WO2006047820A1 - Utilisation de la calgranuline a et b pour la stimulation et l'inhibition de la formation de tissus mineralises - Google Patents

Utilisation de la calgranuline a et b pour la stimulation et l'inhibition de la formation de tissus mineralises Download PDF

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WO2006047820A1
WO2006047820A1 PCT/AU2005/001681 AU2005001681W WO2006047820A1 WO 2006047820 A1 WO2006047820 A1 WO 2006047820A1 AU 2005001681 W AU2005001681 W AU 2005001681W WO 2006047820 A1 WO2006047820 A1 WO 2006047820A1
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polypeptide
vertebrate
polynucleotide encoding
bone
composition
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PCT/AU2005/001681
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WO2006047820A9 (fr
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Carolyn L. Geczy
Hala Zreiqat
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Newsouth Innovations Pty Limited
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Publication of WO2006047820A9 publication Critical patent/WO2006047820A9/fr

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    • 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
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/16—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines

Definitions

  • the present invention relates to methods and compositions for regulating the formation of mineralised tissues in vertebrates.
  • the invention finds particular application in the promotion of bone formation in vivo and the promotion of mineralization on and around implants.
  • the present invention is also applicable to the treatment or prevention of dystrophic mineralization.
  • Mineralized tissues of vertebrates including bone, calcified cartilage, dentine and enamel comprise organic and inorganic (mineral) components. Common to the mineral component of each of these tissues is hydroxyapatite (Ca 1O [PO 4 J 6 [OH] 2 ).
  • the organic phase comprises predominantly collagen but also typically includes a number of non- collagenous proteins.
  • the mineral salts of the skeleton contribute about two-thirds of its weight; the remaining matrix consists of mainly Type I collagen fibrils (90%) and non-collagenous proteins (10%) (Fisher and Termine, 1985).
  • An initial step in the mineralization process in the formation of mineralized tissue is ' the nucleation of hydroxyapatite.
  • osteo-chondral progenitor cells into osteoblasts and chondrocytes is the first step in the formation of bony and cartilage matrix after an insult to the bone. Any failure in the mobilization, proliferation and differentiation of these progenitor cells will lead to failure in the formation of new bone.
  • Hypertrophic chondrocytes are terminally differentiated chondrocytes required for mineralization. Mineral deposition occurs only around hypertrophic chondrocytes. Hypertrophic chondrocytes in the growth plate play a pivotal role in coordinating chondrogenesis and osteogenesis, as they provide a scaffold for subsequent formation of trabecular bone by mineralizing their surrounding matrix, and they may induce a bone collar, the precursor of cortical bone, in the adjacent perichondrium. Osteoblasts are responsible for bone matrix synthesis. They secrete a collagen-rich ground substance essential for later mineralization of hydroxyapatite and other crystals. The collagen strands to form osteoids: spiral fibres of bone matrix. Osteoblasts cause calcium salts and phosphorus to form complexes. These minerals bond with the newly formed osteoid to mineralize the bone tissue.
  • bone formation and/or metabolism may be defective leading to the development of disorders such as osteoporosis, osteoarthritis, osteopetrosis, rheumatoid arthritis, Behcet's Disease, osteogenesis imperfecta (brittle bone disorder), Paget's Disease, hypercalcaemia, rickets and osteomalacia.
  • Bone disorders and bone injuries or trauma may warrant surgery and in some instances the implantation of prosthetic devices and artificial bone.
  • joint arthroplasty has become a common procedure for the treatment of various hip lesions.
  • one of the limitations of this surgery has been the aseptic loosening of the prosthesis resulting from peri-prosthetic bone loss, a condition termed periprosthetic osteolysis.
  • periprosthetic osteolysis a condition termed periprosthetic osteolysis.
  • Calcification is the process of deposition of calcium salts. In the formation of bone this is a normal condition. However in some instances, this is abnormal; for example the calcification of the aortic valve causes narrowing of the passage (aortic stenosis). Ectopic or dystrophic mineralization occurs in a variety of pathological conditions. Calcification of vascular tissue is a common complication of aging, atherosclerosis, diabetes, renal failure and aortic stenosis. Intimal arterial calcification in atherosclerotic plaques causes hardening of the arteries and evidence suggests that this calcification involves both arterial osteoblasts and osteoclasts (Doherty et ah, 2003).
  • Calcification in heart valves can also lead to mechanical failure and clinical failure of prosthetic valves.
  • Dystrophic calcification is frequently one manifestation of a progression of a number of conditions in which inflammation is involved. These include pulmonary calcification and ossification due to various causes such as tuberculosis and cystic fibrosis. Calcification is also common in cancer (including prostate, testicular and ovarian), skin and connective tissue diseases, and in patients receiving heart transplants. Therapies for treating or preventing dystrophic calcification are needed.
  • articular cartilage can withstand an astonishing amount of repetitive physical stress. However, once injured, articular cartilage fails to self-repair even minor defects in its structure. At present, osteoarthritis accounts for half of all chronic conditions in persons over age 65 and it affects 25% of the population over age 60: Current treatment protocols are limited by incomplete integration between the graft and host tissue and the scarcity of suitable donor material. The inability of cartilage for self-repair and the lack of protocols that can reproducibly regenerate durable articular surface provide the rationale for the development of new treatment options based on tissue-engineered cartilage repair.
  • EF-hand calcium binding proteins form a superfamily of calcium binding proteins that contribute to a wide variety of cellular pathways involving calcium as a second messenger or as a sensor.
  • SlOO proteins are a multigene family within this superfamily; 21 members have been identified in the human genome (Ravasi et al., 2004). Most have a high affinity EF-hand calcium binding site and a non-conserved EF hand with reduced affinity for calcium that may be important in mediating extracellular functions. Some SlOO proteins also exhibit high affinity for metal ions such as zinc and magnesium. SlOO proteins are implicated in regulating key intra- and extra-cellular processes including regulation of the cytoskeleton, regulation of calcium homeostasis, cell adhesion, cell proliferation, cell migration, cell differentiation, regulation of kinase activity and pro- inflammatory functions. The diverse functions attributed to different SlOO proteins are likely to depend largely on their binding partner(s), any posttranslational modifications and the metal ions bound (Ravasi et al., 2004).
  • S100A8 and S100A9 are abundant, constitutively expressed, cytoplasmic proteins present in neutrophils and at lower abundance in monocytes.
  • S100A8 and S100A9 are associated with acute and chronic inflammation and host defense and are regulated in macrophages and other cells by pro- and anti-inflammatory cytokines, and by other mediators including vitamin D3, corticosteroids, cAMP, PGE2 and reactive oxygen intermediates.
  • the S100A8/A9 complex has anti-microbial properties, mediated by virtue of its strong zinc-chelating properties.
  • S100A8/A9 also transports unsaturated fatty acids and arachidonic acid.
  • S100A8 and S100A9 may also be involved in myeloid cell differentiation.
  • S100A8 acts as a potent scavenger of oxidants such as hypochlorite and peroxide, generated by activated phagocytes in inflammatory lesions.
  • the extracellular activities of some SlOO proteins are mediated by cellular receptors.
  • the present invention is predicated on the inventors' surprising finding that S100A8 and S100A9 are expressed in hypertrophic chondrocytes, articular chondrocytes, chondroclasts and osteoclasts; S100A8 is also expressed in some osteoblasts. Further, the inventors have surprisingly found that these proteins have the ability to initiate cacification in vitro. Summary of the Invention
  • a method for promoting the formation of mineralised tissue in a vertebrate comprising administering to the vertebrate an effective amount of one or more of the following: an S100A8 polypeptide; an S100A9 polypeptide; a polynucleotide encoding S100A8 operably linked to a promoter; or a polynucleotide encoding S100A9 operably linked to a promoter.
