WO2012151609A1 - Procédé de traitement et de prophylaxie de pathologies osseuses - Google Patents

Procédé de traitement et de prophylaxie de pathologies osseuses Download PDF

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WO2012151609A1
WO2012151609A1 PCT/AU2012/000448 AU2012000448W WO2012151609A1 WO 2012151609 A1 WO2012151609 A1 WO 2012151609A1 AU 2012000448 W AU2012000448 W AU 2012000448W WO 2012151609 A1 WO2012151609 A1 WO 2012151609A1
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glypican
gpc3
gpcl
bone
bmp
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PCT/AU2012/000448
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English (en)
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Barry Crampton Powell
Peter John Anderson
Prem Prakash DWIVEDI
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Women's And Children's Health Research Institute Inc
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Priority to AU2012253204A priority Critical patent/AU2012253204A1/en
Publication of WO2012151609A1 publication Critical patent/WO2012151609A1/fr

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    • 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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present disclosure relates generally to the treatment and prophylaxis of bone pathologies including the augmentation of bone tissue.
  • Osteogenesis is the process of laying down new bone material by osteoblasts. Normal healthy bone tissue generally results from one of two processes: osteogenesis, involving the direct laying down of bone into the mesenchyme and endochrondrial osteogenesis which uses cartilage as a precursor. The integrity of bone tissue relies on a variety of processes and factors including bone mineralization and bone morphogenetic factors.
  • Bone mineralization is essential for bone hardness and strength and involves production of calcium phosphate by bone-forming cells which is laid down within the bone's fibrous matrix or scaffolding. Adverse consequences occur when there is either excess or deficient bone mineralization.
  • Bone morphogenetic proteins are a group of cytokines which have an effect on the formation of bone and cartilage.
  • BMP2 Bone morphogenetic proteins
  • BMP2 4, 6, 7 and 9 have been shown to have roles in ossification and are included in the term "osteogenic BMPs" (He (2005) J Musculoskelet Neuronal Interact 5(%):363-366).
  • Dysregulation of BMP signaling can lead to a range of pathologies affecting the bone.
  • Glypicans are cell surface heparan sulfate proteoglycans. Loss of glypican function can lead to a variety of physiological phenotypes.
  • inactive GPC3 causes Simpson-Golabi Behmel syndrome, which is characterized by pre- and post-natal overgrowth, cleft palate, short broad nose, prognathism, widened nasal bridge and disproportionably large head.
  • GPC3 deficient mice also present with Polydactyly, a common phenotype seen in patients with craniosynostosis syndromes.
  • GPC3 has also been shown to bind FGF2 and overexpression of GPC3 suppressed FGF2-induced cell proliferation in hepatocytes (Midorikawa et al. (2003) Int. J. Cancer I03(4):455-465). The suggested GPC3 interacts in FGF signaling on osteoprogenitors, as FGFR mutations are the common cause of multiple craniosynostosis syndromes.
  • Bone pathologies including bone cancers, fractures, craniosynostosis, osteoporosis and other biochemical or structural deficiencies can cause severe impairment, loss of quality of life and premature death to affected subjects. Surgical or other physical intervention has been the major method of dealing with many of these pathologies. There is a need to be able to treat or prevent or assist in the repair of bone-associated disorders by medicinal intervention.
  • a glypican (ii) a glypican, a glypican functional homolog or a glypican agonist, which glypican inhibits signaling of an osteogenic BMP, in order to inhibit BMP signaling.
  • the glypican is selected from GPC1 and GPC3 and the osteogenic BMP is selected from BMP2, 4, 6, 7 and 9.
  • the cells may be in vivo in a subject such as an animal or mammal including a human.
  • the cells may be ex vivo, such as in in vitro cell culture.
  • a glypican antagonist is used to promote BMP-mediated signaling to thereby promote bone osteogenesis including bone mineralization.
  • the glypican, glypican functional homolog or glypican agonist is used to reduce excessive bone osteogenesis such as in the case where there is excessive bone mineralization.
  • An example of the latter is a dysplasia. This method may also be useful for treating suture-based cranial disorders such as craniosynostosis.
  • An example of a glypican antagonist is an antibody including a synthetic, recombinant or derivative of an antibody.
  • the antibody may also be a humanized antibody.
  • the antibody may be directed to the glypican to inhibit its activity or to prevent its interaction with an osteogenic BMP.
  • Small chemical or proteinaceous inhibitors of the glypican are also contemplated herein.
  • the antagonist is a genetic molecule which inhibits expression of a gene encoding a protein portion of the glypican.
  • a method for promoting bone osteogenesis by administering an effective amount of a glypican antagonist, which glypican inhibits signaling by an osteogenic BMP, for a time and under condition sufficient to reduce glypican inhibition of an osteogenic BMP.
  • the condition being treated is deficient bone mineralization such as resulting from a dysplasia.
  • a method for reducing excessive bone osteogenesis by administering a glypican, glypican functional homolog or glypican agonist which glypican inhibits signaling an osteogenic BMP. This method reduces, for example, excessive bone mineralization.
  • a glypican antagonist in the manufacture of a medicament for the treatment of prophylaxis of a bone pathology in a subject and a use of glypican, a glypican functional homolog or a glypican agonist in the manufacture of a medicament for the treatment of prophylaxis of a bone pathology in a subject.
  • an "osteogenic BMP" includes but is not limited to BMP2, 4, 6, 7 and 9.
  • Particular glypicans contemplated herein are GPC1 and GPC3.
  • Figure 1 is a graphical representation showing that GPCl and GPC3 inhibit BMP2 signaling in human coronal suture cells.
  • Figure 2 is a graphical representation showing that recombinant GPCl and GPC3 inhibit BMP2 signaling in human coronal suture cells.
