WO2004006949A1 - Procedes et compositions destines a reguler la pousse des poils des mammiferes - Google Patents

Procedes et compositions destines a reguler la pousse des poils des mammiferes Download PDF

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WO2004006949A1
WO2004006949A1 PCT/IL2003/000571 IL0300571W WO2004006949A1 WO 2004006949 A1 WO2004006949 A1 WO 2004006949A1 IL 0300571 W IL0300571 W IL 0300571W WO 2004006949 A1 WO2004006949 A1 WO 2004006949A1
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heparanase
cells
hair
mammalian
maturation
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PCT/IL2003/000571
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Eyal Zecharia
Shula Metzger
Tova Chajek-Shaul
Israel Vlodavsky
Michael Elkin
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Hadasit Medical Research Services And Development Ltd.
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Publication of WO2004006949A1 publication Critical patent/WO2004006949A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • 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
    • A61K8/66—Enzymes
    • 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
    • A61Q7/00—Preparations for affecting hair growth
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01166—Heparanase (3.2.1.166)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to regulation of mammalian hair growth. More particularly, the invention relates to enhancement or inhibition of hair growth by modulation of heparanase levels and or expression in cells of keratinocyte lineage and/or cells of mesenchimal origin.
  • the mature hair follicle is a complex mini-organ composed of several concentric cellular cylinders of epithelial origin surrounding the hair shaft and known as inner and outer root sheaths (IRS and ORS) [Paus, R. & Costsarelis, G. NEJM 341: 491-497 (1999)] .
  • Basement membrane (BM) is a specialized extracellular matrix structure organized as a structural lattice of characteristic proteins and polys accharides [Couchman, J.R. du Cros D.L. J. Invest.
  • the anagen phase involves cell proliferation in the proximal follicular epithelium, followed by invasion of the elongating follicle into the subcutaneous tissue, differentiation of the epithelium at the base of the follicle, and formation of hair matrix cells, which proliferate and generate a new hair shaft.
  • a regression phase (catagen) of the hair growth cycle ensues, during which the lower part of the follicle undergoes programmed cell death and involution [Paus & Costsarelis (1999) ibid.].
  • the follicle enters telogen, the resting period. The cycle is then repeated.
  • the ability of hair follicles to constantly renew is ensured by the presence of the multi-potent stem cells which are capable of generating differentiated progeny for the regeneration of the lower follicle and new hair shaft formation at the onset of each new cycle [Janes, S.M. et al. J. Pathol. 197:479-494 (2002)].
  • the bulge region of the ORS located close to the insertion of the arrector pili muscle, has recently been identified as a "niche" for follicular keratinocyte stem cells [Lyle, S. et al. J. Invest. Dermatol. Symp. Proc. 4:296-301(1999); Taylor, G. et al.
  • extracellular matrix ECM
  • BM basement membrane
  • ECM extracellular matrix
  • BM-resident active molecules i.e., growth factors, cytokines and enzymes
  • HS Heparan sulfate
  • HSPs Heparan sulfate proteoglycans
  • ECM extracellular matrix
  • the basic HSPG structure consists of a protein core to which several linear heparan sulfate (HS) chains are covalently O-linked.
  • the polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are modified at various positions by sulfation, epimerization and N-acetylation, yielding clusters of sulfated disaccharides separated by low or non-sulfated regions [Kjellen and Lindahl (1991) ibid.; Bernfield et al. (1999) ibid.; David (1993) ibid.; Iozzo (1998) ibid.].
  • Heparan sulfate binds to and assembles ECM proteins, including fibronectin, laminins, and interstitial collagens, and plays important roles in cell-cell and cell-ECM interactions [Bernfield et al. (1999) ibid.; David (1993) ibid.; Iozzo (1998) ibid.].
  • the HS chains unique in their ability to bind a multitude of proteins, ensure that a wide variety of bioactive molecules (i.e., heparin- binding growth factors, chemokines, lipoproteiris, enzymes) bind to the cell surface and ECM and thereby function in the control of normal and pathological processes, among which are morphogenesis, tissue repair, inflammation, and neovascularization [Bernfield et al. (1999) ibid.; Vlodavsky, I. et al. In Basement membranes: Cellular and molecular aspects (Rohrbach, D.H. Timpl R, eds.) Academic Press, Orlando, FI 327- 343 (1993); Lever, R. et al. Nature Drug Disc. Rev. 1:140-48 (2002)].
  • bioactive molecules i.e., heparin- binding growth factors, chemokines, lipoproteiris, enzymes
  • HS moieties in the epithelial BM is the ability to bind specifically various members of the heparin-binding growth factor family and serve as their extracellular reservoir [Couchman et al. (1995) ibid.; Elkin et al. (2001) ibid.; Folkman, J. et al. Am. J. Pathol. 130:393-400, (1988); Vlodavsky, I. et al. TIBS 16:268-271 (1991); Vlodavsky, I. et al. In Basement membranes: Cellular and molecular aspects (ed. D.H. Rohrbach & R. Timpl) Academic Press Inc., Orlando, FI.
  • HS degradation by mammalian endoglycosidic enzymes was first described in human placenta and rat liver hepatocytes. Since then, heparanase activity has been identified in a variety of normal and malignant cells and tissues [Vlodavsky, I. et al. Invasion & Metastasis 12:112-127 (1992); Parish, CR. et al. Biochim. Biophys. Acta. 1471:M99- M108 (2001); Vlodavsky, I. and Friedmann, Y. J. Clin. Invest. 108:341-347 (2001); Nakajima, M. et al. J. Cell. Biochem. 36:157-163 (1988); Bernard, D.
  • Heparanase cleaves the glycosidic bond with a hydrolase mechanism and is thus distinct from bacterial heparinases and heparitinase, which are called eliminases to indicate their ability to remove the polysaccharide from the core protein in a single step.
  • HS glycosaminoglycan chains are cleaved by heparanase at only a few sites, yielding HS fragments of appreciable size (10-20 sugar units), suggesting that the enzyme recognizes a particular and relatively rare HS structure [Pikas, D.S. et al. J. Biol. Chem. 273:18770-18777 (1998)].
  • the heparanase cDNA contains an open reading frame of 1629 bp encoding a 61.2 kDa polypeptide of 543 amino acids.
  • the mature active 50 kDa enzyme isolated from cells and tissues, has its N-terminus 157 amino acids downstream from the initiation codon, suggesting post-translational processing of the heparanase polypeptide at an unusual cleavage site [Vlodavsky, I. et al. Nat. Med. 5:793-802 (1999 A); Hulett, M.D. et al. Nat. Med. 5:803-809 (1999); Fairbanks, M.B. et al. J. Biol. Chem.
  • Heparanase contains a catalytic ( ⁇ / ⁇ ) 8 TIM-barrel fold (residues 411- 543), characteristic of the clan A glycosyl hydrolase families [Hulett, M.D. et al. Biochemistry 39:15659-15667 (2000)]. Heparanase catalytic mechanism involves two conserved acidic residues, a putative proton donor at Glu 225 and a nucleophile at Glu '343 [Hulett et al. (2000) ibid.].
  • heparanase Because of the potential tissue damage that could result from inadvertent cleavage of HS, heparanase must be tightly regulated, although little is known about the control of its expression, activity, or subcellular localization.
  • the enzyme is synthesized as a pro-enzyme and is localized mostly in perinuclear acidic endosomal and lysosomal granules of fibroblasts and tumor cells and in the tertiary granules of human neutrophils, where it is co-localized with MMP-9 [Nadav, L. et al. J. Cell Sci. 115(10):2179-87 (2002); Mollinedo, F. et al. Biochem. J.
  • heparanase can be membrane- bound. Heparanase immunoreactivity is observed on the surface of various normal and malignant cells [Vlodavsky and Friedmann (2001) ibid.; Friedmann, Y. et al. Am. J. Pathol. 157:1167-1175 (2000)].
