WO2006075350A2 - Produit genique pof1 pour usage cosmetique et/ou therapeutique contre les troubles cutanes - Google Patents

Produit genique pof1 pour usage cosmetique et/ou therapeutique contre les troubles cutanes Download PDF

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Publication number
WO2006075350A2
WO2006075350A2 PCT/IT2006/000012 IT2006000012W WO2006075350A2 WO 2006075350 A2 WO2006075350 A2 WO 2006075350A2 IT 2006000012 W IT2006000012 W IT 2006000012W WO 2006075350 A2 WO2006075350 A2 WO 2006075350A2
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WO
WIPO (PCT)
Prior art keywords
poflb
gene
biologically active
active fragments
encoded product
Prior art date
Application number
PCT/IT2006/000012
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English (en)
Other versions
WO2006075350A3 (fr
Inventor
Daniela Toniolo
Flavio Rizzolio
Original Assignee
Fondazione Centro Dan Raffaele Del Monte Tabor
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Fondazione Centro Dan Raffaele Del Monte Tabor filed Critical Fondazione Centro Dan Raffaele Del Monte Tabor
Publication of WO2006075350A2 publication Critical patent/WO2006075350A2/fr
Publication of WO2006075350A3 publication Critical patent/WO2006075350A3/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair

Definitions

  • the invention concerns the finding that the POFlB gene has a highly specific pattern of expression in stratified epithelia and the encoded protein is specifically expressed in junctions of stratified epithelia.
  • the analysis done indicates that the gene may be involved in epidermis differentiation towards the cornified layers and in cell adhesion.
  • RT-PCR experiments and in situ hybridization in the mouse showed that the gene is highly expressed in epidermis and in few other regions, namely in stratified epithelia of the gut.
  • a polyclonal antibody was produced that demonstrated that this is the case and immunohistochemistry analysis showed that the gene is expressed in differentiated layers of the epidermis and of the hair bulb and in most of the stratified epithelia of the gastric tract from tongue to intestine.
  • the localization in the intestine suggests that the protein interacts with adhesive junctions.
  • the epidermis is a stratified squamous epithelium that has the role to establish a permeability barrier (EPB) between the body and the environment, preventing both the escape of moisture and the entry of toxic molecules.
  • EPB permeability barrier
  • the barrier is provided by the cornified layer, a highly insoluble structure containing cross linked protein of the cell envelope (CE) and lipid lamellae secreted in intracellular spaces, that interconnect with CE and fill up the space between the cornified cells (Hardman et al 1998; Segre, 2003).
  • CE cell envelope
  • lipid lamellae secreted in intracellular spaces that interconnect with CE and fill up the space between the cornified cells
  • Mammalian skin is a self renewing tissue composed of several strata (basal, spinosum, granulosum and corneum) derived from mitotically dividing cells in the basal layer that withdraw from the cell cycle and initiate differentiation. While traveling upward to the skin surface they undergo a linear program of terminal differentiation. In the intermediate layers, cells remain transcriptionally active and synthesize and assemble the cytoskeleton framework. When they reach the granular layer the cells flatten and start to degrade their contents including the nuclei. The CE starts to assemble directly underneath the plasma membrane by sequential incorporation of precursor proteins and lipids containing lamellar bodies. Finally in the stratum corneum the cells become permeable and a calcium influx activates transglutaminases to irreversibly crosslink the CE proteins and to serve as scaffold for lipids extruded from the lamellar bodies.
  • strata basic, spinosum, granulosum and corneum
  • the initial scaffold is composed of plakins, involucrin, loricrin and other proteins.
  • Desmosomes have a role in nucleating the initial scaffold and are gradually dissolved when they reach the granular layer, where tight junctions seem to have an essential role in establishing the epidermal barrier, as shown by the phenotype of the KO mice of claudin-1, a tight junction specific protein (Tsukita and Furuse, 2002; Morita and Miyachi, 2003).
  • a cosmetic product for skin topic use, for skin regeneration and anti-aging and/or for hair and/or hair-epidermis related disorders.
  • POFlB gene it is intended the human gene and any orthologous gene thereof.