  • Figure 3 is a graphical representation showing that recombinant GPCl and GPC3 act together to inhibit BMP2 signaling in human coronal suture cells.
  • Figure 4 is a graphical representation showing that GPCl and GPC3 inhibit BMP2-promoted mineralization in human coronal suture cells and that a combination of GPCl and GPC3 is more effective than either alone.
  • Figure 5 is a photographic representation showing that BMP2 interacts with GPCl and with GPC3 in vitro.
  • Figure 6 is a graphical representation showing that an immunoblockade with GPC3 antibodies increases BMP2 signaling in human coronal suture cells.
  • Figure 7 is a graphical representation showing that an immunoblockade with GPC 1 antibodies increases BMP2 signaling in human coronal suture cells.
  • the present disclosure teaches that certain glypicans inhibit osteogenic BMPs. Reduced BMP signaling leads to poor or reduced osteogenesis and a deficiency in bone mineralization. It is proposed herein to modulate BMP signaling by a selected glypican or its agonists or antagonists.
  • one aspect enabled herein is a method for modulating signaling by an osteogenic BMP in cells associated with bone formation, the method comprising administering to the cells an effective amount of glypican antagonist, which glypican inhibits signaling by an osteogenic BMP, in order to promote BMP signaling or the glypican, glypican functional homolog or glypican agonist in order to inhibit BMP signaling.
  • glypican which inhibits signaling by an osteogenic BMP includes, but is not limited to, GPCl and GPC3.
  • Reference to an "osteogenic BMP" means a BMP which is associated with or which facilitates ossification. Examples include BMP2, 4, 6, 7 and 9.
  • An aspect enabled herein is a method for modulating osteogenic BMP signaling in cells associated with bone formation, the method comprising administering to the cells an effective amount of a GPCl antagonist in order to promote BMP signaling or GPCl , a GPCl functional homolog or a GPCl agonist thereof in order to inhibit BMP signaling.
  • Another aspect enabled herein is a method for modulating osteogenic BMP signaling in cells associated with bone formation, the method comprising administering to the cells an effective amount of a GPC3 antagonist in order to promote BMP signaling or GPC3, a GPC3 functional homolog or a GPC3 agonist thereof in order to inhibit BMP signaling.
  • a GPCl or GPC3 antagonist is used to promote BMP-induced osteogenesis including the promotion of bone mineralization.
  • GPC1 or GPC3, a GPC1 or GPC3 functional homolog or a GPC1 or GPC3 agonist is used to control osteogenesis such as reducing excess bone mineralization.
  • the present disclosure contemplates the use of a glypican which inhibits osteogenic BMP signaling and its agonists and antagonists to ameliorate a bone pathology including bone cancers, bone resorption and repair, fractures, suture-based cranial abnormalities such as craniosynostosis, skeletal disorders, osteoporosis and mineralization abnormalities (including deficient or excessive bone mineralization).
  • a bone pathology includes a single bone pathology, as well as two or more bone pathologies
  • reference to “an aspect” includes reference to a single aspect or two or more aspects
  • reference to “the embodiment” includes single or multiple embodiments; and so on.
  • bone pathology includes any disorder or deficiency in the bone including but not limited to conditions of bone cancer, deficient or excessive bone mineralization or where bone repair is required such as following a fracture, green stick or bone chip, suture-based cranial disorders such as craniosynostosis, cytoskeletal disorders, osteoporosis or other biochemical or structural deficiencies.
  • bone pathology is not to be considered limiting to any one condition, disease or deficiency. One particular condition, however, is abnormal bone mineralization.
  • the term “bone pathology” also refers to a level of bone health.
  • GPC1 and GPC3 inhibit BMP2 and homomultimers and heteromultimers comprising same.
  • a wide variety of conditions which result in loss of bone mineral content is contemplated herein.
  • Subjects with such conditions may be identified through clinical diagnosis utilizing well known techniques.
  • Representative examples of diseases that may be treated included dysplasias, wherein there is abnormal growth or development of bone such as in achondroplasia, cleidocranial dysostosis, enchondromatosis, fibrous dysplasia, Gaucher's disease, hypophosphatemic rickets, Marian's syndrome, multiple hereditary exostoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis, sclerotic lesions, fractures, periodontal disease, pseudoarthrosis and pyogenic osteomyelitis.
  • Another condition contemplated herein includes bone cancer wherein there is abnormal growth of bone cells in bone or other tissue to which bone cells have metastasized.
  • Other conditions contemplated herein include a wide variety of causes of osteopenia (i.e. a condition that causes greater than one standard deviation of bone mineral content or density below peak skeletal mineral content at youth).
  • causes of osteopenia i.e. a condition that causes greater than one standard deviation of bone mineral content or density below peak skeletal mineral content at youth.
  • Representative examples of such conditions include those conditions caused by anemia, steroids; heparin, scurvy, malnutrition, calcium deficiency, idiopathic osteoporosis, congenital osteopenia or osteoporosis, transient regional osteoporosis and osteomalacia.
  • Craniosynostosis refers to the premature fusion of calvarial sutures.
  • the condition may arise from any number of conditions including non-syndromic craniosynostosis or Apert, Beare-Stevenson, Boston, Crouzon, Antley-Bixler, Pfeiffer, Saethre-Chotzen or Muenke syndrome. Over 100 syndromes are associated with craniosynostosis (see Muenke and Wilkie (2000) Craniosynostosis Syndromes 3:61 17- 6146).
  • bone tissue augmentation As indicated above, there may be situations when it is important to assist bone growth or to facilitate bone health maintenance. This is referred to herein as bone tissue augmentation.