  • the heparanase sequence contains a putative hydrophobic transmembrane domain [Hulett et al. (2000) ibid.] and its complete solubihzation from rat liver, platelets and tumor cells, requires the presence of a detergent, indicating association with the cell membrane [Hulett et al. (1999) ibid.].
  • HSPGs are prominent components of blood vessels. In large vessels they are concentrated mostly in the intima and inner media, whereas in capillaries they are found mainly in the subendothelial BM, where they support proliferating and migrating endothelial cells (EC) and stabilize the structure of the capillary wall [Wight, T.N. Arteriosclerosis 9:1-20
  • Angiogenesis represents a coordinated multicellular process involving a wide variety of molecules, including growth factors, extracellular matrix (ECM) components, adhesion receptors and matrix-degrading enzymes [Folkman, J. and Shing, Y. Ad. Exp. Med. Biol. 313:355-364 (1992)].
  • ECM extracellular matrix
  • HSPGs and HSPG-degrading enzymes have lo " hg been implicated in cell invasion, migration, adhesion, differentiation and proliferation [Bernfield et al. (1999) ibid.; David (1993) ibid.; Iozzo (1998) ibid.], all processes that are associated with angiogenesis.
  • Heparin and HS sequester stabilize and protect fibroblast growth factors (FGFs) and vascular endothelial growth factors (VEGFs) from inactivation. Moreover, these molecules can function as low affinity co-receptors that promote the formation of HS-FGF-FGFR complexes, thus facilitating receptor dimerization and signaling [Vlodavsky, I. et al. Cancer Metastasis Rev. 15:177-186 (1996)]. Heparanase releases an active complex of bFGF and an HS fragment from ECM and basement membrane (BM) [Bashkin, P. et al. Biochemistry 28:1737-1743 (1989); Folkman, J. et al. Am. J. Pathol.
  • recombinant 50 kDa heparanase to induce vascularization in vivo was also demonstrated in a wound healing mouse model.
  • Heparanase- induced tissue remodeling and vascularization was also reflected by a significant acceleration of wound closure, measured on day 7 following wounding.
  • the human hair follicle functions as a self-governing differentiated entity which, on average, totally regenerates itself every 3 to 5 years.
  • Each new cycle entails the development of the hair follicle from an active hair shaft production stage (anagen) to quick involution (catagen) to resting stage (telogen) [Stenn, K.S. and Paus, R. Physio. Rev. 81:449-494 (2001)].
  • ECM extracellular matrix
  • organ remodeling especially during the early anagen phase when the growing follicle migrates downwards through the dermis, and during the catagen phase when involution of the hair bulb takes place upwards [Stenn and Paus (2001) ibid.] .
  • hair follicles are avascular. Recent studies indicate that improved foliicle vascularization promotes hair growth and increases hair follicle and hair size [Stenn and Paus (2001) ibid.; Yano, K. J. Clin. Invest. 107:409-417 (2001)]. It is thought that cyclic hair growth depends on the induction of angiogenesis to meet the increased nutritional needs of hair follicles during the anagen phase of rapid cell division. The growing hair follicle is surrounded by blood vessels that arise from the deep dermal vascular plexus.
  • VEGF vascular endothelial growth factor
  • heparanase exerts a potent pro -angiogenic effect in vivo and thereby may function in the control of hair growth and follicle size, as shown by the present invention.
  • Intact BM is inhibitory for growth and sprouting of epithelial cells both in vitro and in vivo.
  • Heparanase-dependent cleavage of BM HSPGs appears to disrupt this physical barrier and promote tissue (i.e., mammary gland) morphogenesis and regeneration [Zcharia, E. et al. Mammary Gland Biology & Neoplasm 6:311-322 (2001)].
  • tissue i.e., mammary gland
  • bFGF HS-bound growth and differentiation factors
  • BM-type molecules such as HSPGs and chondroitin sulfate proteoglycan species distinct from that found in the general dermis [Stenn and Paus (2001) ibid.].
  • the BM itself was thought to be continuous at the junction of the dermal papilla with the hair matrix at all times. Electron microscopoy revealed, however, gaps in the basal lamina just before and during the differentiation of hair matrix cells into inner root sheath and hair cells [Stenn and Paus (2001) ibid.].
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • HGF hepatocyte growth factor
  • PDGF platelet-derived growth factor
  • HS-binding cytokine produced by cells of the dermal papilla KGF
  • KGF keratinocyte growth factor
  • HS has been shown to bind a multitude of active molecules (i.e., heparin-binding growth factors, chemokines, lipoproteins, enzymes) and hence plays a major role in sequestration of these proteins by the BM and ECM [Vlodavsky et al.
  • heparanase is over-expressed in growing hair follicles, and plays a role in degradation of BM and hence facilitates maturation of the hair follicle. Heparanase may thus be involved in critical events in hair growth, including cell migration, BM degradation and follicle vascularization.
  • hair growth involves cellular and molecular events in which heparanase activity appears to be critical, namely: hair keratinocyte stem cell migration, proliferation, and differentiation, as well as regulation by HS-bound growth factors [Paus & Costsarelis (1999) ibid.; Couchman et al. (1995) ibid.; Taylor et al. (2000) ibid.; Oshima (2001) ibid.] .
  • the present invention relates to a method for regulating mammalian hair growth and a method for regulating the differentiation and maturation of mammalian hair follicles and regulating the duration of active growth phase (anagen), both methods of the invention comprising the step of modulating the levels, activity, quantity, concentration and availability of an active heparanase and/or expression of heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin, so as to enhance or inhibit hair growth.
  • the invention further relates to a method for regulating hair growth, (enhancing or reducing), the differentiation and maturation of mammalian hair follicles and regulating the duration of active growth phase (anagen) in a mammalian subject.
  • This method comprises the step of modulating the levels and availability, the concentration, the activity or the quantity of an active heparanase and/or the expression of heparanase in cells of keratinocyte lineage, so as to enhance or inhibit hair growth in said subject.
  • the methods of the invention enable regulation of mammalian hair growth. Regulation according to this embodiment may be by either inducing or reducing differentiation and maturation of mammalian hair follicles and by prolonging or shortening the duration of active growth phase (anagen).
  • the methods of the invention are based on modulation of the levels, activity and availability of active heparanase and/or the expression of heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin that may increase or alternatively decrease the availability, activity and/or expression of heparanase in said cells.
  • a specifically preferred embodiment of the invention relates to a method for enhancing mammalian hair growth and for inducing differentiation and maturation of mammalian hair follicles and enhancing and prolonging the duration of active growth phase (anagen).
  • the growth enhancing method comprises the step of contacting cells of keratinocyte lineage and/or cells of mesenchimal origin with an effective amount of heparanase or any functional fragment thereof or of an expression vector comprising the nucleic acid sequence coding for heparanase or a nucleic acid sequence which induces specific expression of endogenous heparanase in said cells of keratinocyte lineage and/or cells of mesenchimal origin, or of a composition comprising the same, so as to increase the levels and availability of an active heparanase and/or the expression of heparanase in said cells, and thereby, to accelerate differentiation and maturation of hair follicles.
  • the invention relates to a method for enhancing hair growth, differentiation and maturation of mammalian hair follicles and enhancing and prolonging the duration of active growth phase (anagen), in a mammalian subject.
  • this method comprises the step of administering to said subject an effective amount of heparanase or any functional fragment thereof, or of an expression vector comprising the nucleic acid sequence coding for heparanase or a nucleic acid sequence which induces specific expression of endogenous heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin, or of heparanase-expressing or -secreting cells or of a composition comprising the same, so as to increase the levels, activity, availability and/or expression of heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin, and accelerate differentiation and maturation of hair follicles and prolong the anagen phase
  • administration may be by topical, dermal, intradermal or subcutaneous application, to a skin layer of said subject.
  • a mammalian subject may be a human subject, preferably a human who suffers from primary or secondary alopecia, or alternativelly a mammal from which fur is to be obtained.