  • Another object of the invention is the use of the encoded product of the POFlB gene or biologically active fragments thereof for the production of a medicament for skin related disorders, for disorders of the epithelia of the gastrointestinal tract, ior skin regeneration and anti-aging and/or for hair and/or hair-epidermis related disorders.
  • a further object of the present invention is the use of the encoded product of the POFlB gene or biologically active fragments thereof intended to enhance or reduce the process of epidermis differentiation towards the cornified layers and in cell adhesion and/or intended to regulate modification processes undergoing in the granular layer beneath the plasma membrane.
  • Another object of the invention is the use of the encoded product of the POFlB gene or biologically active fragments thereof wherein the gene or polypeptide or the composition is intended to participate in a complex involved in membrane modifications leading to formation of tight junctions in the granular cell layers and in the establishment and maintenance of epidermis permeability barrier.
  • a further object of the present invention is a recombinant protein obtainable by expression of an expression vector pGex-4T3 that contains all or part of a sequence that codes for POFlB.
  • Another object is a recombinant protein obtainable by expression of an eukaryotic expression vector PMT2-HA that contains all or part of a sequence that codes for POFlB.
  • Fig. 1. a: predicted aminoacid sequence of the human POFlB protein and of the mouse poflb protein b: structure prediction and probability of the coiled coil domains.
  • A-C whole sections T: tongue, SG: salivary glands; VF: vibrisse; UpD:upper duodenum; S:stomach; R: rectum; NSE: nasal epithelium; Di:digits; HiL:hilum; Pe:penis; T ⁇ trachea Oe: Oesophagus;.
  • D-E tongue CLxorneified layer
  • IL intemediate layer
  • BL basal layer.
  • F vibrissae
  • G limbs
  • H section of the nose
  • NSE nasal epithelium
  • NC Nasal cavity
  • I external genitalia Pe ⁇ enis
  • J upper intestinal tract
  • tr trachea
  • Oe Oesophagus
  • K-L stomach
  • M intestine
  • b in situ hybridization of sections of adult mouse skin
  • a-e skin sections of adult mouse were fixed by treatment with PFA (paraformaldehyde) included and sliced. Sections of 10 micron were hybridized under standard conditions with the probe described in the text.
  • Fig. 3 Co-Immunostaining using anti POFlB Ab and Ab to a: involucrin (red), b: filaggrin (red); c: desmoglein (green); d: desmoplakin (green); e: desmoglein (green); f: Na-K ATPase (red), a-d are sections of normal human skin, e-f are sections of normal human intestine.
  • Fig. 4 Western Blot analysis using anti POFlB Ab.
  • a Western blot to extracts of COS-7 cells transfected (T) and non transfected (NT) with a POFlB expression vector. 780 and 818 are two different Ab preparations
  • b Western blot analysis of extract from lungs of WT and KO Poflb mice. Anti Actin Ab were used as control. Materials and methods In situ hybridization
  • In situ hybrizdization was done on 18.5 PC mouse embryos and adult mouse skin dissected, fixed in 4% paraformaldehyde (PFA), dehydrated and embedded in paraffin wax for microtome sectioning. Haematoxylin and Eosin was used for staining.
  • In situ hybridisation was performed with standard methods (Rugarli et al., 1993) with antisense RNA probes transcribed from plasmid MAGE: 1280824 containing a the 3'UTR fragment of the mouse Poflb gene.
  • RNA control was also used and it did not hybridized Northern Blot hybridization
  • Commercially available Northern Blots (Human Multiple Tissue Northern Blotl/II, Clontech) were hybridized to a human POFlB probe synthesized by PCR from primers 2 (TCTTTGAAACATCTTTATGGTACTGG) and S
  • POFlB antibodies A full length POFlB human cDNA, containing the cDNA sequence from nt 6 to 2091 was inserted into the PGEX-4T3 vector digested XhoI-EcoRV : POFlB was induced in logaritmically growing cell with 2 mM IPTG at 37 °C/ 200 rpm for 3 hours. Cell were lysed with sample buffer containing 1,5%SDS and extracts were fractionated by 9% gel electrophoresis. The POFlB protein was electroeluted from the gel band and injected into rabbits. Ab were produced by Neosystem (Strasbourg, France) and tested by Elisa. Specificity of the POFlB antibodies