  • pathology does not necessarily mean the treatment of a disease condition. Situations where bone augmentation may be useful in non-disease conditions is in the elderly, young infants, athletes and non-human animals such as horses.
  • bone growth may be promoted in subjects where it is sub-optimal; bone growth may be inhibited in subjects with excessive bone growth; and bone cancer growth can be inhibited.
  • An embodiment particularly enabled herein is a bone pathology associated with deficient or excess BMP-induced signaling.
  • an antagonist of a glypican which inhibits the BMP is administered.
  • the glypican, functional homolog or agonist thereof is administered.
  • a glypican includes purified, naturally occurring forms as well as synthetic forms thereof and forms having a recombinant protein portion.
  • a recombinant or synthetic form may or may not have the same level, extent or pattern of glycosylation or other post-translational modifications as a purified, naturally occurring form. Such forms are useful, for example, to generate antibodies or to screen for small molecule antagonists or agonists.
  • a "glypican agonist” includes any agent which enhances the level or activity of the glypican and/or enhances its interaction with a BMP. An agonist includes the glypican itself.
  • a "glypican antagonist” includes any agent which inhibits glypican activity or glypican-BMP interaction such as an anti-glypican antibody, a small chemical inhibitor of the glypican or a genetic molecule which reduces expression of the protein portion of the glypican.
  • a glypican-BMP complex may also be a useful therapeutic target.
  • BMP means an osteogenic BMP including, but not limited to, BMP2, 4, 6, 7 and 9 as well as monomeric, homodimeric, heterodimeric and other homo- or hetero-multimeric forms.
  • BMP2 may be in the form of a homodimer or in heterodimeric form with BMP7.
  • BMP4 may be a homodimer or a heterodimer with BMP7.
  • a glypican which inhibits BMP2 or 4 or 7 is proposed to inhibit a heterodimer comprising any of BMP2, 4 or 7.
  • GPC1 and GPC3 are used to specifically inhibit BMP2 or homodirrters or heterodimers comprising BMP2.
  • an aspect ' enabled herein is a method for modulating BMP2 signaling including homo- or hetero-multimeric forms comprising BMP2 in cells associated with bone formation, the method comprising administering to the cells an effective amount of a GPCl or GPC3 antagonist in order to promote BMP2 signaling or GPCl or GPC3, a GPCl or GPC3 functional homolog or a GPCl or GPC3 agonist in order to inhibit BMP2 signaling.
  • GPC3 functional homolog includes GPC 1.
  • Another aspect enabled herein is a method for modulating osteogenic BMP signaling in cells associated with bone formation, the method comprising administering to the cells an effective amount of GPCl antagonist in order to promote BMP signaling or GPCl, a GPCl functional homolog or a GPCl agonist thereof in order to inhibit BMP signaling.
  • GPCl functional homolog includes GPC3.
  • agent and like terms including “compound”, “chemical agent”, “pharmacologically active agent”, “medicament”, “active” and “drug” are used interchangeably herein to refer to a chemical compound that induces a desired effect of either inhibiting or promoting glypican function.
  • the terms also encompass pharmaceutically acceptable and pharmacologically active ingredients including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like.
  • agent agents, compounds, chemical agent” "pharmacologically active agent”, “medicament”, “active” and “drug”
  • this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.
  • the aforementioned compounds may specifically modulate the function, activity or level of glypican or its interaction with an osteogenic BMP.
  • the compound is a genetic molecule
  • it may be DNA, RNA, an antisense molecule, a sense molecule, double stranded or single stranded RNA or DNA, short interfering RNA (siRNA), RNA interference (RNAi) or a complex of a nucleic acid and a ribonuclease all of which specifically target a genetic locus or mRNA transcript encoding a protein portion of a glypican.
  • siRNA short interfering RNA
  • RNAi RNA interference
  • Another form of agent binds to the osteogenic BMP thereby preventing the glypican from interacting with the BMP.
  • references to a an "agent”, “compound”, “chemical agent”, “pharmacologically active agent”, “medicament”, “active” and “drug” includes combinations of two or more active agents.
  • a “combination” also includes multi-part such as a two-part composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation.
  • a multi-part pharmaceutical pack may have two or more agents separately maintained.
  • the terms "effective amount” and "therapeutically effective amount” of an agent as used herein mean a sufficient amount of the glypican antagonist or agonist to provide the desired therapeutic or physiological effect or outcome associated with modulating BMP signaling. Undesirable effects, e.g. side-effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount”.
  • the exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact "effective amount”. However, an appropriate "effective amount” in any individual case may be determined by one of ordinary skill in the art using only routine experimentation.
  • the glypican or its antagonist or agonist is provided to an approximately 80kg subject at a dose of from about lOng to 2000mg or the equivalent dose for lighter or heavier subjects. Other dosages may also be used outside this range.
  • the "effective amount" of glypican antagonist or agonist also includes an amount to regulate BMP-induced osteogenesis.
  • a pharmaceutically acceptable carrier excipient or diluent
  • a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction.
  • Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives and the like.
  • a "pharmacologically acceptable" salt, ester, emide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable.
  • Treating" a subject may involve prevention of a condition or other adverse physiological event in a susceptible individual as well as treatment of a clinically symptomatic individual by ameliorating the symptoms of the condition.
  • the condition is associated with BMP signaling in relation to ossification.
  • prophylaxis may also be used.
  • a "subject" as used herein refers to an animal, generally a mammal and more particularly a human who can benefit from the pharmaceutical formulations and methods taught herein. There is no limitation on the type of animal that could benefit from the presently described pharmaceutical formulations and methods. A subject regardless of whether a human or non-human animal may also be referred to as an individual, patient, animal, host or recipient. The compounds and methods enabled herein have applications in human medicine, veterinary medicine as well as in general, domestic or wild animal husbandry.
