activity is carried out conventionally, by colorimetric, fluorometric or chemiluminescence techniques. , among others
  • the biological samples are cells expressing the gene coding for the transcription factor EGR1, and the step c) described above consists in measuring the expression of said gene.
  • the cell used here can be of any type. It may be a cell expressing the EGR1 gene endogenously, such as for example a liver cell, a prostate cell, or even better a skin cell, hair follicle keratinocytes or dermal papilla fibroblasts. . It is also possible to use organs of human or animal origin, for example hair, or hair follicles of vibrissae. It may also be a cell transformed with a heterologous nucleic acid, encoding the transcription factor EGR1, preferably human or mammalian. A wide variety of host cell systems can be used, such as, for example, Cos-7, CHO, BHK, 3T3, HEK293 cells. The nucleic acid can be stably or transiently transfected by any method known to those skilled in the art, for example by calcium phosphate, DEAE-dextran, liposome, virus, electroporation, or microinjection.
  • the expression of the EGR1 gene can be determined by measuring the transcription rate of said gene, or its translation rate
  • transcription rate of a gene is meant the amount of corresponding mRNA produced by translation rate of the gene.
  • a gene is understood to mean the amount of corresponding protein produced.
  • detection markers such as fluorescent agents. , radioactive, enzymatic or other ligands (eg avidme / biotme).
  • the expression of the gene can be measured by real-time PCR or by RNase protection.
  • RNase protection is meant the detection of a known mRNA from the poly (A) RNAs of a tissue that can be done by means of specific hybridization with a labeled probe.
  • the probe is a labeled complementary RNA (for example radioactive or enzymatic) of the messenger to look for. It can be constructed from a known mRNA whose cDNA, after RT-PCR, has been cloned into a phage.
  • RNA-poly (A) of the tissue where the sequence is to be searched is incubated with this probe under slow hybridization conditions in a liquid medium.
  • RNA hybrids are formed between the desired mRNA and the antisense probe.
  • the hybrid medium is then incubated with a mixture of ribonucleases specific for single-stranded RNA, so that only the hybrids formed with the probe can resist this digestion.
  • the digestion product is then deproteinized and repurified, before being analyzed by electrophoresis.
  • the labeled hybrid RNAs are detected for example by autoradiography or chemiluminescence.
  • the translation rate of the gene is evaluated, for example, by immunological assay of the product of said gene.
  • the antibodies used for this purpose may be of polyclonal or monoclonal type. Their production is based on conventional techniques.
  • a polyclonal anti-Early growth response antibody 1 can, inter alia, be obtained by immunizing an animal such as a rabbit or a mouse, using the entire protein. The antiserum is removed and then exhausted according to methods known per se to those skilled in the art.
  • a monoclonal antibody can, inter alia, be obtained by the conventional method of Kohler and Milstein (Nature (London), 256: 495-497 (1975)). Other methods of preparing monoclonal antibodies are also known.
  • the immunoassay can be carried out in solid phase or in homogeneous phase; in a time or in two stages; sandwich method or competitive method, by way of non-limiting examples.
  • the capture antibody is immobilized on a solid phase.
  • solid phase it is possible to use microplates, in particular polystyrene microplates, or particles or solid beads. , paramagnetic beads
  • ELISA assays immunoassays, or any other detection technique can be used to reveal the presence of the antigen-antibody complexes formed.
  • the characterization of antigen / antibody complexes, and more generally isolated or purified but also recombinant proteins (obtained in vitro and in vivo) can be performed by mass spectrometry analysis. This identification is made possible thanks to the analysis (determination of the mass) of the peptides generated by the enzymatic hydrolysis of the proteins (trypsin in general). In general, the proteins are isolated according to the methods known to those skilled in the art. prior to enzymatic digestion.
  • Peptide analysis in the form of a hydrolyzate is carried out by separation of the peptides by HPLC (nano-HPLC) based on their physico-chemical properties (reverse phase).
  • HPLC nano-HPLC
  • the determination of the mass of the peptides thus separated is carried out by ionization of the peptides and either by direct coupling to the mass spectrometer (electrospray mode ESI) or after deposition and crystallization in the presence of a matrix known to those skilled in the art ( analysis in MALDI mode).
  • the proteins are then identified through the use of appropriate software (eg Mascot)
  • the transcription factor EGR1 can be produced according to standard techniques using Cos-7, CHO, BHK, 3T3, HEK293 cells. It can also be produced using microorganisms such as bacteria (e.g. E. coli or B. subtilis), yeasts (e.g. Saccharomyces, Pichia) or insect cells, such as Sf9 or Sf21.
  • bacteria e.g. E. coli or B. subtilis
  • yeasts e.g. Saccharomyces, Pichia
  • insect cells such as Sf9 or Sf21.
  • the subject of the invention is also the use of a modulator of the transcription factor EGR1 that can be obtained according to one of the methods described above for the preparation of a medicament intended for the preventive and / or curative treatment of alopecia.