  • the method further comprises administering one or more divalent cations, such as Zn 2+ and/or Mg 2+ .
  • the mineralised tissue may be bone, calcified cartilage, dentine or enamel.
  • the vertebrate may be a mammal.
  • the mammal may be a human.
  • the S100A8 polypeptide may comprise the amino acid sequence set forth in SEQ ID NO:2.
  • the S100A9 polypeptide may comprise the amino acid sequence set forth in SEQ ID NO:4.
  • the polynucleotide encoding S100A8 may comprise the nucleotide sequence as set forth in SEQ ID NO:1.
  • the polynucleotide encoding S100A9 may comprise the nucleotide sequence as set forth in SEQ ID NO:3.
  • S100A8 and S100A9 on mineralisation may be direct or indirect, for example being mediated via downstream molecules stimulated or inhibited by virtue of the binding of S100A8 and S100A9 with cellular receptors. Accordingly, embodiments of the invention contemplate the use of analogues or mimetics of S100A8 and S100A9 capable of binding to such receptors.
  • composition for promoting the formation of mineralised tissue in a vertebrate comprising one or more of the following: an S100A8 polypeptide; an S100A9 polypeptide; a polynucleotide encoding S100A8 operably linked to a promoter; or a polynucleotide encoding S100A9 operably linked to a promoter.
  • composition may further comprise one or more divalent cations, such as Zn 2+ and/or Mg 2+ .
  • the S100A8 polypeptide may comprise the amino acid sequence set forth in SEQ
  • the S100A9 polypeptide may comprise the amino acid sequence set forth in SEQ ID NO:4.
  • the polynucleotide encoding S100A8 may comprise the nucleotide sequence as set forth in SEQ ID NO:1.
  • the polynucleotide encoding S100A9 may comprise the nucleotide sequence as set forth in SEQ ID NO:3.
  • a method for the treatment or prevention of a bone disorder in a vertebrate comprising administering to the vertebrate an effective amount of one or more of the following: an S100A8 polypeptide; an S100A9 polypeptide; a polynucleotide encoding S100A8 operably linked to a promoter; or a polynucleotide encoding S100A9 operably linked to a promoter.
  • the bone disorder may be osteoporosis, osteopetrosis, rheumatoid arthritis, osteogenesis imperfecta (brittle bone disorder), Paget's Disease, hypercalcaemia, rickets, osteomalacia, or any other disorder of bone metabolism or affecting bone formation and/or integrity.
  • the S100A8 polypeptide may comprise the amino acid sequence set forth in SEQ ID NO:2.
  • the S100A9 polypeptide may comprise the amino acid sequence set forth in SEQ ID NO:4.
  • the polynucleotide encoding S100A8 may comprise the nucleotide sequence as set forth in SEQ ID NO:1.
  • the polynucleotide encoding S100A9 may comprise the nucleotide sequence as set forth in SEQ ID NO:3.
  • compositions for treating or preventing a bone disorder in a vertebrate comprising one or more of the following: an S100A8 polypeptide; an S100A9 polypeptide; a polynucleotide encoding S100A8 operably linked to a promoter; or a polynucleotide encoding S100A9 operably linked to a promoter, optionally together with one or more pharmaceutically acceptable carriers, diluents or adjuvants.
  • the S100A8 polypeptide may comprise the amino acid sequence set forth in SEQ ID NO:2.
  • the S100A9 polypeptide may comprise the amino acid sequence set forth in SEQ ID NO:4.
  • the polynucleotide encoding S100A8 may comprise the nucleotide sequence as set forth in SEQ ID NO:1.
  • the polynucleotide encoding S100A9 may comprise the nucleotide sequence as set forth in SEQ ID NO:3.
  • a method for the treatment or prevention of dystrophic calcification in a vertebrate comprising administering to the vertebrate one or more agents capable of inhibiting the activity of S100A8 and/or S100A9.
  • the dystrophic calcification may be vascular calcification, and may be associated with atherosclerosis, aging, diabetes, renal failure, aortic stenosis or chronic inflammation, such as in tuberculosis or cystic fibrosis.
  • the dystrophic calcification may be associated with other pathological conditions such as failing heart valves, cancer, pulmonary disease or rheumatoid nodule formation.
  • the dystrophic calcification may occur on the surface of artificial prostheses such as artificial heart valves and stents.
  • the one or more agents may be nucleic acid-based, peptide-based or small molecule inhibitors of S100A8 and/or S100A9 or molecules preventing the binding of S100A8 and/or S100A9 to cellular receptors.
  • the one or more agents may be administered to the vertebrate parenterally.
  • the one or more agents may be coated onto an arterial stent for implantation into the vertebrate.
  • a composition for treating or preventing dystrophic calcification in a vertebrate comprising one or more agents capable of inhibiting the expression or activity of S100A8 and/or S100A9, optionally together with one or more pharmaceutically acceptable carriers, diluents or adjuvants.
  • a method for promoting calcification in a vertebrate comprising administering to the vertebrate an effective amount of one or more of the following: an S100A8 polypeptide; an S100A9 polypeptide; a polynucleotide encoding S100A8 operably linked to a promoter; or a polynucleotide encoding S100A9 operably linked to a promoter.
  • the method may further comprise administering one or more divalent cations, such as Zn 2+ and/or Mg 2+ .
  • the method may further comprise administering one or more lipids, such as phosphatidyl serine-containing lipids.
  • a method for reducing calcification in a vertebrate comprising administering to the vertebrate one or more agents capable of inhibiting the activity of S100A8 and/or
  • a ninth aspect of the present invention there is provided a method for diagnosing predisposition to a bone disorder in a vertebrate, the method comprising the steps of: (a) isolating a biological sample from the bone tissue of the vertebrate; and
  • the assay may utilise a compound capable of interacting with the polypeptide(s) such that the interaction can be detected.
  • the assay may involve detecting and/or quantifying S100A8 and/or S100A9 mRNA in the sample. mRNA may be detected and quantified by PCR.
  • the compound capable of interacting with the S100A8 and/or S100A9 polypeptide(s) may be an antibody.
  • a method for promoting mineralization on the surface of an implant in a vertebrate comprising administering to the vertebrate one or more of the following: an S100A8 polypeptide; an S100A9 polypeptide; a polynucleotide encoding S100A8 operably linked to a promoter; or a polynucleotide encoding S100A9 operably linked to a promoter.
  • a method for promoting mineralization on or around the surface of an implant comprising coating the implant with a composition comprising one or more of an S100A8 polypeptide and an S100A9 polypeptide and subjecting the coated implant to conditions suitable for initiating crystallization on the surface of the implant.
  • the composition may further comprise one or more divalent cations.
  • the crystallization may be initiated in vivo following implantation of the coated implant into a vertebrate subject.
  • the crystallization may be initiated in vitro in the presence of a solution comprising calcium ions and phosphate ions.
  • a bioactively coated implant for implantation into a vertebrate wherein the implant is coated with a composition comprising one or more of an S100A8 polypeptide and a S100A9 polypeptide.
  • the composition may further comprise one or more divalent cations, such as Zn 2+ and/or Mg 2+ .
  • the implant may be a prosthetic device for joint replacement or repair (including fixation devices such as screws), alveolar ridge augmentation, orthodontics or other suitable purpose.
  • S100A8 and/or S100A9 in the preparation of a scaffold for tissue engineering, wherein the one or more of an S100A8 polypeptide and a S100A9 polypeptide is incorporated into the scaffold.