  • antibody and in particular an antibody to a particular glypican such as GPC1 or GPC3, as used herein, includes various forms of modified or altered antibodies, such as an intact immunoglobulin, an Fv fragment containing only the light and heavy chain variable regions, an Fv fragment linked by a disulfide bond (Brinkmann et al, ( 1993) Proc. Natl Acad. Sci USA, 90:547-551 ), an Fab or (Fab)'2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody and the like (Bird et al. (1988) Science 242:424-426; Huston et al. (1988) Proc. Nat. Acad. Sci.
  • the antibody may be of animal (especially mouse, rat, sheep or goat) or human origin or may be chimeric (Morrison et al. (1984) Proc. Nat. Acad. Sci. USA, 57:6851-6855) or humanized (Jones et al (1986), Nature 527:522-525).
  • the instant disclosure also encompasses IGNARs (immunoglobulin new antigen receptors).
  • IgNAR is an antibody isotype found only in cartilaginous marine animals (sharks and rays) [Greenberg et al. (1995) Nature 574:168-173; Nuttall et al. (2001) Mol Immunol 55:313-326].
  • the IgNAR response is antigen-driven in the shark, and both immune and naive molecular libraries of IgNAR variable domains have been constructed and successfully screened for antigen-specific binding reagents (Greenberg et al. (1995) supra; Nuttall et al. (2001) supra).
  • IgNAR's are bivalent, but target antigen through a single immunoglobulin variable domain ( ⁇ 14kDa) displaying two complementarity determining region (CDR) loops attached to varying numbers of constant domains (Nuttall et al. (2003) Eur J Biochem 270:3543-3554; Roux et al. (1998) Proc Natl Acad Sci USA 95:11804-11809).
  • CDR complementarity determining region
  • nucleic acid or “oligonucleotide” or grammatical equivalents herein refer to at least two nucleotides covalently linked together and which modulate expression of a gene or allele or locus encoding a protein portion of a glypican.
  • a nucleic acid of the present disclosure is single-stranded or double stranded and will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925 and references therein; Letsinger (1970) J. Org. Chem.
  • nucleic acids include those with positive backbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA, 92:6097); non-ionic backbones (US Patent Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141 and 4,469,863; Angew (1991) Chem. Intl. Ed. English 50:423; Letsinger et al. (1988) supra; Letsinger et al. (1994) Nucleoside & Nucleotide 75:1597; Chapters 2 and 3, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed.
  • test agent refers to an agent that is to be screened in one or more of the assays to identify a glypican antagonist or agonist.
  • the agent can be virtually any chemical including proteinaceous compound. It can exist as a single isolated compound or can be a member of a chemical (e.g. combinatorial) library. In a particular preferred embodiment, the test agent is be a small organic molecule.
  • agent may be replaced with “compound”, “molecule”, “medicament” and the like as listed above.
  • a “gene” includes a genomic gene, allele, locus or a cDNA molecule which encodes a protein portion of a glypican.
  • the terms “gene”, “cDNA”, “nucleic acid molecule” and “nucleotide sequence” may be used interchangeably.
  • a “nucleic acid molecule” may be RNA or DNA.
  • An aspect enabled herein is a method for the treatment or prophylaxis of a bone pathology or reducing the risk of development of a bone pathology, the bone pathology associated with signaling by an osteogenic BMP, in a subject, the method comprising administering io the subject an effective amount of a glypican antagonist, which glypican inhibits BMP signaling, to enhance BMP signaling or the glypican or glypican functional homolog or agonist thereof to inhibit BMP signaling.
  • a glypican antagonist which glypican inhibits BMP signaling
  • Another aspect taught herein is a method for promoting bone growth or health in a subject, the method comprising administering to the subject an effective amount of a glypican antagonist, which glypican inhibits signaling by an osteogenic BMP, which enhances BMP-induced osteogenesis.
  • a method for reducing excess bone mineralization in a subject the method comprising administering to the subject an effective amount of a glypican or glypican functional homolog or an agonist thereof, which glypican inhibits signaling by an osteogenic BMP, to inhibit BMP-induced mineralization.
  • a particular aspect enabled herein is a method for the treatment or prophylaxis of a bone pathology or reducing the risk of development of a bone pathology, the bone pathology associated with osteogenic BMP signaling, in a subject, the method comprising administering to the subject an effective amount of a GPCl or GPC3 antagonist to enhance BMP signaling or GPCl or GPC3 or a GPCl or GPC3 functional homolog or agonist thereof to inhibit BMP signaling.
  • the BMP is BMP2.
  • Another aspect taught herein is a method for promoting bone growth or health in a subject, the method comprising administering to the subject an effective amount of a GPC1 or GPC3 antagonist which enhances BMP-induced osteogenesis.
  • Yet another aspect taught herein is a method for reducing excess bone mineralization in a subject, the method comprising administering to the subject an effective amount of GPC1 or GPC3 or a GPC1 or GPC3 functional homolog or an agonist thereof to inhibit BMP induced mineralization.
  • a method for the treatment or prophylaxis of a bone pathology or reducing the risk of development of a bone pathology, the bone pathology associated with BMP2 signaling, in a subject, the method comprising administering to the subject an effective amount of a GPC1 or GPC3 antagonist to enhance BMP2 signaling or GPC1 or GPC3 or a GPC1 or GPC3 functional homolog or agonist thereof to inhibit BMP2 signaling.
  • Another aspect taught herein is a method for promoting bone growth or health in a subject, the method comprising administering to the subject an effective amount of a GPC1 or GPC3 antagonist which enhances BMP2-induced osteogenesis.