  • a method of preventive and / or curative treatment of alopecia which method comprises administering a therapeutically effective amount of a transcription factor modulator EGR1, to a patient in need of such treatment is described herein.
  • modulators are activators (or inducers) of the transcription factor EGR1.
  • the invention comprises the use of EGR1 transcription factor inducing compounds, such as those identified by the screening method described above, for the preventive and / or curative treatment of alopecia.
  • the modulating compounds are formulated in pharmaceutical compositions, in association with a pharmaceutically acceptable vehicle. These compositions may be administered, for example, enterally, parenterally, or topically. Preferably, the pharmaceutical composition is applied topically orally.
  • pharmaceutical composition may be in the form of tablets, capsules, dragees, syrups, suspensions, solutions, powders, granules, emulsions, suspensions of microspheres or nanospheres or lipid or polymeric vesicles allowing controlled release .
  • the pharmaceutical composition may be in the form of solutions or suspensions for infusion or for injection.
  • the pharmaceutical composition is more particularly intended for the treatment of the skin, the mucous membranes and the scalp and may be in the form of ointments, creams, milks, ointments, powders, soaked swabs, solutions, gels, sprays, lotions or suspensions. It may also be in the form of suspensions of microspheres or nanospheres or lipid or polymeric vesicles or polymeric patches or hydrogels allowing controlled release.
  • This composition for topical application may be in anhydrous form, in aqueous form or in the form of an emulsion.
  • the pharmaceutical composition is in the form of a gel, a cream or a lotion.
  • the composition may comprise an Early Growth Response 1 transcription factor modulator content ranging from 0.001 to 10% by weight, especially from 0.01 to 5% by weight relative to the total weight of the composition.
  • the pharmaceutical composition may further contain inert additives or combinations of these additives, such as:
  • osmotic pressure modifying agents emulsifying agents
  • UV-A and UV-B filters are UV-A and UV-B filters
  • antioxidants such as alpha-tocopherol, butylhydroxyanisole or butylhydroxytoluene, superoxide dismutase, ubiquinol or certain metal chelators.
  • Figure 1 illustrates the induction of passage to anagen by oophorectomy
  • Female mice whose hair follicles in the dorsal region were in telogen on Day 0, were subjected to ovanotomy or not (control) on day 1 of the study
  • a skin sample from the back region of the mice was taken on days 0, 6 and 8 of the study.
  • Figure 1A represents a histological section of skin from the dorsal region of a mouse at day 0 of the study.
  • Figure 1B is a photograph of a histological section of skin from the dorsal region of an ovanectomized mouse at day 7 of the study.
  • Figure IC shows a histological section of skin of the dorsal region of an ovanectomized mouse at day 8 of the study.
  • Figure ID shows a histological section of skin from the dorsal region of a control mouse at day 8 of the study. Histological analysis clearly shows that ovariectomy induced passage to anagen ( Figure
  • FIG. 2 is a table 1 which shows the modulation of the level of expression of the transcription factor EGR1, expressed with respect to Day 0 of the study, in the skin of the dorsal region of ovariectomized mice at day 8 of the study and in the skin of the dorsal region of control mice (telogen phase skin) at day 8 of the study using the Affymetrix chip technology.
  • Female mice with dorsal hair follicles in telogen on Day 0 were ovariotomized on day 1 of the study. Non-ovariectomized mice were kept as a control group.
  • a skin sample from the dorsal region of the mice was taken on days 0 and 8 of the study.
  • RNAs were isolated and gene expression was analyzed by Affymetrix chip technology.
  • Figure 3 is a histogram showing the modulation of the EGR1 transcription factor, in the dorsal region skin of female mice expressed relative to day 0 of the study, during ovariectomy-induced anagen entry.
  • a skin sample from the dorsal region of the mice was performed on days 0, 1, 2, 4, 6 and 8 of the study.
  • RNAs were isolated and gene expression was analyzed by the Affymetrix chip technique. gene expression clearly shows that the EGR1 gene is induced in animals entering anagen.
  • Figure 4 shows the expression, by in situ hybridization, of the EGR1 transcription factor in the hair follicles at the beginning of the skin anagen of the mouse dorsal region.
  • Figure 4A is the photograph of the dark-field image of a section of skin of a mouse, whose hair follicles of the dorsal region are in early anagen, subjected to in situ hybridization using a sense probe of the EGR1 transcription (negative control)
  • Figure 4B is the photograph of the same histological section counter-stained with hematoxylin This photograph (4B) is used to locate on the black background image (4A)
  • FIG. 4C is the photograph of the dark-field image of a section of skin of a mouse, the hair follicles of the dorsal region of which are in early anagen, subjected to in situ hybridization using an antisense probe of EGR1 transcription factor, the histological structures radioactively labeled by the probe are revealed by the accumulation of light spots (silver grains).
  • Figure 4D is the photograph of the same histological section counter-stained with hematoxylin. This photograph (4D) is used to locate on the black background image (4C). Marking areas are indicated by arrows.