  • the scaffold may be coated with a composition comprising S100A8 and/or S100A9 polypeptides.
  • the scaffold may also be seeded with suitable cells.
  • the cells may be, for example, chondrocytes or mesenchymal progenitor cells.
  • the scaffold may be a polymer scaffold.
  • composition may further comprise one or more divalent cations, such as Zn 2+ and/or Mg 2+ .
  • a fourteenth aspect of the present invention there is provided the use of S100A8 and/or S100A9 in the preparation of a scaffold for tissue engineering, wherein the scaffold is seeded with cells engineered so as to express S100A8 and/or S100A9.
  • polypeptide means a polymer made up of amino acids linked together by peptide bonds.
  • polynucleotide refers to a single- or double-stranded polymer of deoxyribonucleotide bases, ribonucleotide bases or known analogues of natural nucleotides, or mixtures thereof.
  • treating and “treatment” refer to any and all uses which remedy a disorder or disease state or symptoms, prevent the establishment of a disorder or disease, or otherwise prevent, hinder, retard, or reverse the progression of a disorder or disease or other undesirable symptoms in any way whatsoever.
  • the term "effective amount” includes within its meaning a non-toxic but sufficient amount of an agent or compound to provide the desired therapeutic or preventative effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount”. However, for any given case, an appropriate "effective amount” may be determined by one of ordinary skill in the art using only routine experimentation. In the context of this specification, the terms "activity" as it relates to S100A8 and
  • S100A9 means any cellular function, action, effect or influence exerted directly or indirectly by S100A8 and S100A9 in relation to mineralization, calcification, crystallization or bone formation, either by a nucleic acid sequence or fragment thereof encoding the S100A8 and S100A9 proteins, or by the S100A8 and S100A9 proteins themselves or any fragment thereof. Accordingly, included within the definition of "activity” is the effects and actions of S100A8 and S100A9 on the activities of other molecules, for example downstream molecules inhibited or induced by virtue of interactions between S100A8 and S100A9 with their cellular receptors.
  • inhibitor refers to any agent capable
  • the inhibitor may operate to prevent transcription, translation, post-transcriptional or post-translational processing or otherwise inhibit the activity or S100A8 or S100A9 in any way, via either direct or indirect action.
  • the inhibitor may for example be nucleic acid, peptide, any other suitable chemical compound or molecule or any combination of these. io Additionally, it will be understood that in indirectly impairing the activity of S100A8 or S100A9, the inhibitor may affect the activity of other cellular molecules that in turn act as regulators of S100A8 and S100A9 activity. Similarly, the inhibitor may effect the activity of molecules which are themselves subject to regulation or modulation by S100A8 or S100A9.
  • Fig. 1 Expression of SlOO A8 in bone marrow stromal stem cells.
  • Fig. 2 Immunohistochemical staining of pre-osteogenic cells.
  • FIG. 3 Immunohistochemical staining of mouse chondrocytes.
  • A Positive staining of hypertrophic chondrocytes (arrows) for S100A8.
  • B Negative staining of hypertrophic chondrocytes (arrows) for S100A9.
  • C Positive staining of calcified chondrocytes (arrows) for S100A8.
  • D Von Kossa staining (black) of calcified areas.
  • E Positive staining of articular chondrocytes (arrows) for S100A8.
  • Fig. 4. A. Immunohistochemical staining of mouse osteoclasts for S100A8 and S100A9. Darkened regions indicate positive staining. OC, osteoclasts.
  • B Inset of A showing positive staining for S100A8 only.
  • Fig. 6 Immunohistochemical staining demonstrating expression of S100A8 in human embryonic osteoblasts (A), preosteogenic cells (A) and osteoclasts (B).
  • FIG. 7. Immunohistochemical staining demonstrating S100A8 (A) and S100A9 (B) expression in mouse embryonic chondrocytes.
  • Fig. 8. Detection of S 100A9 (A) and S 100A8 (B) by Western blotting of calcifying microvesicles from human calcified arteries.
  • Sl and S2 are samples isolated from calcified arteries from two patients undergoing bypass surgery. Recombinant S100A9 and S100A8 are shown in the right hand lane of the respective gels as standards. Molecular weight marker sizes (kDa) are indicated (arrows).
  • A anti-S100A8 reactivity in a normal artery.
  • B typical anti-S100A8 reactivity in an area of neovascularization. Large arrows indicate S100A8 + cells around a neovessel; small arrows show S100A8 + EC.
  • C arrows show S100A8 + FC (rose) of macrophage origin (CD68 + , brown).
  • S100A9 was located in EC (small arrows), inflammatory cells (large arrows) and covering the ECM (stars) in areas of neovascularization.
  • E in situ hybridization showing S100A9 mRNA in an area of plaque neovascularization.
  • S100A9-mRNA + EC small arrows
  • macrophage-like cells and FC large arrows
  • Fig. 10 Co-localization of S100A9 (brown) with calcified deposits (violet) in early (A) and advanced (B-E) atherosclerotic plaques stained with anti-S100A9.
  • A S100A9 was localized intra- and extracellularly. Numerous S100A9 + cells showed illdefined borders suggesting their destruction.
  • B low magnification micrograph showing S100A9 + cells located in an intimal area containing large calcified deposits.
  • C and D show details of B.
  • some S100A9 + cells are closely attached to calcified deposits (asterisk).
  • E S100A9 + cells exhibiting ill defined contours (straight arrows) apposed with calcified deposits (asterisk); anti-S100A9 immunoreactivity is evident extracellularly (open arrow).
  • Fig. 11 Scanning electron micrographs illustrating in vitro crystallization in the presence of: (A) S100A8 and S100A9; (B) S100A8 and S10A9 with 0.2mM ZnCl 2 ; (C) S100A8 and 0.2mM ZnCl 2 ; and (D) S100A9 and 0.2mM ZnCl 2 .
  • Fig. 12. ALP activity ( ⁇ mol/mg) expressed by primary human osteoblast-like cells cultured on tissue culture plastic for 3 days (3D) and 7 days (7D) in the presence of S100A8, S100A9 or S100A8+S100A9.
  • a method for promoting the formation of mineralized tissue in a vertebrate comprising administering to the vertebrate an effective amount of one or more of the following: an S100A8 polypeptide; an S100A9 polypeptide; a polynucleotide encoding S100A8 operably linked to a promoter; or a polynucleotide encoding S100A9 operably linked to a promoter.
  • the invention also provides for compositions for promoting the formation of mineralized tissue in a vertebrate wherein the composition comprises one or more of the following: an S100A8 polypeptide; an S100A9 polypeptide; a polynucleotide encoding S100A8 operably linked to a promoter; or a polynucleotide encoding S100A9 operably linked to a promoter.
  • an S100A8 polypeptide an S100A9 polypeptide
  • a polynucleotide encoding S100A8 operably linked to a promoter or a polynucleotide encoding S100A9 operably linked to a promoter.
  • Part of the mineralisation process is the formation of calcifying microvesicles that contain lipids, and proteins that co-ordinate calcium binding and enzymes that generate phosphate, leading to the formation of calcium phosphates.
  • S100A8 and/or S100A9 may aggregate and bind lipid and other proteins as well as calcium and zinc. S100A8 and S100A9 may also interact with receptors on cells in the tissue environment to initiate and propagate generation of other proteins involved in the mineralisation process.
  • disorders of bone metabolism or disorders affecting bone formation, growth or integrity may also be treated using methods and compositions according to embodiments of the invention.
  • Such disorders may include, but are not limited to, osteoporosis, osteomalacia, osteopetrosis, rheumatoid arthritis and Behcet's Disease.