  • the invention further provides for a method for reducing or inhibiting mammalian hair growth, reducing differentiation and maturation of mammalian hair follicle and reducing and shortening the duration of active growth phase (anagen), by reducing the amount of heparanase.
  • inhibition of hair growth comprises the step of contacting cells of keratinocyte lineage and/or cells of mesenchimal origin with an effective amount of heparanase inhibitor or of an anti-sense oligonucleotide targeted to heparanase or of a construct comprising an anti-sense nucleic acid sequence of heparanase, or of heparanase specific ribozyme, or of a composition comprising the same, so as to decrease the levels and availability of active heparanase and/or expression of heparanase in said cells, and inhibit differentiation and maturation of hair follicles.
  • the invention relates to a method for reducing or even inhibiting hair growth, differentiation and maturation of hair follicles and reducing and shortening the duration of active growth phase (anagen) in a mammalian subject.
  • the method of inhibiting hair growth is based on reducing the quantity and concentration of heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin of said subject and comprises the step of administering to the treated subject an effective amount of a heparanase inhibitor or of an anti-sense oligonucleotide targeted to heparanase or of a construct comprising an anti-sense nucleic acid sequence of heparanase, or of heparanase specific ribozyme, or of a composition comprising the same, so as to decrease the levels, activity, availability and/or expression of heparanase in said cells, and thereby inhibit differentiation and maturation of
  • Administration may be by applying topically, dermaily, intradermally or subcutaneously, an effective amount of heparanase inhibitor or of an anti-sense oligonucleotide targeted to heparanase or of a construct comprising an anti-sense nucleic acid sequence of heparanase, or of heparanase specific ribozyme, or of a composition comprising the same, so as to " decrease the availability, activity and/or expression of heparanase, and inhibit differentiation and maturation of hair follicle.
  • the methods of the invention are intended for treating a mammalian subject, for example, a human subject suffering from extensive hair growth or a pet mammal, a laboratory mammal or a mammal used for food consumption.
  • the invention further relates to the use of heparanase or any functional fragment thereof as an agent in the regulation of the differentiation and maturation of mammalian hair follicles and in the regulation of the duration of active growth phase (anagen) and thereby regulating mammalian hair growth. Still further, the invention relates to the use of heparanase as an agent for enhancing or reducing differentiation and maturation of hair follicles and prolonging or shortening the duration of active growth phase (anagen) in a mammalian subject and thereby enhance or reduce the hair growth of said subject.
  • the invention relates to the use of heparanase in the treatment of a mammalian subject to enhance hair growth, differentiation and maturation of hair follicles and enhance and prolong the duration of active growth phase (anagen) in said subject. Further, the invention provides for the use of heparanase or any functional fragment thereof in the preparation of a composition for enhancing differentiation and maturation of mammalian hair follicles and enhancing and prolonging the duration of active growth phase (anagen) and thereby enhancing mammalian hair growth.
  • the second aspect of the invention relates to a composition for inducing differentiation and maturation of mammalian hair follicles and enhancing and prolonging the duration of active growth phase (anagen), thereby enhancing mammalian hair growth.
  • the invention further relates to a composition for the treatment of a mammalian subject suffering from primary or secondary alopecia, to enhance hair growth.
  • compositions of the invention may comprise as an active ingredient any one of heparanase or any functional fragment thereof, an expression vector comprising the nucleic acid sequence coding for heparanase or any functional fragments thereof or a nucleic acid sequence which induces specific expression of endogenous heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin and heparanase-expressing or -secreting cells.
  • the heparanase used as an active ingredient in the composition of the invention may be any one of recombinant heparanase or of a natural source.
  • the invention further provides for a composition for reducing mammalian hair growth, a composition for reducing differentiation and maturation of mammalian hair follicle and shortening the duration of growth active phase (anagen) and a composition for treating a mammalian subject to reduce hair growth.
  • compositions act in reducing the heparanase levels, quantity, activity or availability of an active heparanase and/or its expression in cells of keratinocyte lineage and/or in cells of mesenchimal origin, and comprise as an active ingredient any one of heparanase inhibitor, an anti-sense oligonucleotide targeted to heparanase, a construct comprising an anti-sense nucleic acid sequence of heparanase and heparanase specific ribozyme.
  • compositions of the invention optionally further comprise pharmaceutically or veterinarily acceptable additive carrier, excipient or diluent.
  • compositions of the invention may be in the form of an aqueous solution, gel, cream, paste, lotion, spray, emulsion, shampoo, suspension, powder, dispersion, salve or ointment.
  • compositions of the invention are intended for treating a mammalian subject, for example a human or a domestic mammal, preferably domestic mammals, from which fur is to be obtained.
  • DNA Quantitative PCR amplification was preformed on DNA extracted from four GO lines of transgenic mice over-expressing the human heparanase gene. Specific primers were used to detect the transgene sequences. Total DNA was assessed by using specific primers against ribosomal protein L19 genomic sequence.
  • IB Expression of the human heparanase protein in various tissues of the hpa transgenic vs. control mice. Lysates from the indicated tissues of control (-) and transgenic mice (+) were subjected to SDS-PAGE and
  • the processed (50 kDa) active enzyme is detected in all tissues examined.
  • the latent 65 kDa enzyme is detected primarily in the gut and heart.
  • IC Immunohistochemical staining of heparanase in liver, heart and colon of transgenic mice. Immunostaining with monoclonal antibodies against human heparanase was preformed with paraffin embedded tissue specimens derived from transgenic (left) and control (right) animals.
  • Tissue lysates were prepared from lung, brain and heart. Similar amounts of protein from control and transgenic animals were incubated with sulfated
  • control is very low compared to heparanase activity in tissues derived from the transgenic animals.
  • Tg. transgenic
  • cont. control
  • Hpa heparanase
  • the secondary antibody used for the confocal microscopy was labeled with cy-3 (Cy-3-conjugated goat anti-mouse IgG (1:100), Jackson, Bar-Har- bor,ME).
  • AVD average vessel density
  • T. af. inj. d
  • Cont. control
  • Tg. transgenic mice
  • FIG. 3A-3B Heparanase activity in wound fluid.
  • Wound fluid was collected 40 h after skin incision and analyzed for heparanase activity and protein.
  • 3A Wound fluid collected from control and hpa transgenic mice was incubated (18 h, 37°C, Ph 6.0) with sulfate labeled ECM. Degradation fragments released into the incubation medium were analyzed by gel filtration, as described in experimental procedures.
  • 3B The wound fluids were subjected to Western blot analysis using antibodies directed against i) the human heparanase (mAb 130); ii) both the human and mouse heparanases (polyclonal P9 Ab); and iii) basic FGF.
  • FIG 4 Expression of heparanase in resting vs. growing hair follicles.
  • Normal, 8 week old C57/BL mice were treated with "SallyHansen" hair remover wax strip kit.
  • Eight days after treatment (late anagen), skin segments were processed for histological examination and sections subjected to immunostaining with anti-mouse heparanase antibodies (P9 Ab).
  • Skin taken from untreated mice (telogen phase) served as control.
  • Intense immunostaining of heparanase was noted in the actively growing (anagen) hair follicles (bottom) vs. a relatively weak staining, concentrated primarily in the base of the follicle, in skin taken from mice in telogen (top).
  • C57BL/6 mouse dorsal skin of defined hair cycle stages (telogen skin before depilation, day 0; early anagen, 4 days post depilation; late anagen, 9 days post depilation) was harvested and stained with anti-heparanase antibody (red staining).
  • 5B Early anagen skin: heparanese expressions begins in the ORS of the follicle invading subcutaneous tissue (arrowhead).
  • 5C Peak anagen skin: heparanase-expressing cells could be easily detected in the outer root sheath of the growing follicles (arrowheads).
  • 5D Heparanase enzymatic activity was analyzed in skin samples collected on day 0, 4 and 9 post-depilation. Skin lysates, normalized for equal protein, were incubated (24h, pH 6.0, 37°C) with " sulfate labeled ECM. Labeled degradation fragments released into the incubation medium were analyzed by gel filtration on sepharose 6B, as described in Elkin et al (2001) ibid.