  • Figure 5 shows the expression, by in situ hybridization, of the EGR1 transcription factor in the late-aged hair follicles of the mouse dorsal region skin.
  • Figure 5A is the photograph of the dark-field image of a section of the skin of a mouse, whose hair follicles of the dorsal region are in late anagen, subjected to in situ hybridization using a sense probe of the transcription factor EGR1 (negative control).
  • Figure 5B is the photograph of the same histological section counter-stained with hematoxylin.
  • FIG. 5B is used to locate on the black background image
  • Figure 5C is the photograph of the black background image of a section of skin of a mouse, including the hair follicles of the region dorsal are in late anagen, subjected to in situ hybridization using an EGR1 transcription factor antisense probe, the histological structures radiolabelled by the probe are revealed by the accumulation of light spots (silver grains).
  • Figure 5D is the photograph of the same histological section counter-stained with hematoxylin. This photograph (5D) is used to locate on the black background image (5C). Marking areas are indicated by arrows. In situ hybridization analysis clearly shows that transcripts are specifically expressed in anagen hair follicles.
  • EXPERIMENTAL DATA Example 1: Expression of EGR1 during entry into anagen induced by rovariectomy by Affymetrix chip technology.
  • RESULTS 42 day old female C57BL / 6 mice with dorsal hair follicles in telogen (Chase, 1954) were ovariectomized or not on day 1 of the study. Oophorectomy performed during the telogen phase induces a massive entry of hair follicles from the anagenic dorsal region within one week (Chanda, 2000.) while the hair follicles of the dorsal region of the control animals are still in telogen. skin were made in the dorsal region at day 0, 1, 2, 4, 6 and 8 of the study Part of the sample was used to confirm the passage to anagen by histological analysis The other part of the sample was used to perform transcriptome analysis with Affymetrix chip technology.
  • RNA expression was analyzed on an Affymetrix station (microfluidic module, hybridization oven, scanner, computer) according to the supplier's recommendations.
  • Affymetrix station microfluidic module, hybridization oven, scanner, computer
  • total RNAs isolated from tissues are transcribed into cDNA.
  • biotin-labeled cRNAs are synthesized using T7 polymerase and a biotin-conjugated NTP precursor. The cRNAs are then fragmented into small fragments. All molecular biology steps are controlled using Agilent's "Lab on a chip" system to confirm the effectiveness of enzyme reactions.
  • the Affymetrix chip is hybridized with the biotinylated cRNA, rinsed and then fluorescently labeled using a streptavidin conjugated fluorophore.
  • the chip is scanned and the results are calculated using the MAS5 software provided by Affymetrix.
  • An expression value is obtained for each gene as well as an indication of the presence or absence of the value obtained.
  • the calculation of the significance of the expression is based on the analysis of the signals that are obtained following the hybridization of the cRNA of a given gene with a perfectly matched oligonucleotide ("perfect match") versus an oligonucleotide that contains a mutation ("single mismatch”) in the central region of the oligonucleotide.
  • the transcription factor EGR1 is expressed in the telogen phase and in the anagen phase of the hair cycle. Differential analysis between the expression at the telogenic stage at (JO) and the anagen (O8 ovariectomized) shows that the expression is stronger (factor 1.4) in early anagen compared with the telogen stage. In control mice, the expression of the transcription factor EGR1 is not induced but reduced compared to the beginning of study.
  • FIG. 3 The kinetics of expression of the transcription factor EGR1 during the entry anagen phase following ovariectomy indicates that in the first days after ovariectomy the expression of the factor EGR1 is reduced.
  • EGR1 expression is strongly induced compared to previous days. . This induction is well correlated with the entry into the anagen phase, since in the control animals whose hair follicles of the dorsal region remained in telogen, the expression of the EGR1 gene is not induced.
  • Sense and antisense probes were prepared from the transcription factor EGR1 by incubation of the linearized gene (2 ⁇ g) with 63 ⁇ Ci of [ 35 S] UTP (1250 Ci / mmol, NEN, Massachusetts, USA) in the presence of RNA. T7 or T3 polymerase. In situ hybridization was performed on formaldehyde-fixed mouse tissue wrapped in paraffin. Sections (4 ⁇ m wide) were then deparaffinized in toluene and rehydrated in an alcohol gradient. After drying, the different sections were incubated in prehybridization buffer for two hours.
  • Hybridization was carried out overnight in hybridization buffer (prehybridization buffer with 10mM DTT and 2X10 6 cpm RNA / 35 S labeled) at 53 ° C.
  • the excess probe was removed and the sections were slanted in LM1 photographic emulsion (Amersham Biosciences, UK) and exposed in the dark at 4 ° C for at least one month.
  • the sections were then developed and counter-stained with hematoxylin and eosin. Following the incubation in the presence of a photographic emulsion, the histological structures radiolabelled by the probe are revealed (accumulation of silver grains). A specific signal is shown by positive labeling with the antisense probe ( Figure 4B and Figure 5B) and the absence of labeling with the sense probe ( Figure 3A and Figure 4A) used as a negative control.