  • osteoporosis osteomalacia
  • osteopetrosis osteopetrosis
  • rheumatoid arthritis rheumatoid arthritis
  • Behcet's Disease Behcet's Disease.
  • a predominant mineral component of bone is hydroxyapatite.
  • the biological apatites in bone are not pure hydroxyapatite but contain trace ions including magnesium (Mg 2+ ) and zinc (Zn 2+ ).
  • Mg 2+ deficiency significantly and progressively diminishes bone formation leading to osteoporosis.
  • Zn 2+ also stimulates bone formation and Zn 2+ deficiency may be involved in the development of osteoporosis.
  • SlOO proteins have been attributed many functions, often associated with their ability to bind calcium and zinc (Raftery et al, 1996). Accordingly, methods and compositions of the present invention contemplate the use of divalent cations such as Zn 2+ and Mg 2+ in addition to S100A8 and S100A9.
  • a vertebrate in need of treatment is administered with an effective amount of S100A8 and/or S100A9 polypeptides.
  • the vertebrate to be treated is a human, and accordingly, the S100A8 and S100A9 polypeptides are the human S100A8 and S100A9 polypeptides.
  • the precise sequences of the S100A8 and S100A9 used according to the methods and compositions of the present invention may vary depending on a number of factors, for example the species of vertebrate to be treated, such that the S100A8 and S100A9 sequences are selected so as to be derived from the species to be treated.
  • Embodiments of the invention also contemplate the administration of polynucleotides encoding S100A8 and/or S100A9.
  • the polynucleotides are typically each operably linked to a promoter such that the appropriate polypeptide sequences are produced following administration of the polynucleotides to the vertebrate.
  • the polynucleotides may be administered to subjects in a vector.
  • the vector may be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion of foreign sequences, their introduction into eukaryotic cells and the expression of the introduced sequences.
  • the vector is a eukaryotic expression vector and may include expression control and processing sequences such as a promoter, an enhancer, ribosome binding sites, polyadenylation signals and transcription termination sequences.
  • the polynucleotides may be located on separate nucleic acid constructs or on the same construct. In embodiments in which the polynucleotides are located on the same construct, they may be operably linked to the same of different promoters.
  • the nucleic acid component(s) to be administered may comprise naked DNA or may be in the form of a composition, together with one or more pharmaceutically acceptable carriers.
  • the S100A8 polypeptide may have the amino acid sequence as set forth in SEQ ID NO:2.
  • the nucleotide sequence of the polynucleotide encoding S100A8 may be as set forth in SEQ ID NO.l or display sufficient sequence identity thereto to hybridise to the sequence of SEQ ID NO:1.
  • the nucleotide sequence of the polynucleotide may share at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 96%, 97%, 98% or 99% identity with the sequence set forth in SEQ ID NO:1.
  • the S100A9 polypeptide may have the amino acid sequence as set forth in SEQ ID NO:4.
  • the nucleotide sequence of the polynucleotide encoding S100A9 may be as set forth in SEQ ID NO: 3 or display sufficient sequence identity thereto to hybridise to the sequence of SEQ ID NO:3.
  • the nucleotide sequence of the polynucleotide may share at least 50%, 60%, 70%, 80%, 85%, 90%, 96%, 97%, 98% or 99% identity with the sequence set forth in SEQ ID NO:3.
  • polypeptide and “polynucleotide” as used herein are fragments and variants thereof.
  • fragment refers to a nucleic acid or polypeptide sequence that encodes a constituent or is a constituent of full-length S100A8 and S100A9 proteins.
  • polypeptide the fragment possesses qualitative biological activity in common with the full-length protein.
  • nucleic acid sequence variants encode polypeptides which possess qualitative biological activity in common.
  • polypeptide sequence variants also possess qualitative biological activity in common. Further, these polypeptide sequence variants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • a variant polypeptide may include analogues, wherein the term "analogue” means a polypeptide which is a derivative of S100A8 or S100A9, which derivative
  • polypeptide 5 comprises addition, deletion, substitution of one or more amino acids, such that the polypeptide retains substantially the same function as native S100A8 or S100A9.
  • conservative amino acid substitution refers to a substitution or replacement of one amino acid for another amino acid with similar properties within a polypeptide chain (primary sequence of a protein). For example, the substitution of the charged amino acid Q glutamic acid (GIu) for the similarly charged amino acid aspartic acid (Asp) would be a conservative amino acid substitution.
  • Ectopic or dystrophic mineral deposition occurs in a number of pathological conditions. For example atherosclerotic plaques frequently become calcified leading to a s potential increase in the likelihood of adverse cardiac events. Calcification in heart valves can lead to mechanical failure and clinical failure of prosthetic valves. Dystrophic mineral deposition is also often a feature of many inflammatory conditions including pulmonary calcification and ossification due to various causes such as tuberculosis and cystic fibrosis. Calcification is also common in cancers (including prostate, testicular and Q ovarian cancer), skin and connective tissue diseases, and in patients receiving heart transplants.
  • the present invention also provides for the inhibition of S100A8 and S100A9 in methods and compositions for the treatment of ectopic or dystrophic calcification.
  • a suitable inhibitor may act directly or indirectly on S100A8 and/or S100A9.
  • the inhibitor may act to impair or prevent cellular or intracellular import and/or export of S100A8 or S100A9, may decrease the stability of S100A8 or S100A9 or may block any one or more of a number of other actions of these proteins, such as transcriptional activation of downstream acting genes.
  • a suitable inhibitor Q may exert its inhibitory effect on S100A8 or S100A9 activity via its interaction with or effect on a regulator of S100A8 or S100A9 or a cellular receptor(s) of S100A8 or S100A9.
  • the inhibitor may be a peptide-based inhibitor such as an antibody.
  • Suitable antibodies include, but are not limited to polyclonal, monoclonal, 5 chimeric, humanised, single chain, Fab fragments, and an Fab expression library.
  • Antibodies may be prepared from discrete regions or fragments of the S100A8 or S100A9 polypeptides.
  • An antigenic S100A8 or S100A9 polypeptide contains at least about 5, and preferably at least about 10, amino acids.
  • an anti-S100A8 or anti-S100A9 monoclonal antibody may be prepared using the hybridoma technology described in Antibodies-A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, N. Y. (1988), the disclosure of which is incorporated herein by reference.
  • an anti-S100A8 or anti-S100A9 monoclonal antibody typically containing Fab portions
  • an anti-S100A8 or anti-S100A9 monoclonal antibody typically containing Fab portions
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include the hybridoma technique originally developed by Kohler et al., 1975, Nature, 256: 495-497, as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al, in Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., (1985)).
  • Immortal, antibody-producing cell lines can be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., "Hybridoma Techniques” (1980); Hammerling et al., “Monoclonal Antibodies and T-cell Hybridomas” (1981); Kennett et al., “Monoclonal Antibodies” (1980).
  • a means of producing a hybridoma from which the monoclonal antibody is produced a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunised with a recognition factor-binding portion thereof, or recognition factor, or an origin-specific DNA-binding portion thereof.
  • Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the present recognition factor and their ability to inhibit specified transcriptional activity in target cells.
  • a monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity.
  • the culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium.
  • the antibody-containing medium is then collected.
  • the antibody molecules can then be further isolated by well-known techniques.
  • S100A8 or S100A9 there are various procedures known in the art which may be used for the production of polyclonal antibodies to S100A8 or S100A9, or fragments or analogues thereof.
  • various host animals can be immunized by injection with S100A8 or S100A9 polypeptides, or fragments or analogues thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc.