  • Figure 6A-6D Isolation, culture, characterization and heparanase activity of clonogenic keratinocytes from rat vibrissa bulge, representing hair step cell population
  • 6C Pattern of keratin expression by clonogenic keratinocytes. The cells were washed with PBS and lysed in RIPA buffer. The lysates were analyzed by Western blot with antibodies against keratin (K) 5, 6, 10, 14, and 15. Note the presence of keratinocyte stem cell marker K 15 and keratins 5, 6 and 14, also known to be expressed by bulge stem cells; and absence of K10, which is an early marker of terminal differentiation.
  • 6D Cultured bulge clonogenic keratinocytes were lysed and tested for heparanase activity using the assay described in Fig. 5D.
  • Skin samples were harvested from the dorsal area of Hpa-transgenic and control mice at defined stages of the first postnatal hair cycle and processed for histological analysis. Note the increased length, number and depth of invasions into subcutaneous tissue of hair follicles in Hpa- transgenic mice through the anagen phase, at days 12 and 16 (asterisks), and the prolonged duration of the anagen phase in Hpa-Tg mice. Abbreviations: P.n. d. (postnatal day), Hpa-Tg. (heparanase-transgen), cont (control).
  • Figure 9A-9C Accelerated hair re-growth in HPA-transgenic mice after chemotherapy-induced alopecia
  • mice Control and Hpa-transgenic mice were depilated and treated with cyclophosphamide (CYP, 2 injections of 125mg kg of body weight, on experimental days 1 and 3). Histology of day 15 * showing involution and regression of hair follicles (magnitude of x200 and x400).
  • Tumor cells over-expressing heparanase were previously shown to elicit neovascularization, as also reflected by stimulated tissue vascularity, granulation and wound repair in response to exogeneously added recombinant heparanase [Elkin (2001) ibid.]. These effects were attributed to heparanase mediated release of active HS-bound bFGF and VEGF; release of accessory HS degradation fragments that promote FGF-receptor binding and activation; and to stimulation of endothelial cell invasion and degradation of the subendothelial basal lamina [Vlodavsky (2001) ibid.; Elkin (2001) ibid.].
  • heparanase over-expressing transgenic mice Using heparanase over-expressing transgenic mice, the inventors have now demonstrated an accelerated rate of hair growth following perturbation (wound, shaving) of the skin.
  • a high expression of the heparanase protein was noted in anagen vs. telogen follicles in normal mice that were treated with a hair remover wax vs. untreated mice, respectively (Figs. 4 and 5). It was previously reported that skin keratinocytes are among the few normal cell types that express high levels of heparanase [Bernard D. et al. J. Invest. Dermatol. 117:1266-73 (2001)], although the role of heparanase in the control of hair growth was not investigated.
  • the inventors relate the accelerated growth of hairs primarily to the increased vascularization of the perturbed skin, observed and quantified using histological examination of tissue sections and magnetic resonance imaging (MRI) of the wound area in control vs. hpa-transgenic mice.
  • MRI magnetic resonance imaging
  • heparanase degrades BM and thereby mobilize HS-bound growth factors [Bashkin (1989) ibid.; Elkin (2001) ibid.]. Disruption, of BM associated with neovascularization was in fact demonstrated in the mammary gland of heparanase over- expressing transgenic mice.
  • Heparanase has been previously shown by us to release ECM-resident growth and angiogenic promoting factors and to stimulate cell migration through a reconstituted BM [Bashkin (1989) ibid.; Elkin (2001) ibid.; Goldshmidt (2002) ibid.].
  • the present invention ascribes a role of heparanase in promoting hair follicle growth and maturation due to its combined effect on cell migration, ECM degradation and neovascularization, all known to participate in hair follicle cycling and growth.
  • the present invention relates to a method for regulating mammalian hair growth, comprising the step of modulating the levels, activity, availability and/or the expression of heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin, so as to enhance or inhibit hair growth.
  • regulating mammalian hair growth is effected by regulating the differentiation and maturation of hair follicles and regulating the duration of the active growth phase (anagen).
  • This method comprises the step of modulating the levels, activity and availability of an active heparanase, e.g. the concentration or quantity and/or the expression of heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin, so as to accelerate and promote or inhibit differentiation and maturation of hair follicles.
  • keratinocyte lineage cells comprised within the outer and inner root sheath of the follicle (ORS and IRS, respectively) and hair matrix cells.
  • the keratinocte lineage cells according to the invention are the ORS cells.
  • Outer Root sheath (ORS) keratinocytes may be characterized by the expression of keratins 5 6, 14, 16, 17.
  • Most preferred ORS cells are cells comprised within a specific region of ORS, called the bulge, where hair keratinocyte stem cells reside. The specific marker for these stem cells is keratin 15 [see for example ref: Lyle, S. et al. J. Cell Sci. 111:3179-3188 (1998)].
  • inner root sheath (where no heparanase expression was observed, at least in normal follicles) comprises three layers of keratinocytes: Henle's layer, Huxley's layer and the cuticule of the IRS [Fuchs, E. Annu. Rev. Cell. Dev. Biol. 11:123-153 (1995)].
  • cells of mesenchimal origin is particularly meant dermal papilla cells.
  • the invention further relates to a method for enhancing or reducing hair growth, the differentiation and maturation of hair follicles and regulation of the duration of the active growth phase (anagen) in a mammalian subject in need.
  • This method comprises the step of modulating the levels, activity, availability, the concentration or the quantity of an active heparanase and/or the expression of heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin, so as to enhance or inhibit hair growth in said subject.
  • the methods of " the invention enable regulation of mammalian hair growth. Regulation according to this embodiment may be either by inducing or by reducing differentiation and maturation of mammalian hair follicle and by prolonging or shortening the duration of the active growth phase (anagen).
  • the methods of the invention are based on modulation of the levels, activity and availability of active heparanase and/or the expression of heparanase that may increase or alternatively decrease the availability and/or expression of heparanase in cells of a keratinocyte lineage and/or in cells of mesenchimal origin.
  • Hair loss and baldness are common phenomena in mammals including humans. Hair loss may be naturally occurring (primary alopecia) or it may be induced by chemical or physical agents (secondary alopecia). Hair loss may also result from specific disease states, such as mange or formation of scar tissue from bites, and with increasing age. Hair loss is a common condition in healthy adult male humans, and occurs also in adult female humans.
  • a specifically preferred embodiment of the invention relates to a method for enhancing mammalian hair growth.
  • this method comprises the step of contacting cells of keratinocyte lineage and/or cells of mesenchimal origin with an effective amount of heparanase or any functional fragment thereof- or of an expression vector comprising the nucleic acid sequence coding for heparanase or a nucleic acid sequence which induces specific expression of endogenous heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin, or of a - composition comprising the same, so as to increase the levels, activity and availability of active heparanase and/or the expression of heparanase in said cells, and thereby, to accelerate differentiation and maturation of hair follicles.
  • the invention relates to a method for inducing differentiation and maturation of mammalian hair follicles and enhancing and prolonging the duration of the active growth phase (anagen) in a mammalian subject in need of such treatment.
  • Such method comprises the step of administering to said subject an effective amount of heparanase or any functional fragments thereof or of an expression vector comprising the nucleic acid sequence coding for heparanase or a nucleic acid sequence which induces specific expression of endogenous heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin, or of heparanase-expressirlg or -secreting cells or of a composition comprising the same, so as to increase the levels, activity, availability and/or expression of heparanase in said cells of said subject, and thereby accelerate differentiation and maturation of hair follicles.
  • said cells of keratinocyte lineage may be cells comprised within the outer and inner root sheath of the follicle (ORS and IRS, respectively) and hair matrix cells.
  • the keratinocte lineage cells are the ORS cells.
  • said cells of mesenchimal origin may be dermal papilla cells.
  • the methods of the invention are intended for treating a mammalian subject, for example, a human suffering from hair loss and baldness.