  • Yet another aspect taught herein is a method for reducing excess bone mineralization in a subject, the method comprising administering to the subject an effective amount of GPC1 or GPC3 or a GPC1 or GPC3 functional homolog or an agonist thereof to inhibit BMP2-induced mineralization.
  • the BMP may be in homomultimeric or heteromultimeric form.
  • the instant disclosure teaches the inhibition of a glypican which inhibits signaling by an osteogenic BMP in order to treat or prevent a bone pathology' or to augment bone tissue.
  • a bone pathology results from or is exacerbated by deficient bone mineralization.
  • a GPC 1 or GPC3 antagonist is used to enhance BMP2 signaling.
  • the use of the glypican itself or an agonist thereof to dampen or otherwise reduce osteogenic BMP signaling is where a bone pathology results from or is exacerbated by excess bone mineralization.
  • a genetic construct comprising a nucleic acid molecule which encodes the protein portion of a glypican, which glypican inhibits osteogenic BMP signaling, to elevate glypican levels or a nucleic acid molecule which inhibits expression of the protein portion of the glypican (such as an antisense molecule, a molecule which induces sense suppression, a double stranded R A molecule, ribozyme, etc).
  • Diagnostic protocols include genetic and protein based assays aimed at determining the level of expression of the protein portion of a glypican or glypican activity or osteogenic BMP activity.
  • a method of screening for an agent which modulates the level or activity of the glypican includes contacting a test cell containing a gene- encoding the protein portion of the glypican with a test agent and detecting a change in the expression level or activity of the glypican in the test cell as compared to the expression or activity of glypican in a control cell where a difference in expression level of glypican protein or the activity of glypican in the test cell and the control cell indicates that the agent may modulate the symptoms of a bone pathology.
  • the bone pathology is one resulting from aberrant osteogenic BMP signaling.
  • control is a negative control cell contacted with the test agent at a lower concentration than the test cell.
  • expression level of the protein portion of the glypican is detected by measuring the level of the glypican mRNA in the cell and/or the level of glypican protein product is detected by determining the level of protein in the biological cell.
  • the pharmacophore Once the pharmacophore has been found, its structure is modeled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a . range of sources, e.g. spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process.
  • a . range of sources e.g. spectroscopic techniques, x-ray diffraction data and NMR.
  • Computational analysis, similarity mapping which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms
  • other techniques can be used in this modeling process.
  • Modeling can be used to generate agents which interact with the linear sequence or a three-dimensional configuration.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted onto it can conveniently be selected so that the therapeutic agent is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the agent is peptide-based
  • further stability can be achieved by cyclizing the peptide, increasing its rigidity.
  • the agents found by this approach can then be screened to see whether they have the target property, or to what extent they can modulate the activity of a target protein or modulation expression of a target gene.
  • Agents are also contemplated by the present disclosure which regulate expression of a gene encoding the protein portion of a glypican. This could involve, inter alia, providing gene function to a cell such as in gene therapy, or, it could involve inhibiting gene function Using gene silencing constructs including antisense oligonucleotides or expression constructs.
  • a target nucleic acid sequence or a part of a nucleic acid sequence such as a nucleic acid sequence capable of regulating nucleic acid expression may be introduced into a cell in a vector such that the nucleic acid sequence remains extrachromosomal. In such a situation, the nucleic acid sequence will be expressed by the cell from the extrachromosomal location.
  • Vectors for introduction of a nucleic acid sequence both for recombination and for extrachromosomal maintenance are known in the art and any suitable vector may be used.
  • Methods for introducing nucleic acids into cells such as electroporation, calcium phosphate co-precipitation and viral transduction are known in the art.
  • viruses have been used as nucleic acid transfer vectors or as the basis for preparing nucleic acid transfer vectors, including papovaviruses (e.g. SV40, Madzak et al. (1992) J Gen Virol 75: 1533-1536), adenovirus (Berkner (1992) Curr Top Microbiol Immunol 755:39-66; Berkner et al. (1988) BioTechniques (5:616-629; Gorziglia and Kapikian (1992) J Virol 66:4407-4412; Quantin et al. (1992) Proc Natl Acad Sci USA 59:2581-2584; Rosenfeld et al.
  • papovaviruses e.g. SV40, Madzak et al. (1992) J Gen Virol 75: 1533-1536
  • adenovirus e.g. SV40, Madzak et al. (1992) Curr Top Microbiol Immunol 755:39-66;
  • herpesviruses including HSV and EBV (Margolskee (1992) Curr Top Microbiol Immunol 158:67-95; Johnson et al. (1992) J Virol (56:2952-2965; Fink et al. (1992) Hum Gene Ther 3:1-19; Breakefield and Geller (1987) Mol Neurobiol 7:339-371 ; Freese et al. (1990) Biochem Pharmaco. 40:2189-2199; Fink et al. (1996) Ann Rev Neurosci 79:265- 287), lentiviruses (Naldini et al.
  • Non- viral nucleic acid transfer methods are known in the art such as chemical techniques including calcium phosphate co-precipitation, mechanical techniques, for example, microinjection, membrane fusion-mediated transfer via liposomes and direct DNA uptake and receptor-mediated DNA transfer.
  • Viral-mediated nucleic acid transfer can be combined with direct in vivo nucleic acid transfer using liposome delivery, allowing one to direct the viral vectors to particular cells.
  • the retroviral vector producer cell line can be injected into particular tissue. Injection of producer cells would then provide a continuous source of vector particles.
  • the present disclosure further contemplates the introduction of antisense and sense molecules such as polynucleotide sequences, which are useful in silencing transcripts of the glypican protein gene. Ribozymes, micro RNAs, synthetic RNAi, DNA-derived RNAi as well as double stranded RNAs may also be introduced. Both pre-transcriptional and post-transcriptional. gene silencing is contemplated including antisense silencing. Furthermore, polynucleotide vectors containing all or a portion of a glypican protein gene locus may be placed under the control of a promoter in an antisense or sense orientation and introduced into a cell. Expression of such an antisense or sense construct within a cell interferes with target transcription and/or translation.