  • the S100A8 or S100A9 polypeptides or fragments or analogues thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • various adjuvants may be used to increase the immunological response, including but not limited to Freund's (complete and incomplete), cellulose acetate, mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • Freund's complete and incomplete
  • mineral gels such as aluminium hydroxide
  • surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol
  • BCG Bacille Calmette-Guerin
  • Corynebacterium parvum bacille Calmette-Guerin
  • Assays for immunospecif ⁇ c binding of antibodies may include, but are not limited to, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays, Western blots, precipitation reactions, agglutination assays, complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, and the like (see, for example, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.
  • Antibody binding may be detected by virtue of a detectable label on the primary anti-S100 antibody.
  • the anti- Si 00 antibody may be detected by virtue of its binding with a secondary antibody or reagent which is appropriately labelled.
  • a variety of methods are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • Antibodies can be used in diagnostic methods and kits that are well known to those of ordinary skill in the art to detect qualitatively or quantify S100A8 or S100A9 in tissue.
  • osteoblasts grown from small pieces of vertebral bone harvested from healthy female patients under 15 years of age
  • osteoclasts generated from normal human blood monocytes
  • the inhibitor may be a nucleic acid-based inhibitor.
  • embodiments of the invention may utilise antisense technology to inhibit the expression of S100A8 or S100A9 by blocking translation of the proteins.
  • Antisense technology takes advantage of the fact that nucleic acids pair with complementary sequences. Suitable antisense molecules can be manufactured by chemical synthesis or, in the case of antisense RNA, by transcription in vitro or in vivo when linked to a promoter, by methods known to those skilled in the art.
  • antisense oligonucleotides typically of 18-30 nucleotides in length, may be generated which are at least substantially complementary across their length to a region of either the S100A8 or S100A9 nucleotide sequence. Binding of the antisense oligonucleotide to its complementary cellular nucleotide sequences may interfere with transcription, RNA processing, transport, translation and/or mRNA stability. Suitable antisense oligonucleotides may be prepared by methods well known to those of skill in the art and may be designed to target and bind to regulatory regions of the nucleotide sequence or to coding (exon) or non-coding (intron) sequences.
  • antisense oligonucleotides will be synthesized on automated synthesizers. Suitable antisense oligonucleotides may include modifications designed to improve their delivery into cells, their stability once inside a cell, and/or their binding to the appropriate target. For example, the antisense oligonucleotide may be modified by the addition of one or more phosphorothioate linkages, or the inclusion of one or morpholine rings into the backbone (so-called 'morpholino' oligonucleotides).
  • RNA interference An alternative antisense technology, known as RNA interference (RNAi), may be used, according to known methods in the art (for example WO 99/49029 and WO 01/70949, the disclosures of which are incorporated herein by reference), to inhibit the expression of S100A8 or S100A9.
  • RNAi refers to a means of selective post- transcriptional gene silencing by destruction of specific mRNA by small interfering RNA molecules (siRNA). The siRNA is generated by cleavage of double stranded RNA, where one strand is identical to the message to be inactivated. Double-stranded RNA molecules may be synthesised in which one strand is identical to a specific region of the S100A8 or S100A9 mRNA transcript and introduced directly.
  • siRNA small interfering RNA molecules
  • dsDNA can be employed, which, once presented intracellularly is converted into dsRNA.
  • Methods for the synthesis of suitable molecule for use in RNAi and for achieving post- transcriptional gene silencing are known to those of skill in the art.
  • a further means of inhibiting expression of S100A8 or S100A9 maybe achieved by introducing catalytic antisense nucleic acid constructs, such as ribozymes, which are capable of cleaving S100A8 or S100A9 mRNA transcripts and thereby preventing the production of wildtype protein.
  • Ribozymes are targeted to and anneal with a particular sequence by virtue of two regions of sequence complementarity to the target flanking the ribozyme catalytic site. After binding the ribozyme cleaves the target in a site-specific manner.
  • S100A8 and S100A9 inhibition is not only achievable by use of nucleic acid-based and peptide-based inhibitors.
  • Chemical inhibition for example using small molecule inhibitors is also contemplated and a number of alternative approaches to achieving S100A8 and S100A9 inhibition may be used in the methods and compositions of the present invention.
  • Any other agent, and means of inhibiting activity which is suitable for achieving inhibition of S100A8 or S100A9 is also included within the scope of the present invention.
  • nucleic-acid based inhibitors of S100A8 and/or S100A9 such as DNAzymes or ribozymes may be coated onto the surface of arterial stents for administration to patients suffering from atherosclerosis or on prosthetic heart valves.
  • the inhibitor may reduce plaque that may have accumulated in the patients or may serve to prevent or retard the development of such plaque, or help maintain patency of heart valves.
  • compositions and routes of administration are provided.
  • Molecules and agents of the invention may be administered in the form of compositions in which the composition also comprises one or more pharmaceutically acceptable carriers, diluents, excipients or adjuvants.
  • Compositions may be administered either therapeutically or preventively, hi a therapeutic application, compositions are administered to a patient already suffering from a disorder, in an amount sufficient to cure or at least partially arrest the disorder and its complications.
  • the composition should provide a quantity of the compound or agent sufficient to effectively treat the patient, hi therapeutic applications the treatment may be for the duration of the disorder.
  • the therapeutically effective dose level for any particular patient will depend upon a variety of factors including: the disorder being treated and the severity of the disorder; activity of the compound or agent employed; the composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of sequestration of the agent or compound; the duration of the treatment; drags used in combination or coincidental with the treatment, together with other related factors well known in medicine.
  • an effective dosage is expected to be in the range of about O.OOOlmg to about lOOOmg per kg body weight per 24 hours; typically, about O.OOlmg to about 750mg per kg body weight per 24 hours; about O.Olmg to about 500mg per kg body weight per 24 hours; about O.lmg to about 500mg per kg body weight per 24 hours; about O.lmg to about 250mg per kg body weight per 24 hours; about l.Omg to about 250mg per kg body weight per 24 hours.
  • an effective dose range is expected to be in the range about l.Omg to about 200mg per kg body weight per 24 hours; about l.Omg to about lOOmg per kg body weight per 24 hours; about l.Omg to about 50mg per kg body weight per 24 hours; about l.Omg to about 25mg per kg body weight per 24 hours; about 5.0mg to about 50mg per kg body weight per 24 hours; about 5.0mg to about 20mg per kg body weight per 24 hours; about 5.0mg to about 15mg per kg body weight per 24 hours.
  • an effective dosage may be up to about 500mg/m 2 .
  • an effective dosage is expected to be in the range of about 25 to about 500mg/m 2 , preferably about 25 to about 350mg/m 2 , more preferably about 25 to about 300mg/m 2 , still more preferably about 25 to about 250mg/m 2 , even more preferably about 50 to about
  • compositions may be prepared according to methods which are known to those of ordinary skill in the art and accordingly may include a pharmaceutically acceptable carrier, diluent and/or adjuvant. These compositions can be administered by standard routes. In general, the compositions may be administered by the parenteral (e.g., intravenous, intraarterial, intraspinal, subcutaneous or intramuscular), oral or topical route.
  • parenteral e.g., intravenous, intraarterial, intraspinal, subcutaneous or intramuscular
  • oral or topical route e.g., oral or topical route.
  • the carriers, diluents and adjuvants must be "acceptable” in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof.
  • Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glyco
  • compositions of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection.
  • a formulation suitable for oral ingestion such as capsules, tablets, caplets, elixirs, for example
  • an ointment cream or lotion suitable for topical administration
  • an eye drop in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation
  • parenteral administration that is, subcutaneous, intramuscular or intravenous injection.