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EP08858303A 2007-11-26 2008-11-26 Bei der behandlung von alopecia verwendete egr1-modulatoren Withdrawn EP2242849A2 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR0759323A FR2924128A1 (fr) 2007-11-26 2007-11-26 Modulateurs de egr1 dans le traitement de l'alopecie
PCT/FR2008/052131 WO2009071841A2 (fr) 2007-11-26 2008-11-26 Modulateurs de egr1 dans le traitement de l'alopécie

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EP2242849A2 true EP2242849A2 (de) 2010-10-27

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EP08858303A Withdrawn EP2242849A2 (de) 2007-11-26 2008-11-26 Bei der behandlung von alopecia verwendete egr1-modulatoren

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CA (1) CA2706672A1 (de)
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EP2519653A4 (de) * 2009-12-31 2013-07-10 Univ Columbia Verfahren zur detektion und regulierung von kreisrundem haarausfall und genkohorten davon
FR3143040A1 (fr) * 2022-12-12 2024-06-14 L'oreal Méthode de pronostic et/ou de diagnostic d’une perte de densité capillaire

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IL139936A0 (en) * 1998-06-02 2002-02-10 Glaxo Group Ltd Gene therapy method
US20030124554A1 (en) * 1999-12-01 2003-07-03 Martin Braddock Screening method for compounds capable of modularing egr-1-regulated expression
AU2003235768A1 (en) * 2002-01-07 2003-07-24 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften Molecular trichogram

Non-Patent Citations (2)

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Title
DATABASE MEDLINE [online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; November 2005 (2005-11-01), RENDL MICHAEL ET AL: "Molecular dissection of mesenchymal-epithelial interactions in the hair follicle.", Database accession no. NLM16162033 *
PLOS BIOLOGY NOV 2005 LNKD- PUBMED:16162033, vol. 3, no. 11, November 2005 (2005-11-01), pages e331, ISSN: 1545-7885 *

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WO2009071841A2 (fr) 2009-06-11
US20100260736A1 (en) 2010-10-14
FR2924128A1 (fr) 2009-05-29
WO2009071841A3 (fr) 2009-10-15
CA2706672A1 (fr) 2009-06-11

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