  • the full length human POFlB cDNA containing the cDNA sequence from nt 6 to 2091, was cloned in the eukaryotic expression vector PMT2- HA. Plasmid DNA was purified by Qiagen Plasmid Midi Kit and 5 ⁇ gr were trasfected into COS-7 cell by calcium phosphate. After 24 hrs cell were collected and lysed in sample buffer containing 1,5%SDS Transfected and non transfected cell extracts were fractionated by 9% polyacrylamide gel and transferred to nitrocellulose filters for Western blot. Ab to POFlB were diluted 1 :300 to 1 : 5000. Irnmunostaining
  • the Poflb mouse gene was isolated from a PAC mouse library made from DNA of 129/Sv mice.
  • a targeting vector was prepared that contained 6.6kb of the Poflb gene corresponding to 1.1 kb at the 5' end, the first two exons and 5.5 kb dowstream in the first intron.
  • the POFlB gene encodes a protein 589 aa long that has no homology to previously described proteins. Comparison to the database showed that the C terminal half of the predicted protein has similarity to the C-terminal portions of myosins: accordingly, POFlB is a soluble protein and its C terminal half is compatible with the presence of a coiled coil domain, similar to that described for intermediate filaments (Fig. 1). Expression of POFlB gene in mouse and human
  • POFlB seems to be a gene encoding a protein involved in differentiation of some vertebrates stratified epithelia. Tissue distribution of the POFlB protein A full length human POFIB gene sequence was cloned in a E coli expression vector. The POFlB protein was produced and injected in rabbits, to prepare polyclonal antibodies (Ab). The antiserum specificity was tested in COS-7 cells transiently transfected with an eukaryotic expression vector carrying the full length POFlB construct: by Western blot the Ab interacted with only one band of the expected size, demonstrating high specificity (Fig. 4a).
  • the antibody was used for immunohistochemistry analysis of human and mouse tissues and the results in the two species were similar. Frozen sections of normal human and mouse skin were co-immunostained with anti POFlB antibodies and antibodies to known epidermis proteins: some of the results are shown in Fig. 3.
  • the POFlB protein was highly expressed in the granular cell layer of the skin as it co-localized with involucrin and filagrin, two proteins known to participate in the cell membrane modifications leading to CE and localized to the granular cell layer (Fig. 3 a and b).
  • POFlB appears close to the plasma membrane: since it is a soluble protein and has no predicted membrane spanning hydrophobic domains it is likely to participate in the membrane modification processes undergoing in the granular layer beneath the plasma membrane.
  • Fig. 3 c and d it is also shown that POFlB does not colocalize with the desmosome proteins desmoglein and desmoplakin that are abundant in the membrane of the cells in the stratum spinosums but very reduced in the granular layer.
  • the POFlB gene encodes a protein specifically expressed in several epithelia and upregulated in the granular layers of the skin.
  • the protein predicted structure and the colocalization experiments indicate that it may participate in a complex involved in the membrane modifications leading to formation of the CE and in particular that it may be part of a complex involved in the formation of the tight junctions in the granular cell layers and in the establishment and maintenance of the EBP. Construction of a mouse carrying a KO of the P of Ib gene
  • mice were constructed by inserting a neomycin cassette into the second exon of the gene, downstream from the ATG, to interrupt translation of the protein. Recombinat clones were screened and injected into blastocists. Chimeras were crossed to C57BL/6J mice and backcrossed 5 times. Further backcrossing is ongoing. Recombinant clones were controlled by Western Blot using the anti POFlB Ab on proteins extracted from lung (Fig.4b). The mice appeared normal and fertile.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dermatology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Birds (AREA)
  • Gerontology & Geriatric Medicine (AREA)
  • Cosmetics (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne l'utilisation du produit codé du gène POFlB ou de ses fragments biologiquement actifs pour l'élaboration d'un produit cosmétique pour usage topique sur la peau, pour la régénération de la peau et la lutte contre le vieillissement cutané de la peau, ainsi que les troubles capillaires et/ou les troubles liés simultanément aux cheveux et au derme.
PCT/IT2006/000012 2005-01-13 2006-01-11 Produit genique pof1 pour usage cosmetique et/ou therapeutique contre les troubles cutanes WO2006075350A2 (fr)