  • non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.
  • suitable carriers, diluents, excipients and adjuvants for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin.
  • these oral formulations may contain suitable flavouring and colourings agents.
  • the capsules When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which delay disintegration.
  • Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.
  • Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents.
  • Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol.
  • Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine.
  • Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar.
  • Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate.
  • Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring.
  • Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten.
  • Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite.
  • Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc.
  • Suitable time delay agents include glyceryl monostearate or glyceryl distearate.
  • Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier.
  • suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof.
  • Suspensions for oral administration may further comprise dispersing agents and/or suspending agents.
  • Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate or acetyl alcohol.
  • Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or - laurate, polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate and the like.
  • the emulsions for oral administration may further comprise one or more emulsifying agents.
  • Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.
  • Methods for preparing parenterally administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., hereby incorporated by reference herein. Implants and biomaterials
  • Prosthetic devices are commonly used as implants, for example in the treatment of bone injuries, bone disorders or for cosmetic reasons.
  • Orthopaedic surgery may call for the use of artificial joints such as hips or bones.
  • Artificial bone may be used to strengthen bone weakened due to bone disorders or fractures.
  • Orthodontic surgery may call for the use of artificial crowns or replacement dental roots.
  • the present invention provides for the preparation of bioactive coatings wherein implants are coated with a composition comprising an S100A8 and/or S100A9 polypeptide.
  • a composition comprising an S100A8 and/or S100A9 polypeptide.
  • coating a S100A8/S100A9 heterodimer complex onto biomaterials for skeletal tissue application may be a way forward to enhance bone formation.
  • the proteins may be, for example, covalently bound to the implant surface by aminopropyltriethoxysilane chemistry.
  • the coating will cover the metaphyseal/diaphyseal junction of the implant surface to provide an implant surface with increased biocompatibility of the prosthesis with the surrounding host bone. The formation of new bone around the implant will result in better fixation and improved long term performance of the implant.
  • Coating biomaterials with S100A8 or S100A9 may regulate crystallization, growth, morphology, and assembly of inorganic crystals and a molecular recognition at organic- inorganic interfaces.
  • Implants to which the present invention is applicable may be formed of any suitable implantable substance. Suitable substances will be well known to those skilled in the art and include metals, alloys such as titanium, ceramics, hardened plastics, polyethylene and carbon fibre reinforced resins.
  • Coatings of the invention may be combined with other suitable compounds such as, for example, collagen or bone growth factors.
  • suitable compounds such as, for example, collagen or bone growth factors.
  • such compounds may induce the growth of bone cells and/or encourage bone ingrowth into the implant surface.
  • divalent cations play a critical role in bone remodelling and skeletal development.
  • the present inventors have previously shown that surface chemistry modification of metallic implants with divalent cations alter the molecular component of implant surface and promote optimal osteogenesis (Zreiqat et al, 1999; 2002). Accordingly, methods and compositions of the present invention contemplate the use of divalent cations such as Zn 2+ and Mg 2+ in addition to S100A8 and S100A9 in the production of bioactive coatings and the promotion of mineralization on and around implants.
  • the present invention also contemplates the administration of S100A8 and/or S100A9 polypeptides or polynucleotides to patients with implants to encourage bone formation and, for example, bone ingrowth, on the surface of the implant.
  • a problem associated with the long term used of artificial prostheses such as artificial heart valves and stents is the tendency for calcification to occur on the surface of the prostheses. Accordingly, methods and compositions of the present invention provide for the inhibition of such calcification using agents, as described herein, that are capable of inhibiting the expression or activity of SlOO A8 and/or S100A9. Tissue engineering
  • Tissue engineering has the potential to provide functional substitutes of lost or damaged native tissues by an integrated use of cells, biomaterial scaffolds, and biochemical and physical regulatory factors.
  • tissue engineering is in the regeneration of cartilaginous tissue.
  • Articular cartilage which provides joints with a nearly frictionless weight distributing surface for transferring forces between bones, has a very limited ability to repair itself with a biomechanically functional tissue.
  • Current treatment options include chondral shaving, subchondral drilling, microfracturing of the subchondral plate, and mosaicplasty.
  • optimal therapy to predictably restore a durable articular cartilage surface is yet to be defined.
  • autologous chondrocytes or mesenchymal progenitor cells have been isolated from a small biopsy, expanded in vitro and then re ⁇ injected into the defect embedded in gel-like materials.
  • the newly formed tissue degenerates with time and that the cartilage layer tends to thin, indicating an uncertain long-term outcome.
  • cartilaginous implants engineered in vitro may be less vulnerable to the mechanical loads and metabolic changes at the site of implantation, as compared to isolated cells, and thus promote enhanced tissue repair.
  • Scaffold requirements for cartilage tissue engineering include: biocompatibility, biodegradation over an appropriate time scale, and high porosity with large interconnected pores to facilitate mass transport, infiltration of cells, and interstitial flow of fluid (Freed and Vunjak-Novakovic, 2000).
  • the scaffold should also provide a surface for cell attachment, a structural and logistic template for tissue formation and its subsequent integration with the host tissue, and adequate mechanical properties to support mechanotransduction during cultivation and load bearing following implantation of the engineered graft.
  • the present invention contemplates the use of S100A8 and S100A9 to coat the surface of biomaterials used as scaffolds for tissue engineering.
  • the scaffolds are preferably also seeded with cells, such as chondrocytes or mesenchymal progenitor cells.
  • the scaffold may be seeded with cells, such as chondrocytes, engineered so as to express or overexpress S100A8 and/or S100A9.
  • chondrocytes from hyaline cartilage The differentiated phenotype of chondrocytes from hyaline cartilage is gradually lost during expansion in monolayers. Chondrocytes can reexpress their differentiated phenotype by transfer into an environment that prevents cell flattening, but serially passaged cells never completely recover their chondrogenic potential. Chondrocytes expanded in the presence of S100A8 and/or S100A9 may maintain their phenotype in response to environmental changes. After seeding onto three-dimensional polymer scaffolds, chondrocytes expanded in the presence of S100A8 and/or S100A9 formed cartilaginous tissue that may be histologically and biochemically comparable to that obtained using primary chondrocytes.
  • mice were used at the ages stated and were either wild-type CDl mice (+/+) or mice heterozygous for A8 (+/-)generated as described by Passey et al. (1999). Tissue preparation
  • Tissue harvested from A8 +/+ or A8 +/" mice at 2, 4, 8 and 15 weeks of age were immediately fixed in either 4% w/v paraformaldehyde or cold 95% alcohol for paraffin or resin embedding respectively.
  • Human tissue samples were collected from small pieces of rib or vertebrae harvested from healthy patients under 15 years of age and were processed for either paraffin or resin embedding. Permission to use discarded human tissue was granted by the Human Ethics Committee of the University of New South Wales (No. 97043).
  • tissue for paraffin embedding was decalcified at 4°C in 15% ethylenediaminetetra-acetic acid di-sodium salt (EDTA)/0.5% paraforrnaldehyde/phosphate-buffered saline (PBS) pH 8.0, while slowly rotated.
  • the decalcifying solution was changed daily over 7 days. Decalcified samples were dehydrated in ascending solutions of ethanol and embedded in paraffin.
  • Resin (hydroxyethylmethacrylate)-embedded and fixed tissues were treated with an intermediate infiltrating solution 95% Historesin (Leica kit, Heidelberg, BRD), followed by total infiltration through two changes of cold 100% Historesin as described (Laboux O, 2003). Tissue was then embedded in a HistoMold and polymerization completed at RT.