  • antisense and sense molecules such as polynucleotide sequences, which are useful in silencing transcripts of the glypican protein gene. Ribozymes, micro RNAs, synthetic RNAi, DNA-derived RNA
  • the engineered genetic molecules encode oligonucleotides and similar species for use in modulating the expression of the glypican protein, i.e. the oligonucleotides induce pre-transcriptional or post-transcriptional gene silencing.
  • the constructs may encode inter alia micro RNA, dsRNA, hairpin RNAs, RNAi, siRNA or DNA.
  • target gene is used for convenience to encompass DNA encoding the target gene product, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA.
  • a method for treatment or prophylaxis of diseases or conditions characterized by being or causing a bone pathology associated with the level or extent of osteogenic BMP signaling comprising administering to a subject an agent capable of regulating glypican protein expression or activity or enhancing glypican protein expression or activity.
  • This method includes promoting bone growth or overall health.
  • the agents taught herein may be combined with one or more pharmaceutically acceptable carriers and/or diluents to form a pharmacological composition.
  • Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts to, e.g., stabilize, or increase or decrease the absorption or clearance rates of the pharmaceutical compositions of the disclosure.
  • Physiologically acceptable compounds can include, e.g., carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the peptides or polypeptides, or excipients or other stabilizers and/or buffers.
  • Detergents can also be used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers.
  • physiologically acceptable carriers and formulations for peptides and polypeptide are known to the skilled artisan and are described in detail in the scientific and patent literature, see e.g., Remington's Pharmaceutical Sciences (1990) 18 th Edition, Mack Publishing Company, Easton, PA ("Remington's").
  • Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms.
  • Various preservatives are well known and include, e.g., phenol and ascorbic acid.
  • a pharmaceutically acceptable carrier including a physiologically acceptable compound depends, for example, on the route of administration of the modulatory agent of the disclosure and on its particular physio-chemical characteristics.
  • Administration of the agent, in the form of a pharmaceutical composition may be performed by any convenient means known to one skilled in the art. Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeal ⁇ , intravenously, intraperitoneal ly,. subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly, intrathecally, intracereberally, intranasally, orally, rectally, patch and implant. A slow or sustained release product is also contemplated herein.
  • the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
  • the active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier, see, e.g, International Patent Publication Number WO 96/11698.
  • Agents of the present disclosure when administered orally, may be protected from digestion. This can be accomplished either by complexing the agent with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the agent in an appropriately resistant carrier such as a liposome.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as ⁇ bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the agents in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the agent may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension.
  • suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
  • the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
  • the agents When the agents are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
  • penetrants appropriate to the barrier to be permeated can be used for delivering the agent.
  • penetrants are generally known in the art e.g. for transmucosal administration, bile salts and fusidic acid derivatives/ In addition, detergents can be used to facilitate permeation.
  • Transmucosal administration can be through nasal sprays or using suppositories e.g. Sayani and Chien (1996) Crit Rev Ther Drug Carrier Syst 73:85-184.
  • the agents are formulated into ointments, creams, salves, powders and gels.
  • Transdermal delivery systems can also include patches.
  • the agents of the disclosure can be delivered using any system known in the art, including dry powder aerosols, liquids delivery systems, air jet nebulizers, propellant systems, and the like, see, e.g., Patton (1998) Nat Biotech 76:141- 143; product and inhalation delivery systems for polypeptide macromolecules by, e.g., Dura Pharmaceuticals (San Diego, CA), Aradigm (Hayward, CA), Aerogen (Santa Clara, CA), Inhale Therapeutic Systems (San Carlos, CA), and the like.
  • the pharmaceutical formulation can be administered in the form of an aerosol or mist.
  • the formulation can be supplied in finely divided form along with a surfactant and propellant.
  • the device for delivering the formulation to respiratory tissue is an inhaler in which the formulation vaporizes.
  • Other liquid delivery systems include, for example, air jet nebulizers.
  • agents contemplated herein can also be administered in sustained delivery or sustained release mechanisms, which can deliver the formulation internally.
  • sustained delivery or sustained release mechanisms which can deliver the formulation internally.
  • biodegradable microspheres or capsules or other biodegradable polymer configurations capable of sustained delivery of an agent can be included in the formulations of the instant disclosure (e.g. Putney and Burke (1998) Nat Biotech 16: 153-157).
  • the pharmaceutical formulations comprising agents contemplated herein are incorporated in lipid monolayers or bilayers such as liposomes, see, e.g., US Patent Nos 6,1 10,490; 6,096,716; 5,283,185 and 5,279,833.
  • the disclosure also teaches formulations in which water-soluble modulatory agents of the disclosure have been attached to the surface of the monolayer or bilayer.
  • peptides can be attached to hydrazide-PEG-(distearoylphosphatidyl) ethanolamine-containing liposomes (e.g. Zalipsky et al. (1995) Bioconjug Chem 6:705-708).
  • Liposomes or any form of lipid membrane such as planar lipid membranes or the cell membrane of an intact cell e.g. a red blood cell, can be used.
  • Liposomal formulations can be by any means,, including administration intravenously, transdermally (Vutla et al. (1996) J Pharm Sci #5:5-8), transmucosally or orally.
  • the specification also provides pharmaceutical preparations in which the agents of the disclosure are incorporated within micelles and/or liposomes (Suntres and Shek (1994) J Pharm Pharmacol 46:23-2%; Woodle et al. (1992) Pharm Res 9:260-265).