  • a mammalian subject for example, a human suffering from hair loss and baldness.
  • methods for enhancing hair growth according to the invention are particularly useful for mammals, preferably domestic mammals, and most preferably, mammals from which fur is to be obtained. Hair growth in domestic animals is of economic concern, both from cosmetic standpoint in pets and show animals, and in production of fiber and felts used in the textile and garment industries.
  • Such mammals may be sheep, alpaca, camels, llama and angora goats and angora rabbits.
  • the invention relates to a method for treating a mammalian subject which suffers from primary or secondary alopecia, to enhance hair growth, differentiation and maturation of mammalian hair follicles and enhance and prolong the duration of active growth phase (anagen).
  • this method of treatment comprises the step of administering to said subject an effective amount heparanase or any functional fragment thereof, or of an expression vector comprising ' the nucleic acid sequence coding for heparanase or a nucleic acid sequence which induces specific expression of endogenous heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin, or of heparanase-expressing or - secreting cells or of a composition comprising the same, so as to increase the levels, activity, availability and or expression of heparanase in said cells of a keratinocyte lineage and/or cells of mesenchimal origin, and accelerate differentiation, maturation of hair follicle and prolong the anagen phase.
  • administration may be by applying topically, dermally, intradermally or subcutaneously, an effective amount of heparanase or any functional fragment thereof, or of an expression vector comprising the nucleic acid sequence coding for heparanase or a nucleic acid sequence which induces specific expression of endogenous heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin or of heparanase expressing or secreting cells or of a composition comprising the same, to the skin of said subject.
  • a main function of mammalian hair is to provide environmental protection. However, that function has largely been lost in humans, in whom hair is kept or removed from various parts of the body essentially for cosmetic reasons. For example, it is generally preferred to have hair on the scalp but not on the face.
  • Various procedures have been employed to remove unwanted hair including shaving, electrolysis, depilatory creams or lotions, waxing, plucking, and therapeutic antiandrogens. These conventional procedures generally have associated drawbacks.
  • the invention further provides for a method for reducing or inhibiting mammalian hair growth by reducing the amount of heparanase.
  • inhibition of hair growth comprises the step of contacting cells of keratinocyte lineage and/or cells of mesenchimal origin with an effective amount of heparanase inhibitor or of an anti-sense oligonucleotide targeted to heparanase or of a construct comprising an anti-sense nucleic acid sequence of heparanase, or of heparanase specific ribozyme, or of a composition comprising the same, so as to decrease the levels, activity and availability of an active heparanase and/or expression of heparanase in said cells, and inhibit differentiation and maturation of hair follicles.
  • the invention provides for a method for reducing differentiation and maturation of mammalian hair follicles and decreasing and shortening the duration of active growth phase (anagen) in a mammalian subject.
  • the method of the invention comprises the step of administering to said subject a effective amount of any one of heparanase inhibitor, of an anti-sense oligonucleotide targeted to heparanase or of a construct comprising an anti-sense nucleic acid sequence of heparanase, of heparanase specific ribozyme, and of a composition comprising the same, so as to decrease the levels, activity, availability or the concentration of an active heparanase and/or expression of heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin of said subject, " and thereby inhibit differentiation and maturation of hair follicles.
  • the invention relates to a method for treating a mammalian subject to reduce or even inhibit hair growth, differentiation and maturation of mammalian hair follicles and decrease and shorten the duration of active growth phase (anagen).
  • the method of inhibiting hair growth is based on reducing the quantity and concentration of heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin of said subject and therefore comprises the step of administering to said subject an effective amount of a heparanase inhibitor or of an anti-sense oligonucleotide targeted to heparanase or of a construct comprising an anti-sense nucleic acid sequence of heparanase, or of heparanase specific ribozyme, or of a composition comprising the same, so as to decrease the levels, activity, availability and/or expression of heparanase in said cells, and thereby inhibit differentiation and maturation of
  • Administration may be by applying topically, dermally, intradermally or subcutaneously, an effective amount of heparanase inhibitor or of an anti- sense oligonucleotide targeted to heparanase or of a construct comprising an anti-sense nucleic acid sequence of heparanase, or of heparanase specific ribozyme, or of a composition comprising the same, so as to decrease the levels, activity, availability and/or expression of heparanase in cells of a keratinocyte lineage and/or in cells of mesenchimal origin, and inhibit differentiation and maturation of hair follicles.
  • the methods of the invention are intended for treating a mammalian subject, for example, a human in need of treatment for reducing hair growth, particularly hair growth that results from abnormal or diseased conditions.
  • a mammalian subject for example, a human in need of treatment for reducing hair growth, particularly hair growth that results from abnormal or diseased conditions.
  • methods for reducing hair growth according to the invention are useful for a pet mammal, a laboratory mammal or a mammal used for food consumption.
  • the invention further relates to the use of heparanase or any functional fragment thereof as an agent in the regulation of the differentiation and maturation of hair follicles and in the regulation of the duration of the active growth anagen phase and thereby regulating mammalian hair growth.
  • the invention relates to the use of heparanase as an agent in the treatment of a mammalian subject to regulate hair growth, differentiation, maturation of hair follicles and regulate the duration of the active growth phase (anagen) and thereby enhance or reduce the hair growth of said subject.
  • heparanase for regulation of mammalian hair growth that may be any one of enhancing and reducing differentiation and maturation of mammalian hair follicles, and prolonging or shortening the duration of the active growth phase (anagen).
  • the invention relates to the use of heparanase as an agent for enhancing differentiation and maturation of hair follicles and enhancing and prolonging the duration of active growth phase (anagen), thereby enhancing mammalian hair growth.
  • the invention relates to the use of heparanase as , an agent for enhancing hair growth, differentiation and maturation of hair follicles and enhancing and prolonging the duration of active growth phase (anagen), in a mammalian subject in need.
  • the invention relates to the use of heparanase in the treatment of a mammalian subject suffering from primary or secondary alopecia, to enhance hair growth, differentiation and maturation of hair follicles and to enhance and prolong the duration of active growth phase (anagen), in said subject.
  • the invention further provides for the use of heparanase or any functional fragment thereof in the preparation of a composition for enhancing differentiation and maturation of hair follicles and enhancing and prolonging the duration of active growth ' phase (anagen), thereby enhancing mammalian hair growth.
  • the invention relates to the use of heparanase in the preparation of a composition for the treatment of a mammalian subject suffering from primary or secondary alopecia, to enhance hair growth, differentiation and maturation of hair follicles and enhance and prolong the duration of active growth phase (anagen), and thereby enhance the hair growth of said subject.
  • the second aspect of the invention relates to a composition for enhancing mammalian hair growth.
  • the composition of the invention may comprise as an active ingredient heparanase or any functional fragment thereof, an expression vector comprising the nucleic acid sequence coding for heparanase or any functional fragments thereof or a nucleic acid sequence which induces specific expression of endogenous heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin or heparanase- expressing or -secreting cells.
  • the invention relates to a composition for inducing differentiation and maturation of hair follicles and enhancing and prolonging the duration of active growth phase (anagen), and thereby enhancing mammalian hair growth.
  • This composition comprises as an active ingredient any one of heparanase or any functional fragment thereof, an expression vector comprising the nucleic acid sequence coding for heparanase or any functional fragments thereof and a nucleic acid sequence which induces specific expression of endogenous heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin and heparanase-expressing or -secreting cells.
  • compositions for the treatment of a mammalian subject " to enhance hair growth, differentiation and maturation of hair follicles and enhance and prolong the duration of active growth phase (anagen) in said subject which composition comprises as an active ingredient heparanase or any functional fragment thereof, an expression vector comprising the nucleic acid sequence coding for heparanase or any functional fragments thereof or a nucleic acid sequence which induces specific expression of endogenous heparanase in cells of keratinocyte lineage and/or in cells of mesenchimal origin or heparanase-expressing or -secreting cells.