  • Paraffin and resin sections (4 ⁇ m) were cut with a microtome (Leitz 121, Germany) or a heavy-duty microtome (Reichert-Jung, Leica, Germany), respectively, collected on Chrome alum-gelatin coated glass slides and air-dried at RT. Immunohistochemistry
  • Immunostaining was with rabbit polyclonal IgG raised against A8 and A9 as described (Iismaa et ah, 1994; Kocher et ah, 1996). De-paraffinized and resin-embedded etched sections (incubated in acetone for 20 minutes), were blocked with 2% bovine serum albumin (BSA) in HBSS for 1 h before primary Abs were applied at the appropriate concentrations (pre-determined by serial dilution), and sections incubated overnight in a humidifying chamber at 4 0 C.
  • BSA bovine serum albumin
  • TBSl Tris-buffered saline 1
  • secondary biotin-labeled antibody Multi Link, swine anti- immunoglobulins, Dako E0453, Denmark; l:200v/v
  • Alkaline phosphatase was determined at pH 9.2 using the naphthol phosphate method (Chayen and Bitensky, 1981) with fast red violet LB base as coupler. Resin- embedded sections were stained in Von-Kossa solution (1% AgNO 3 /H 2 O) and incubated under UV light for 5-10 minutes before air drying. Sections were mounted in Eukitt (Zeis, BRD).
  • mice The physiological consequences of heterozygous deletion of A8+/- to bone were evaluated in mice (8 and 15 weeks ;6 males, 6 females), compared to A8+/+ mice).
  • Tibiae, and femurs were dissected free of soft tissue and prior to three-point bending tests using an MTS 858 Bionix Testing Machine (MTS Systems Corporation, Minneapolis, Minn.) at 2 rnm/min until failure.
  • Force displacement data will be generated using MTS 858 Bionix machine (MTS Systems) to calculate the three-point bending used at 2 mm/min until failure occurs. Samples were tested while immersed in normal saline at room temperature, with a support span of 10 mm.
  • n 12 each age and each type immersed in 22% potassium hydroxide were placed on a shaker at room temperature for 1 hr. Muscle and soft tissue were removed in 15% potassium hydroxide for 1.5 hrs and mice washed in distilled water. For de-fatting, skeletons were immersed in a solution of 10% xylene, 45% chloroform, 45% EtOH for 10 min then fixed in 95% ethanol for 5 min to dry the bone. The dry weight of the bone was recorded.
  • ISH In situ hybridization
  • ISH performed essentially as described in detail elsewhere (Zreiqat et ah, 1996) was used to detect S100A8 and A9 mRNA.
  • cDNA sequences were checked using the Genebank Database using the Blast-Align program
  • GPDH glyceraldehyde-3 -phosphate dehydrogenase
  • Tissue sections (4 ⁇ m thick) on chrome-gelatin coated slides were de-paraffinized, 100 ⁇ L photobiotin-labeled cDNA plasmid added and slides placed in a humidified chamber for 22 h at 42 0 C before post-hybridization stringency washing as described (Zreiqat et ah, 1996).
  • Anti-CD3 T-cell marker; Sigma
  • anti-CD68 monocyte/macrophage marker
  • anti-von-Willebrand factor EC marker
  • fascin dendritic cell (DC) marker
  • Primary antibodies were biotinylated equine antimouse, caprine anti-rabbit, and rabbit anti-caprine (Vector Laboratories, Burlingame, CA) IgGs.
  • primary antibodies were omitted, or sections treated with pre-/nonimmune rabbit IgG. Control sections were negative. Immunostaining in sections stained with Alizarin red S, which identifies atherosclerotic calcification, was difficult, so calcified deposits were visualized by counterstaining with Mayer's hematoxylin.
  • Binding affinity of S100A8 and S100A9 proteins to hydroxy apatite Recombinant SlOO protein was prepared as described (Iismaa et at, 1994). S100A8, S100A9 or a combination of both were resuspended in buffer solution (1 mM KPO4, 500 mM NaCl, pH 7.4) and incubated with 100 ⁇ l hydroxyapatite (Sigma) in a final volume of 1 ml at 37 0 C rocking incubator for 1 h in the presence or absence of 1 mM ZnSO 4 .
  • the supernatant was then separated from the hydroxyapatite by centrifugation at 4000 x g for 2 min and levels of protein concentration remained in the supernatant analysed by BCA assay (Sigma) and expressed as absorbance values at 562 nm.
  • Amount of SlOO protein bound to the hydroxyapatite was determined using anti- Si 00A8 or A9 IgG. Briefly, 10 ⁇ l of the hydroxyapatite was blocked with 0.5% skim milk (w/v in TBS), incubated with 10 ⁇ g/ml anti A8 or A9 IgG for 2 h at room temperature then captured with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Bio-Rad, Hercules, CA).
  • HRP horseradish peroxidase
  • ECL enhanced chemiluminescence
  • Dual-colour fluorescence-activated cell sorting was employed to determine the pattern of expression of the stromal precursor cell marker STRO-I and the osteoblastic marker ALP on cultured BMSSC (using alkaline phosphatase antibody B4-78).
  • RNA isolated directly from BMSSC and freshly isolated chondrocytes liberated from human cartilage following collagenase digestion was used as a control.
  • S100A8 is expressed in BMSSC but not the chondrocytes. It appears that S100A8 is down regulated in cultured chondrocytes.
  • the BMSSC population demonstrated a heterogeneous but highly reproducible pattern of S100A8 expression following subdivision by FACS based on STRO-1/ALP expression (Fig. IB).
  • the subfractions Rl to R4 show different expression patterns for STRO-I and ALP as follows: Rl STRO-1 + /ALF, R2 STRO-1 + /ALP + , R3 STRO-17ALP " , R4 STRO-IVALP + .
  • the most immature pre-osteogenic stromal population (Rl; STRO- I + / ALP " , 31%) was found to express high levels of S100A8 in contrast to the more committed pre-osteoblastic cell fraction (R2; STRO-1 + /ALP + , 10%).
  • Fig. 1C represents semiquantative RT-PCR on each STRO-1/ALP subfraction based on S 100A8: ⁇ -actin ratio.
  • STRO- 1 + /ALP ' cells are the most primitive followed by STRO " 1 + /ALP + then STRO-IVALP + and STRO-IVALP " .
  • Fig. IA BMSSC were positive for S100A8 expression. Immunohistochemistry confirmed that S100A8 is expressed in pre-osteogenic cells in mouse tissue (Fig. 2A) and human tissue (Fig. 2C). In contrast, S100A9 was not detected in RT-PCR analysis of BMSSC (Fig. IA) nor by immunohistochemistry in mouse pre- osteogenic cells (Fig. 2B).
  • S100A8 was found to be expressed in areas of differentiating hypertrophic chondrocytes (zone of maturing cartilage) (Fig3A), while S100A9 was absent in this zone (Fig. 3B). In contrast, both S100A8 and S100A9 were present in the zone of calcifying cartilage. This is indicated in Fig. 3C for S100A8. (Data not shown for S100A9).
  • Fig. 3D shows staining of the calcified areas of bone (in black) using Von Kossa staining.
  • S100A8 was also found to be expressed in human hypertrophic chondrocytes (data not shown).
  • S100A8 and S100A9 were also found to be expressed in articular chondrocytes (Fig. 3E for S100A8; data not shown for S100A9).