Applications Claiming Priority (2)

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US64354705P 2005-01-13 2005-01-13
US60/643,547 2005-01-13

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WO2006075350A2 true WO2006075350A2 (fr) 2006-07-20
WO2006075350A3 WO2006075350A3 (fr) 2006-11-02

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001010382A2 (fr) * 1999-08-06 2001-02-15 Genzyme Corporation Compositions et procedes se rapportant a claudine-7
WO2001080879A2 (fr) * 2000-04-20 2001-11-01 Smithkline Beecham Biologicals S.A. Compositions
EP1312615A2 (fr) * 2001-11-16 2003-05-21 Eisai Co., Ltd Protéines de la famille jeap, constituants des jonctions serrées des glandes exocrines

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001010382A2 (fr) * 1999-08-06 2001-02-15 Genzyme Corporation Compositions et procedes se rapportant a claudine-7
WO2001080879A2 (fr) * 2000-04-20 2001-11-01 Smithkline Beecham Biologicals S.A. Compositions
EP1312615A2 (fr) * 2001-11-16 2003-05-21 Eisai Co., Ltd Protéines de la famille jeap, constituants des jonctions serrées des glandes exocrines

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
ANONIMOUS: "Human protein atlas"[Online] page 1, XP002391486 Retrieved from the Internet: URL:http://hpr.se/tissue_profile.php?antib ody_id=2033> [retrieved on 2006-07-17] *
BIONE S ET AL: "Mutation analysis of two candidate genes for premature ovarian failure, DACH2 and POF1B" HUMAN REPRODUCTION (OXFORD), vol. 19, no. 12, December 2004 (2004-12), pages 2759-2766, XP002391484 ISSN: 0268-1161 cited in the application *
BIONE SILVIA ET AL: "X chromosome genes and premature ovarian failure" SEMINARS IN REPRODUCTIVE MEDICINE, vol. 18, no. 1, 2000, pages 51-57, XP008066786 ISSN: 1526-8004 cited in the application *
BRANDNER JOHANNA M ET AL: "Expression and localization of tight junction-associated proteins in human hair follicles." ARCHIVES OF DERMATOLOGICAL RESEARCH, vol. 295, no. 5, September 2003 (2003-09), pages 211-221, XP002391485 ISSN: 0340-3696 *
CHOON JIN OOI ET AL: "REGULATION OF TIGHT JUNCTION PROTEINS IN HUMAN SUBJECTS WITH INFLAMMATORY BOWEL DISEASE" GASTROENTEROLOGY, ELSEVIER, PHILADELPHIA, PA, US, vol. 118, no. 4, April 2000 (2000-04), page AGAA795, XP008022521 ISSN: 0016-5085 *
CITI S ET AL: "Tight junction proteins" BIOCHIMICA ET BIOPHYSICA ACTA. MOLECULAR CELL RESEARCH, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 1448, no. 1, 19 November 1998 (1998-11-19), pages 1-11, XP004277870 ISSN: 0167-4889 *
LACOMBE ARNAUD ET AL: "Disruption of POF1B Binding to Nonmuscle Actin Filaments Is Associated with Premature Ovarian Failure." AMERICAN JOURNAL OF HUMAN GENETICS. JUL 2006, vol. 79, no. 1, July 2006 (2006-07), pages 113-119, XP008066778 ISSN: 0002-9297 *
MOORE-OLUFEMI S D ET AL: "Gut edema alters expression of tight junction proteins" JOURNAL OF THE AMERICAN COLLEGE OF SURGEONS, COLLEGE, CHICAGO, IL, US, vol. 199, no. 3, September 2004 (2004-09), page 39, XP004595028 ISSN: 1072-7515 *
SEGRE J: "Complex redundancy to build a simple epidermal permeability barrier" CURRENT OPINION IN CELL BIOLOGY 2003 UNITED KINGDOM, vol. 15, no. 6, 2003, pages 778-784, XP002391483 ISSN: 0955-0674 cited in the application *
TSUKITA S ET AL: "Claudin-based barrier in simple and stratified cellular sheets" CURRENT OPINION IN CELL BIOLOGY 01 OCT 2002 UNITED KINGDOM, vol. 14, no. 5, 1 October 2002 (2002-10-01), pages 531-536, XP002391482 ISSN: 0955-0674 cited in the application *
WALSH-REITZ M M ET AL: "AMP-18 PROTECTS BARRIER FUNCTION OF COLONIC EPITHELIAL CELLS: ROLE OF TIGHT JUNCTION PROTEINS" AMERICAN JOURNAL OF PHYSIOLOGY: GASTROINTESTINAL AND LIVER PHYSIOLOGY, AMERICAN PHYSIOLOGICAL SOCIETY, US, vol. 289, July 2005 (2005-07), pages G163-G171, XP008055706 ISSN: 0193-1857 *

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