  • the heparanase used as the active ingredient in the compositions of the invention may be any one of recombinant heparanase or of a natural source.
  • the compositions of the invention are intended for the treatment of a mammalian subject, for example, a human subject suffering from primary or secondary alopecia or a mammal from which fur is to be obtained.
  • the invention further provides for a composition for reducing mammalian hair growth.
  • a composition for reducing mammalian hair growth acts in reducing the heparanase levels, activity, quantity or availability of an active heparanase and/or its expression in cells of keratinocyte lineage and/or in cells of mesenchimal origin, and comprises as an active ingredient a heparanase inhibitor, an anti-sense oligonucleotide targeted to heparanase, a construct comprising an anti-sense nucleic acid sequence of heparanase or heparanase specific ribozyme.
  • the invention provides for a composition for reducing differentiation and maturation of mammalian hair follicles and reducing and shortening the duration of active growth phase (anagen), comprising as active ingredient a heparanase inhibitor, an anti-sense oligonucleotide targeted to heparanase, a construct comprising an anti-sense nucleic acid sequence of heparanase or heparanase specific ribozyme.
  • active growth phase anagen
  • the invention provides for a composition for treating a mammalian subject to reduce hair growth, differentiation and maturation of mammalian hair follicles and reduce and shorten the duration of active growth phase (anagen).
  • a composition for treating a mammalian subject comprises as active ingredient a heparanase inhibitor, an anti-sense oligonucleotide targeted to heparanase, a construct comprising an anti-sense nucleic acid sequence of heparanase or heparanase specific ribozyme.
  • compositions of the invention optionally further . comprise pharmaceutically or veterinarily acceptable additive carrier, excipient or diluent.
  • the compositions of the invention may be in the form of an aqueous solution, gel, cream, paste, lotion, spray, shampoo, suspension, emulsion, powder, dispersion, salve or ointment.
  • the compositions of the invention may be absorbed in a carrier which can be affixed to the skin.
  • compositions of the invention are intended for use and treating a mammalian subject, for example a human suffering from hair loss and baldness or alternatively, use or treatment for reducing hair growth particularly hair growth that results from abnormal or diseased conditions.
  • a mammalian subject for example a human suffering from hair loss and baldness or alternatively, use or treatment for reducing hair growth particularly hair growth that results from abnormal or diseased conditions.
  • the methods for enhancing hair growth according to the invention are useful for mammals, preferably domestic mammals, from which fur is to be obtained. Hair growth in domestic animals, as mentioned above, is of economic concern, both from cosmetic standpoint in pets and show animals, " and in production of fiber and felts used in the textile and garment industries.
  • Such mammals as non-limiting example, may be sheep, alpaca, camels, llama and angora goats and angora rabbits.
  • mice over-expressing the heparanase gene have been previously generated by the inventors, and were described elsewhere [Zcharia et al. (2001) ibid.]. Briefly, the full-length human heparanase cDNA in PSI plasmid was digested with EcoRI and Xbal and subcloned into pCAGGS plasmid at the EcoRI-Xbal sites at the right orientation and under the constitutive control of the chicken ⁇ -actin promoter expressed in all tissues.
  • the plasmid was digested with Sall-Pctl and the resulting linear fragment containing the CMV enhancer, ⁇ -actin promoter, hpa cDNA and a sequence encoding the ⁇ -globin poly A was purified (agarose gel followed by a millipore column). The purified fragment was applied for microinjection to fertilized eggs of C57B1 x Balb/c strain, to produce transgenic mice over-expressing the hpa cDNA in all tissues. Genomic DNA was extracted from tail tips and transgenic mice were selected using a PCR amplification assay.
  • Bovine corneal EC were cultured as described [Vlodavsky, I. Current Protocols in Cell Biology. John Wiley & Sons, New York, N.Y., (1999 B) pp. 10.4.1- 10.4.14 (supplement 1)].
  • the subendothelial ECM produced by these cells was exposed by dissolving the cell layer with PBS containing 0.5% Triton X-100 and 20 mM NH4OH, followed by four washed in PBS [Vlodavsky (1999 B) ibid.].
  • the ECM remained intact, free of cellular debris and firmly attached to the entire area of the tissue culture dish [Vlodavsky (1999 B) ibid.].
  • corneal endothelial cells were cultured in the presence of Na2[ ⁇ S] ⁇ 4 (Amersham), added (25 ⁇ Ci/ml) on day 1 and 5 after seeding [Vlodavsky et al. Invasion & Metastasis 12:112-127 (1992); Vlodavsky (1999 A) ibid.; Vlodavsky (1999 B) ibid.].
  • Tail fragments were incubated overnight at 55 °C in a lysis buffer (8 M urea, 0.2 M Tris-HCl, 0.4 M NaCl, 20 mM EDTA, 1 % N-Laurylsarcosine, 10 ⁇ g/ml proteinase K).
  • a lysis buffer 8 M urea, 0.2 M Tris-HCl, 0.4 M NaCl, 20 mM EDTA, 1 % N-Laurylsarcosine, 10 ⁇ g/ml proteinase K.
  • the dissolved tissue underwent phenol extraction and ethanol precipitation, to obtain highly purified genomic DNA.
  • the integration of the human heparanase cDNA [GenBank Accession no. AF144325] in the mouse genome was verified by PCR using two sets of primers.
  • the first couple was designed to amplify the 5' region of the transgene. It included a ⁇ -actin promoter specific primer designated 5'- pCAGGS: 5'-TCTAGAGCCTCTGCTAACCA-3' (also denoted by SEQ ID NO: 1) and human hpa specific primer, designated Hpa-300: 5'- TGACTTGAGATTGCCAGTAACTTC-3' (also denoted by SEQ ID NO: 2).
  • the second primers set was designed to amplify the 3' region of the transgene.
  • Hpa-830 5'-CTGTCCAACTCAATGGTCTAACTC-3' (also denoted by SEQ ID NO:3)
  • a primer specific to the plasmid derived 3'-untranslated region designated 3'pCAGGS: 5'-ATAGCCAGCCTGCACCTGA-3' (also denoted by SEQ ID NO: 4).
  • PCR conditions were as follows: 2 minutes at 95°C followed by 33 cycles of 15 seconds at 95°C, 1 minute at 58°C and 1 minute at 72°G
  • Tissue samples primary tumor, liver, lung were fixed with 4% formaldehyde in PBS, embedded in paraffin and sectioned (5 ⁇ m sections). Following deparaffinization and rehydration, tissue sections were washed (3x) with PBS and stained with hematoxyline/eosine or Mallory stains. Tissue sections were then washed mounted with 90% glycerol in PBS and visualized with a Zeiss axioscope microscope [Friedmann, Y. et al. Am. J. Pathol. 157:1167-1175 (2000)]. .
  • Immunohistochemistry was performed as described [Friedmann et al. (2000) ibid.]. Briefly, 5 ⁇ m sections were deparaffinized and rehydrated. Tissue was then denatured for 3 minutes in a microwave oven in citrate buffer (0.01 M, pH 6.0). Blocking steps included successive incubations in 0.2% glycine, 3% H 2 0 2 in methanol and 5% goat serum. Sections were incubated with a monoclonal (mAb 130) anti-human heparanase antibody diluted 1:3 in PBS, or with DMEM supplemented with 3.3 % horse serum as control, followed by incubation with HRP-conjugated goat anti-mouse IgG antibody (Jackson).
  • Polyclonal (P9) anti-heparanase antibodies directed against a synthetic peptide corresponding to amino acids 155-162 of the 65 kDa human heparanase, were used to detect the mouse heparanase.
  • the preparation and specificity of these antibodies were previously described and demonstrated [Friedmann Y. et al. (2000) ibid.; Vlodavskyet al. (1999 A)]. Color was developed using Zymed AEC substrate kit (Zymed) for 10 minutes, followed by counter stain with Mayer's hematoxylin [Friedmann et al. (2000) ibid.; Vlodavsky (1999 A)].