  • Liposomes and liposomal formulations can be prepared according to standard methods and are also well known in the art see, e.g., Remington (1990) supra; Akimaru et al. (1995) Cytokines Mol Ther 7: 197-210; Alving et al. (1995) Immunol Rev 74.5:5-31 ; Szoka and Papahadjopoulos (1980) Ann Rev Biophys Bioeng P:467-508, US Patent Nos. 4, 235,871, 4,501,728 and 4,837,028. [0106]
  • the pharmaceutical compositions taught herein can be administered in a variety of unit dosage forms depending upon the method of administration. Dosages for typical pharmaceutical compositions are well known to those of skill in the art.
  • Such dosages are typically advisorial in nature and are adjusted depending on the particular therapeutic context, patient tolerance, etc.
  • the amount of agent adequate to accomplish this is defined as the "effective amount”.
  • the dosage schedule and effective amounts for this use i.e. the "dosing regimen” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, pharmaceutical formulation and concentration of active agent, and the like.
  • the mode of administration also is taken into consideration.
  • the dosage regimen must also take into consideration the pharmacokinetics, i.e. the pharmaceutical composition's rate of absorption, bioavailability, metabolism, clearance, and the like. See, e.g., Remington (1990) supra; Egleton and Davis (1997) Peptides 75: 1431 -1439; Langer (1990) Science 249: 1527-1533.
  • the agents and/or pharmaceutical compositions defined and taught herein may be co-administered with one or more other agents.
  • Reference herein to "co-administered” means simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes.
  • Reference herein to "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of agents and/or pharmaceutical compositions. Coadministration of the agents and/or pharmaceutical compositions may occur in any order.
  • diagnostic and/or prognostic assays are contemplated herein as well as reagents useful for identifying the presence of a disease or condition, or the propensity to develop a disease or condition, or to determine the severity of a disease or condition or the stage of progression of a disease or condition, such disease and conditions are characterized by being a bone pathology associated with BMP signaling. These include deficient or excess bone mineralization.
  • a method of diagnosing or predicting the development of a bone pathology in a subject comprising isolating a sample from a potentially affected bone or bone tissue from the subject, the sample comprising genetic material encoding the protein portion of a glypican, which glypican inhibits signaling by an osteogenic BMP or a protein or BMP protein and determining the pattern of expression of the genetic material or protein wherein an up-regulation of glypican expression or protein activity or a reduction in BMP activity is indicative of deficient BMP signaling and a reduction of glypican expression or protein activity or enhanced BMP2 activity is indicative of excessive BMP signaling.
  • the levels are compared to normal control levels or statistically determined normal levels.
  • the disclosure extends to antibodies or their antigen binding fragments specific for a particular glypican such as GPC1 or GPC3.
  • Antibodies may be polyclonal or monoclonal.
  • Polyclonal antibodies to the glypican can be prepared using methods well-known to those of skill in the art (see, for example, Green et al. (1992) Immunochemical Protocols (Manson ed) ⁇ -5 Williams et al. (1995) DNA Cloning 2: Expression Systems, 2 nd Ed., Oxford University Press). Although polyclonal antibodies are typically raised in animals such as rats, mice, rabbits, goats, or sheep, a target polypeptide antibody of the present disclosure may also be derived from a subhuman primate antibody. General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al, International Patent Publication No.
  • variable region domain may be of any size or amino acid composition and will generally comprise at least one hypervariable amino acid sequence responsible for antigen binding embedded in a framework sequence.
  • variable (V) region domain may be any suitable arrangement of immunoglobulin heavy (VH) and/or light (VL) chain variable domains.
  • VH immunoglobulin heavy
  • VL light chain variable domains.
  • the V region domain may be monomeric and be a VH or V L domain where these are capable of independently binding antigen with acceptable affinity.
  • V region domain may be dimeric and contain VH-VH, VH-VL, or VL-VL, dimers in which the VH and VL chains are non-covalently associated (abbreviated hereinafter as F v ).
  • the chains may be covalently coupled either directly, for example via a disulphide bond between the two variable domains, or through a linker, for example a peptide linker, to form a single chain domain (abbreviated herein after as scF v ).
  • variable region domain may be any naturally occurring variable domain or an engineered version thereof.
  • engineered version is meant a variable region domain that has been created using recombinant DNA engineering techniques.
  • engineered versions include those created for example from natural antibody variable regions by insertions, deletions or changes in or to the amino acid sequences of the natural antibodies.
  • Particular examples of this type include those engineered variable region domains containing at least one CDR and optionally one or more framework amino acids from antibody and the remainder of the variable region domain from a second antibody.
  • the variable region domain may be covalently attached at a ' C -terminal amino acid to at least one other antibody domain or a fragment thereof.
  • variable region domain may be linked to an immunoglobulin CH I domain or a fragment thereof.
  • VL domain may be linked to a C domain or a fragment thereof.
  • the antibody may be a Fab fragment wherein the antigen binding domain contains associated VH and VL domains covalently linked at their C-termini to a CHI and CK domain respectively.
  • the CH I domain may be extended with further amino acids, for example to provide a hinge region domain as found in a Fab fragment, or to provide further domains, such as antibody CH2 and CH3 domains.
  • Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR).
  • CDR peptides can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA or antibody-producing cells (see, for example, Larrick et al. (1991) Methods: A Companion to Methods in Enzymology 2:106; Courtneay-Luck,(1995) Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al (eds), Cambridge University Press:166 and Ward et al. (1995) Monoclonal Antibodies: Principles and Applications Birch et al., Wiley-Liss, Inc.: 137.