  • c Expression ofSJOOA ⁇ and S100A9 in osteoclasts (OC):
  • S100A8 and S100A9 were both found to be strongly expressed in osteoclasts in vivo as determined by immunohistochemistry (see Fig..4 for S100A8; data not shown for S100A9). In view of the role of osteoclasts in bone resorbtion, these findings indicate a role for S100A8 and S100A9 in the remodeling process during bone development. The expression of S100A8 and S100A9 by osteoclasts in vitro was also determined. Osteoclasts were generated in vitro from normal human blood monocytes. Human monocytes isolated from mononuclear cells from Red Cross donor buffy coats by differential centrifugation were allowed to adhere to the discs for 1 hour.
  • the population attached to the tissue culture plates consists of >96% monocytes.
  • Monocytes were cultured on tissue culture plates for 7 days in DMEM supplemented with 10% FCS 5 25ng/ml M-CSF, 10 "7 M dexamethasome and 10 "8 M vitamin D3.
  • 50 ⁇ g of recombinant RANKL, TNFa and IL-IB were added on day 7 and culture continued until day 21.
  • Mature osteoclasts are routinely observed to form by 11-12 days and these give rise to hundreds of resorption pits on dentine by day 14.
  • RANKL is an osteoclast differentiating factor.
  • IL-l ⁇ , TNF- ⁇ and other cytokines upregulate RANK-RANKL interaction that results in enhanced formation of mature osteoclasts. Further, TNF- ⁇ may stimulate osteoclast formation in the absence of RANKL activity.
  • S100A9 (Fig. 5A) and S100A8 (Fig. 5B) were expressed in the preosteoclastic cells. Expression of S100A8 and S100A9 were determined relative to expression of HPRT (as a control).
  • Embryonic expression ofS100A8 and S100A9 in bone cells As shown in Fig. 6 S100A8 was found to be expressed at the mRNA level in human embryos in osteoblasts (Fig. 6A), presosteogenic cells (Fig. 6B) and in osteoclasts (Fig. 6C), indicating that S100A8 was produced by these cells rather than being deposited in these cells. Embryonic mouse tissue indicated further the expression of S100A8 and S100A9 by hypertrophic chondrocytes at the mRNA level (Fig. 7).
  • S100A8 +/" mice (8 weeks) exhibited small but significantly increased thickness compared to the S100A8 +/+ mice.
  • Differences in the cortical bone thickness indicate that there has been some disturbance to the normal process of bone remodelling.
  • Increased cortical bone may also indicate that the process involved in the formation of trabecular bone has been altered causing to a significant decline in the amount of trabecular bone and hence in crease in cortical bone diameter.
  • Dystrophic calcification is common in many inflammatory disorders but most prominent in athersclerosis and arterial calcification and in kidney diseases.
  • the inventors have now found high expression of S100A8 and S 1009 in human atheroma, particularly around areas of calcification. Some of these have the appearance of hypertrophic chondrocytes and others show that calcifying microvesicles are the precursors to arterial calcification.
  • the protein-adsorbed plates were inserted into a channel connecting two halves of an electrolytic cell, one compartment containing calcium buffer (165 mM NaCl, 10 mM HEPES, 2.5 mM CaCl 2 , ⁇ H7.4), the other phosphate buffer (165 mM NaCl, 10 mM HEPES, ImM NaH 2 PO 4 , ⁇ H7.4), the ionic concentrations and ionic strength of which were physiological.
  • the ions passed over the plate surface; buffers were changed regularly to maintain a constant ionic environment. Calcification experiments were conducted for 2 weeks with and without the addition of Zn 2+ .
  • the concentration of ZnCl 2 added was protein/ZnCl 2 is 1:10 molar ratio (w/w).
  • electrophoresis plates were washed with nano-pure water (17M) to remove residual deposited salts, air-dried and used for further characterization by scanning electron microscopy (SEM).
  • SEM scanning electron microscopy
  • the glass plates were sputter-coated with Au/Pd and observed using a Cambridge Stereoscan 120 SEM.
  • both S100A8 and S100A9 have the ability to influence and alter crystallization.
  • the number of mineral particles deposited on S100A9 appears greater those than deposited on S100A8.
  • the calcium phosphate deposited had unique characteristic morphologies; S100A9 generated a highly porous morphology without crystallinity, whereas A8 produced a more crystalline, plate-like morphology. Characteristics were similar in the presence of Zn 2+ .
  • the S100A8/A9 complex had a smoother surface morphology indicating finer deposits (Fig. HA and B).
  • the morphology of the apatite generated by the S100A8/A9 complex was markedly influenced by Zn 2+ (Fig. HB), apparently influencing protein assembly resulting in a more crystalline appearance.
  • HBDC Human bone-derived cells
  • ⁇ -MEM ⁇ -minimal essential media
  • HBDC HBDC were seeded into individual wells of 24-well plates in the full culture medium. S100A8, S100A9, or S100A8+S100A9 were added in triplicate to the plates at a final concentration of 1 ⁇ M per well and HBDC were left to grow for 3 and 7 days. On day 5 medium was changed and fresh proteins were prepared and added to the cells. At the predetermined time point lysates were collected from the cells stored at -8O 0 C until assay was conducted.
  • alkaline phosphatase was assayed by the use of an ALP determination kit (Diagnostic kit, Thermotrace, Australia) as the release of p- nitrophenol from ⁇ -nitrophenolphosphate, thereby providing an indicator of osteoblast differentiation. Briefly, after homogenization of the cell pellet in 100 ⁇ of Tris pH 8.0 and lysis by ultrasonification for 4 min, 10 /xl of the suspension were mixed with 100 ⁇ l of ⁇ -nitrophenolphosphate solution and incubated at 37 °C for 15 min. The reaction was stopped by addition of 1 ml 0.05N NaOH.
  • the colour change was measured spectrophotometrically at 410 nm (Hitachi U-2000 Spectrophotometer) and the amount of enzyme released by the cells was quantified by comparison with a standard curve.
  • the standard curve was made from the absorbance measurement of various known concentrations of p-nitrophenol standard solution (Diagnostic kit 104-LL, Sigma), which were measured in a similar manner as above. ALP levels were normalized to cellular numbers at the end of the experiment (expressed as ⁇ mol/min/ ⁇ g).

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Abstract

La présente invention a trait à un procédé visant à stimuler la formation de tissus minéralisés chez un vertébré, ledit procédé comprenant l’administration audit vertébré d’une quantité efficace d’un ou plusieurs des éléments suivants : un polypeptide S100A8 ; un polypeptide S100A9 ; un polynucléotide codant pour le polypeptide S100A8 lié de manière fonctionnelle à un promoteur ; ou un polynucléotide codant pour le polypeptide S100A9 lié de manière fonctionnelle à un promoteur. La présente invention concerne également un procédé visant à traiter ou à prévenir un trouble osseux chez un vertébré, ledit procédé comprenant l’administration audit vertébré d’une quantité efficace d’un ou plusieurs des éléments suivants : un polypeptide S100A8 ; un polypeptide S100A9 ; un polynucléotide codant pour le polypeptide S100A8 lié de manière fonctionnelle à un promoteur ; ou un polynucléotide codant pour le polypeptide S100A9 lié de manière fonctionnelle à un promoteur.
PCT/AU2005/001681 2004-11-01 2005-11-01 Utilisation de la calgranuline a et b pour la stimulation et l'inhibition de la formation de tissus mineralises WO2006047820A1 (fr)

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WO2014071128A1 (fr) * 2012-11-02 2014-05-08 The Brigham And Women's Hospital, Inc. Vésicules matricielles dérivées de macrophages : un autre mécanisme de microcalcification dans les plaques d'athérosclérose
US9623078B2 (en) 2012-10-25 2017-04-18 Genomix Co., Ltd. Method for treating cardiac infarction using HMGB1 fragment
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