  • Functionality and maturation of the neovasculature are determined from gradient echo images acquired during the inhalation of air, air-C02 (95% air and 5% CO2), and oxygen-C02 (95% oxygen and 5% CO2; carbogen), as described [Goldshmidt (2002) ibid.; Abramovitch, R. et al. Cancer Res. 59:5012-5016 (1999)]. Four images are acquired at each gas mixture. Other experimental details are as reported previously [Goldshmidt (2002) ibid.; Abramovitch et al. (1999) ibid.].
  • VF Vascular function
  • VD vasodilation
  • MRI data were analyzed on a PC computer using IDL software (Research Systems Inc., Colorado).
  • Vascular function (VF) was derived from images acquired during inhalation of carbogen and air-C02, using the equation described in previous studies [Goldshmidt (2002) ibid.; Abramovitch, R. et al. (1999) ibid.]. VF measures the capacity of erythrocytes to deliver oxygen from the lungs to each pixel in the image [Goldshmidt (2002) ibid.; Abramovitch et al. (1999) ibid.].
  • Vasodilation (VD) was derived from air and air-C0 2 images, as described [Goldshmidt (2002) ibid.; Abramovitch et al.
  • Transgenic animals over-expressing heparanase provide a model system for testing, for example, the role of heparanase in angiogenesis and hair follicle growth, in response to wounding and hair depilation, respectively.
  • Angiogenesis is evaluated in tissue sections by histological analysis of vessel density per microscopic field and/or by magnetic resonance imaging (MRI) analysis of vascular density, functionality and maturation [Abramovitch et al. (1999) ibid.].
  • MRI magnetic resonance imaging
  • mice Four Go founder mice were obtained, bearing a different copy number of the human hpa cDNA in their genome as revealed by a PCR reaction specific for the human hpa cDNA (Fig. IA). These founders were mated with C57BL mice to create Fl mice and those were mated among themselves to create F2 mice. Homozygous F2 mice from each Go line were identified by Southern blot analysis and " quantitative PCR assay. Homozygosity was verified by mating with C57BL mice, where all the pups are supposed to be positive heterozygous. All founder transgenic mice were back crossed with C57BL mice in order to establish C57BL transgenic mice with a pure genetic background. The heparanase transgenic mice are fertile and show no signs of abnormality.
  • heparanase protein expressed in various organs was verified by Western blot analysis of tissue extracts derived from F2 homozygous transgenic and control mice (Fig. IB). Immunohistochemical staining of tissue sections revealed a high expression of the human heparanase protein in tissues derived from the transgenic mice, but not control mice (Fig. IC). Measurements of heparanase activity in tissue extracts revealed a much higher activity in the transgenic vs. control mice in all tissues examined (Fig. ID).
  • Example 2 Example 2
  • heparanase The effect of heparanase on normal neovascularization in response to a cutaneous wound was investigated using control vs. heparanase trasgenic mice. Briefly, 1cm long full thickness dermal incisions were formed by fine surgical scissors and closed by cyano acrylate. Mice [10 control &12 hpa (heparanase) transgenic mice] were examined on days 1, 3 and 7 after incision. The extent of neovascularization was evaluated by MRI analysis of the average vascular density (AVD) and functionality as a function of time, as described [Goldshmidt (2002) ibid.] . A four-fold increase in AVD was noted 24 h after wounding in hpa transgenic vs.
  • a four-fold increase in AVD was noted 24 h after wounding in hpa transgenic vs.
  • FIG. 2A Gross examination of the wound area revealed a markedly increased vascularity in the transgenic vs. control mice on day 7 after wounding (Fig. 2B). Skin reepithelialization and wound vascularization were also examined in histological sections. Mallory staining of sections derived from the wound area revealed a markedly increased vascularity in the transgenic vs. control mice on day 5 after wounding (Fig. 2C). The increased vascularity was also reflected by an inflammatory response, resulting in a delay of wound healing.
  • the heparanase enzyme is normally localized in late endosomes and lysosomes. Immunolocalization of heparanase in the wound area using confocal microscopy, revealed that the enzyme was preferentially expressed on the surface of endothelial cells lining angiogenic blood vessels (Fig. 2D) and dermal connective tissue cells. These results emphasize the significance of heparanase in tissue repair, angiogenesis and inflammation.
  • the wound fluid collected 40 h after injury was analyzed for heparanase activity and bFGF content. As demonstrated in Figure 3A, heparanase activity was 10 fold higher in the wound fluid of hpa-tg (heparanase transgenic mice) vs. control mice. Western blot analysis revealed higher amounts of bFGF in the hpa-tg wound fluid, suggesting a possible mechanism for heparanase as an angiogenic factor (Fig. 3B).
  • Heparanase as described above, degrading the heparan sulfate (HS) scaffold of the ECM, is involved in cell migration, angiogenesis and tissue remodeling [Parish, CR. et al. Biochim. Biophys. Acta.. 1471:M99-M108 (2001); Vlodavsky, I. and Friedmann, Y. J Clin. Invest. 108:341-347 (2001)] and hence, similar to other ECM-degrading enzymes [Bergers, G. et al. Nature Cell Biol. 2:737-744 (2000); Goodman, L/V. and Ledbetter, S.R. J. Cell. Physiol. 151:41-49, (1992)], is likely to play a role in the regulation of hair growth. Therefore, the effect of heparanase on mammalian hair growth was next examined.
  • mice Normal, 8 week old, C57/BL mice were treated with "SallyHansen” hair remover wax strip kit. Eight days after treatment (late anagen), skin segments were processed for histological examination and sections stained with either Masson-trichrome, or subjected to immunostaining with anti- mouse heparanase antibodies. Skin taken from mice in telogen served as control.
  • a marked increase in the number and density of hair follicles was noted in the skin of the treated (anagen) vs. untreated control (telogen) mice. Whereas most of the hair follicles of the control skin were small and localized in the epidermis and dermis, the majority of follicles in the treated mice were fully mature, originating from the dermis and hypodermis. Intense immunostaining of heparanase was noted in the actively growing (anagen) hair follicles. In contrast, a relatively weak staining, concentrated primarily in the base of the follicle was seen in skin taken from mice in telogen. The observed over-expression of heparanase in growing hair follicles, suggests a possible involvement of the enzyme in hair growth and maturation.
  • the inventors next analyzed in more detail the temporal and spatial distribution of heparanase-expressing cells during depilation-induced hair growth cycle. Therefore, C57BL/6 mouse dorsal skin of defined hair cycle stages (telogen skin before depilation, day 0; early anagen, 4 days post depilation; late anagen, 9 days post depilation) was harvested and stained with anti-heparanase antibody. As shown in Figs. 5A-C and 6A, a specific subset of hair follicular keratinocytes expresses the heparanase enzyme in a highly coordinated manner during the hair growth cycle.
  • keratinocytes are located in the ORS, in the vicinity of the bulge region, a follicular stem cell niche [Lyle et al. (1999) ibid.; Taylor et al. (2000) ibid.; Oshima (2001) ibid.]. Heparanase enzymatic activity was analyzed in skin samples collected on day 0' 4' and 9 post depilation. Skin lysates, normalized for equal protein, were incubated with sulfate labeled ECM. Fig. 5D indicates that heparanase expression and activity in this region could only be detected through early to peak anagen.
  • rat vibrissa follicle clonogenic -keratinocytes were next isolated using a procedure described in Oshima et al. (2001) ibid, and Kobayashi et al. (1993) ibid. It should be noted that these cells are closely related, if not identical, to the bulge-residing stem cells, and therefore, served as a model [Oshima et al. (2001) ibid.].
  • the cells were further characterized as an immediate progeny of the stem cells (according to the expression pattern of characteristic keratins, (Fig. 6C), and shown to exhibit heparanase protein expression and enzymatic activity when cultured in vitro (Fig. 6A, D).