  • Monoclonal antibodies are also contemplated herein which are specific for the glypican such as GPC1 or GPC3. Monoclonal antibodies can be generated utilizing a variety of techniques. Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler and Milstein (1975) Nature 256: 495-499 and Coligan et al. (1991 ) Current Protocols in Immunology 1, John Wiley & Sons; Picksley et al. (1995) DNA Cloning 2: Expression Systems, 2 nd Edition, Glover et al (eds), page 93 Oxford University Press).
  • Figure 1 shows that increased GPCl and GPC3 gene expression leads to inhibition of BMP2-induced signaling in human cranial cells.
  • Cells were co-transfected with a BMP- responsive luciferase reporter (pID183-Luc), a control luciferase vector and GPCl, GPC3 or green fluorescent protein (GFP) gene expression vectors as indicated and, 24 hours later, BMP2 was added at lOOng/ml. Cell extracts were assayed 24 hours later by Dual Luciferase assay.
  • * P ⁇ 0.05 compared to BMP2 and BMP2/GFP treatments. Error bars represent mean + SD.
  • N 3. All proteins were from R&D Systems.
  • FIG. 2 shows that increasing amounts of recombinant GPCl and GPC3 protein inhibit BMP2-induced signaling in human cranial cells.
  • Cells were co-transfected with a BMP-response luciferase reporter (pID183-Luc), a control luciferase vector and 24 hours later BMP2 as added at lOOng/ml and recombinant GPCl or GPC3 at concentrations shown (ng/ml) or albumin at 500ng/ml.
  • Cell extracts were assayed 24 hours later by Dual Luciferase assay.
  • * P ⁇ 0.05 compared to BMP2 and BMP2/albumin treatments. Error bars represent mean + SD.
  • N 3. All proteins were from R&D Systems.
  • Recombinant GPCl and GPC3 protein act together to inhibit BMP2 induced signaling
  • FIG. 3 shows that increasing amounts of recombinant GPCl and GPC3 protein inhibit BMP2-induced signaling in human cranial cells.
  • Cells were co-transfected with a BMP-response luciferase reporter (pID183-Luc), a control luciferase vector and 24 hours later BMP2 as added at lOOng/ml and recombinant GPCl or GPC3 at concentrations shown (ng/ml).
  • Cell extracts were assayed 24 hours later by Dual Luciferase assay.
  • * P ⁇ 0.05 compared to BMP2, BMP2 + 50ng/ml GPC3 and BMP2 + lOng/ml GPCl . Error bars represent mean + SD.
  • N 3. All proteins were from R&D Systems.
  • FIG. 4 shows that an approximately equimolar amount (i.e. similar number of molecules) of GPCl or GPC3 inhibits BMP2-mediated mineralization of human cranial suture cells and that combined addition of GPCl and GPC3 were more effective than either protein alone.
  • FIG. 5 shows that (A) GPCl and BMP2 physically interact with each other in vitro and that (B) GPC3 and BMP2 physically interact with each other in vitro.
  • Equimolar amounts of pairwise combinations of recombinant BMP2, GPCl , GPC3 or albumin were incubated in vitro, immunoprecipitated (IP) and Western blotted (WB) as shown.
  • Different GPCl and GPC3 antibodies (ab) were used for immunoprecipitation and western blot to enhance specificity.
  • the non-specific isotype antibodies did not immunoprecipitate GPCl, GPC3 or BMP2, indicating that the precipitations observed with the GPCl and GPC3 antibodies were specific.
  • Western blots with albumin antibodies were negative.
  • FIG. 6 shows that immunoblockade of GPC3 by specific antibodies targeted against GPC3 protein increases BMP2 signaling activity in human cranial cells.
  • Human coronal suture cells were transfected with BMP reporter. 24 hours later 2 ⁇ g, 25 ⁇ g or 5( ⁇ g/ml GPC3 antibody or 5( g/ml IgG isotype (non-specific antibody control) was added, followed 3 hours later by BMP2. Extracts assayed 24 hours later.
  • * P ⁇ 0.05 compared to indicated treatments. Error bars represent mean + SD.
  • N 3.
  • FIG. 7 shows that immunoblockade of GPC1 by specific antibodies targeted against GPC1 protein increases BMP2 signaling activity in human cranial cells.
  • Human coronal suture cells were transfected with BMP reporter. 24 hours later 5( ⁇ g/ml GPC1 antibody or 50 g/ml IgG isotype (non-specific antibody control) was added, followed 3 hours later by BMP2. Extracts assayed 24 hours later.
  • * P ⁇ 0.05 compared to indicated treatments. Error bars- represent mean + SD.
  • N 3

Abstract

La présente description concerne généralement le traitement et la prophylaxie de pathologies osseuses par modulation de la signalisation ostéogénique de la protéine morphogénétique osseuse (BMP) à l'aide de modulateurs glypicane.
PCT/AU2012/000448 2011-05-06 2012-04-30 Procédé de traitement et de prophylaxie de pathologies osseuses WO2012151609A1 (fr)

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US9862764B2 (en) 2014-12-19 2018-01-09 Novartis Ag Compositions and methods for antibodies targeting BMP6
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Publication number Priority date Publication date Assignee Title
JP2017504812A (ja) * 2014-01-17 2017-02-09 ミノミック インターナショナル リミティド 診断用の細胞表面前立腺癌抗原
US9862764B2 (en) 2014-12-19 2018-01-09 Novartis Ag Compositions and methods for antibodies targeting BMP6
US10683346B2 (en) 2014-12-19 2020-06-16 Novartis Ag Compositions and methods for antibodies targeting BMP6
US11530261B2 (en) 2014-12-19 2022-12-20 Novartis Ag Compositions and methods for antibodies targeting BMP6
US11434269B2 (en) 2016-06-15 2022-09-06 Novartis Ag Methods for treating disease using inhibitors of bone morphogenetic protein 6 (BMP6)

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