  • the wound area of the transgenic mice was covered with hair to a much higher extent than that of the control mice. Therefore, the inventors further analyzed the role of heparanase in hair growth using the heparanase transgenic mice. As shown by Fig. 7, eight week-old control mice and transgenic mice over-expressing the heparanase gene were shaved and compared for the rate of hair growth. A difference in hair growth was noted as early as 5 days after shaving. Ten days later the shaved area of the transgenic mice was fully covered with hair, while that of the control mice was devoid of growing hair (Fig. 7A), reflecting an accelerated rate of hair re-growth.
  • Fig. 7C immunostaining with anti-heparanase antibody revealed increased levels of heparanase protein in hair follicles from heparanase transgenic vs. control mice. Moreover, increased expression of heparanase in the transgenic mice was found to be correlated with increased (about 3 fold) blood vessel size and density (Fig. 7D).
  • Fig. 8 skin samples were harvested from the dorsal area of heparanase-transgenic and control mice at defined stages of the first postnatal hair cycle and processed for histological analysis.
  • the heparanase transgenic mice exhibited increased length, number and depth of invasion into subcutaneous tissue of hair follicles, through the anagen phase, at days 12 and 16 (asterisks, Fig. 8).
  • heparanase-over-expresing transgenic mouse model established in the inventors laboratory, it was found by the present invention, that heparanase has a profound promoting effect on physiologic hair growth (Figs. 7, 8).
  • the inventors therefore analyzed whether heparanase induces hair re-growth after chemotherapy-induced alopecia.
  • Control and heparanase-transgenic mice were depilated and treated with cyclophosphamide (CYP, 2 injections of 125mg/kg of body weight, on experimental days 1 and 3), to imitate severe chemotherapy- induced alopecia. By experimental day 15, the mice " in both groups showed complete alopecia on the dorsal skin.
  • FIG. 9A Histologically, involution and regression of hair follicles were easily detected at that stage (Fig. 9A). At experimental day 30, intense hair growth was evident both by histology (Fig. 9B) and gross examination (Fig. 9C) in heparanase transgenic, but not in the control mice.
  • heparanase enzymatic activity expressed by follicular bulge-derived stem cells and their immediate progeny, may enhance hair growth through facilitation of stem cell migration (by means of degrading extracellular barriers for cell movement), and through release of HS-bound hair growth factors, sequestered in the follicular BM.
  • bFGF HS-bound growth factors involved in hair growth regulation
  • heparanase in bulge keratinocyte stem cell migration was next analyzed utilizing the above mentioned in vitro migration assay for clonogenic keratinocytes isolated from the bulge and representing the stem cell population (Fig. 6B). Isolated cells are added to the upper compartment of 48-well boyden chambers and allowed to migrate through porous polycarbonate membrane, coated with reconstituted basement membrane preparation (matrigel), which mimics in this assay the naturally occurring extracellular barriers for cell migration (ECM, BM). As shown by Fig. 6C, bulge clonogenic keratinocytes express heparanase and are capable of migration through matrigel-coated membrane.
  • the inventors performed the migration assay in the presence of increasing concentrations of specific inhibitors of heparanase enzymatic activity, in order to verify the causative involvement of heparanase in hair keratinocyte stem cell migration. It should be noted that this hair clonogenic keratinocyte migration assay may also be helpful in testing the activity of other candidate compounds affecting the hair stem cell migration, which is currently viewed as a key event in the hair growth [Taylor et al. (2000) ibid.; Oshima (2001) ibid.].
  • HS-bound growth factors i.e., KGF, bFGF
  • BaF 3 lymphoid cell line
  • FGFR2 Illb for KGF, FGFRl for bFGF HS-bound growth factors
  • Isolated bulge clonogenic keratinocytes are incubated on dishes coated with basement membrane-like extracellular matrix (BM-like ECM), prepared as described in Couchman et al. (1995) ibid, and Goldshmidt, O. et al. J. Biol. Chem.
  • mice alopecia model 8-week-old c57bl/6 mice with all follicles in the telogen phase [Shirai, A. et al. J. Dermatol. Sci. 25:213-218, (2001)].
  • mice 24 - 26 months old.
  • heparanase-expressing vector The heparanase expressing adeno -vector was constructed using the AdEasy System (Q-BIOgene). Briefly, an EcoRl- Notl 1.7 kb fragment, containing the entire open reading frame of heparanase, was cloned into the compatible sites of the p Shuttle -CMV vector (cat No.: AES1020).
  • the resultant plasmid is linearized, using Pmel restriction enzyme, and transfected to adenovirus packaging cell line 293 (cat No.:AES0503). After 7 to 12 days the cell condition medium which contains the infectious virus is collected and used for infections. Mice with all the follicles in the telogen phase (8 week old females) or mice with chemotherapy-induced alopecia are locally infected (local injection of the virus) (back skin) with the heparanase-expressing or control adeno- vector. Heparanase expression in the hair follicle is evaluated by a ctivirt, Western blot and immunohistochemistry analysis. Hair growth rate and accompanying changes in peri-follicular area (i.e. angiogenes) are assessed by gross and histology examination [Sato, N. et al. (2001) J. of the Nat. Can. Inst. 93(24): 1858-1864].

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Abstract

L'invention concerne un procédé destiné à réguler la pousse des poils des mammifères s'exerçant par régulation de la différenciation et de la maturation des follicules pileux et par régulation de la durée de la phase de croissance active (anagène). Le procédé de l'invention comprend les étapes consistant à moduler les taux, l'activité et/ou l'expression de l'héparanase dans des cellules de lignage kératinocyte ou dans des cellules d'origine mésenchymateuse, de manière à améliorer ou inhiber la pousse des poils. L'invention concerne également des procédés de traitement de sujets souffrant d'alopécie primaire ou secondaire, ainsi que des compositions et des utilisations de l'héparanase dans la régulation de la pousse des poils de mammifères.
PCT/IL2003/000571 2002-07-11 2003-07-10 Procedes et compositions destines a reguler la pousse des poils des mammiferes WO2004006949A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005118808A1 (fr) * 2004-06-01 2005-12-15 Hadasit Medical Research Services & Development Ltd. Molecules d'acide nucleique servant d'inhibiteurs puissants d'heparanase, leurs compositions et leurs methodes d'utilisation
US8445445B2 (en) 2009-09-15 2013-05-21 Five Prime Therapeutics, Inc. Method of promoting hair growth using FGFR4 extracellular domains
US8685931B2 (en) 2009-12-17 2014-04-01 Five Prime Therapeutics, Inc. Hair growth methods using FGFR3 extracellular domains
US9475871B2 (en) 2010-11-15 2016-10-25 Five Prime Therapeutics, Inc. Treatment of cancer with elevated dosages of soluble FGFR1 fusion proteins

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002019962A2 (fr) * 2000-09-11 2002-03-14 Insight Strategy And Marketing Ltd. Utilisations therapeutiques et cosmetiques des heparanases
WO2002047654A2 (fr) * 2000-12-14 2002-06-20 L'oreal Composition cosmetique comprenant de l'heparanase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002019962A2 (fr) * 2000-09-11 2002-03-14 Insight Strategy And Marketing Ltd. Utilisations therapeutiques et cosmetiques des heparanases
WO2002047654A2 (fr) * 2000-12-14 2002-06-20 L'oreal Composition cosmetique comprenant de l'heparanase

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005118808A1 (fr) * 2004-06-01 2005-12-15 Hadasit Medical Research Services & Development Ltd. Molecules d'acide nucleique servant d'inhibiteurs puissants d'heparanase, leurs compositions et leurs methodes d'utilisation
US8445445B2 (en) 2009-09-15 2013-05-21 Five Prime Therapeutics, Inc. Method of promoting hair growth using FGFR4 extracellular domains
US8685931B2 (en) 2009-12-17 2014-04-01 Five Prime Therapeutics, Inc. Hair growth methods using FGFR3 extracellular domains
US9475871B2 (en) 2010-11-15 2016-10-25 Five Prime Therapeutics, Inc. Treatment of cancer with elevated dosages of soluble FGFR1 fusion proteins

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