WO2012151609A1 - Method of treatment and prophylaxis of pathologies of the bone - Google Patents

Abstract

The present disclosure relates generally to the treatment and prophylaxis of bone pathologies by modulating osteogenic bone morphogenetic protein (BMP) signalling using glypican modulators.

Description

METHOD OF TREATMENT AND PROPHYLAXIS OF PATHOLOGIES OF THE BONE

FILING DATA

[0001] This application is associated with and claims priority from US Provisional Patent Application No. 61/483,512, filed on 6 May, 201 1, entitled "Method of treatment and prophylaxis of pathologies of the bone", the entire contents of which, are incorporated herein by reference.

FIELD

[0002] The present disclosure relates generally to the treatment and prophylaxis of bone pathologies including the augmentation of bone tissue.

BACKGROUND

[0003] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[0004] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country. [0005] Osteogenesis (or ossification) 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. [0006] 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.

[0007] Bone morphogenetic proteins (BMPs) are a group of cytokines which have an effect on the formation of bone and cartilage. Of the BMP molecules, 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.

[0008] Gene expression products associated with a bone pathology or healthy bone tissue were identified in International Patent Application No. PCT/AU2007/000055, the contents of which are incorporated herein by reference. One family of factors identified in the context of a suture-based cranial disorder is the glypican family which includes glypican 1 (GPC1) and glypican 3 (GPC3).

[0009] Glypicans are cell surface heparan sulfate proteoglycans. Loss of glypican function can lead to a variety of physiological phenotypes. For example, 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. [0010] 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.

SUMMARY

[0011] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any other element or integer or method step or group of elements or integers or method steps.

[0012] It must be noted that, as used in the subject specification, the singular forms "a"* "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a BMP" includes a single BMP, as well as two or more BMPs; reference to "an antagonist" includes a single antagonist, as well as two or more antagonists; reference to "the disclosure" includes a single reference to "the disclosure" includes a single and multiple aspects taught by the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term "invention". All such aspects are enabled within the width of the present invention.

[0013] A method is provided for modulating signaling by an osteogenic BMP in cells associated with bone formation by the administration to the cells of an effective amount of:

(i) an antagonist of a glypican, which glypican inhibits signaling of an osteogenic BMP, in order to promote BMP signaling; or

(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. [0014] In an embodiment, the glypican is selected from GPC1 and GPC3 and the osteogenic BMP is selected from BMP2, 4, 6, 7 and 9.

[0015] The cells may be in vivo in a subject such as an animal or mammal including a human. Alternatively, the cells may be ex vivo, such as in in vitro cell culture.

[0016] A glypican antagonist is used to promote BMP-mediated signaling to thereby promote bone osteogenesis including bone mineralization. Alternatively, 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.

[0017] 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. Alternatively, the antagonist is a genetic molecule which inhibits expression of a gene encoding a protein portion of the glypican.

[0018] A method is also provided herein 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. In an example, the condition being treated is deficient bone mineralization such as resulting from a dysplasia. [0019] Also taught herein is 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. [0020] Also provided herein is the use of 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. [0021] As indicated above, an "osteogenic BMP" includes but is not limited to BMP2, 4, 6, 7 and 9. Particular glypicans contemplated herein are GPC1 and GPC3. BRIEF DESCRIPTION OF THE FIGURES

[0022] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0023] Figure 1 is a graphical representation showing that GPCl and GPC3 inhibit BMP2 signaling in human coronal suture cells. [0024] Figure 2 is a graphical representation showing that recombinant GPCl and GPC3 inhibit BMP2 signaling in human coronal suture cells.

[0025] Figure 3 is a graphical representation showing that recombinant GPCl and GPC3 act together to inhibit BMP2 signaling in human coronal suture cells.

[0026] 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. [0027] Figure 5 is a photographic representation showing that BMP2 interacts with GPCl and with GPC3 in vitro.

[0028] Figure 6 is a graphical representation showing that an immunoblockade with GPC3 antibodies increases BMP2 signaling in human coronal suture cells.

[0029] Figure 7 is a graphical representation showing that an immunoblockade with GPC 1 antibodies increases BMP2 signaling in human coronal suture cells. DETAILED DESCRIPTION

[0030] 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.

[0031] Consequently, 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. [0032] Reference to a "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. [0033] 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. [0034] 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.

[0035] In an embodiment, a GPCl or GPC3 antagonist is used to promote BMP-induced osteogenesis including the promotion of bone mineralization.

[0036] In another embodiment, 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.

[0037] The present disclosure, therefore, 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).

[0038] As used herein, singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "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.

[0039] The term "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. The term "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. Hence, modulating the level of osteogenic BMP signaling via antagonism or agonism using a selected glypican is proposed to be useful in the treatment or prophylaxis of the bone pathology. For example, GPC1 and GPC3 inhibit BMP2 and homomultimers and heteromultimers comprising same.

[0040] 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. [0041] 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.

[0042] 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). 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.

[0043] The term "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).

[0044] 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. The term "pathology", therefore, 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. Furthermore, 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.

[0045] An embodiment particularly enabled herein is a bone pathology associated with deficient or excess BMP-induced signaling. Hence, where there is a deficiency in osteogenic BMP signaling, an antagonist of a glypican which inhibits the BMP is administered. Where there is an excess of BMP signaling, the glypican, functional homolog or agonist thereof is administered.

[0046] Reference to "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.

[0047] Reference to a "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. For example, 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. Hence, a glypican which inhibits BMP2 or 4 or 7 is proposed to inhibit a heterodimer comprising any of BMP2, 4 or 7.

[0048] In an embodiment, GPC1 and GPC3 are used to specifically inhibit BMP2 or homodirrters or heterodimers comprising BMP2.

[0049] Consequently, 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.

[0050] An example of a GPC3 functional homolog includes GPC 1.

[0051] 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.

[0052] An example of a GPCl functional homolog includes GPC3. [0053] The term "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. When the terms "agent", "compound", "chemical agent" "pharmacologically active agent", "medicament", "active" and "drug" are used, then it is to be understood that 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. Insofar as 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. Another form of agent binds to the osteogenic BMP thereby preventing the glypican from interacting with the BMP.

[0054] Reference 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. For example, a multi-part pharmaceutical pack may have two or more agents separately maintained.

[0055] 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. In an example, 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.

[0056] The "effective amount" of glypican antagonist or agonist also includes an amount to regulate BMP-induced osteogenesis.

[0057] By a "pharmaceutically acceptable" carrier, excipient or diluent is meant 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.

[0058] Similarly, 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.

[0059] "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. In promoting overall bone health, or preventing bone damage, the term "prophylaxis" may also be used.

[0060] 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.

' .

[0061] The term "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. USA, 55:5879-5883). 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).

[0062] The instant disclosure also encompasses IGNARs (immunoglobulin new antigen receptors). An 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). The small size, and thermodynamic and chemical stability of IgNAR variable domains (VNARS), offer distinct advantages over conventional antibodies. Furthermore, the small V_NAR size enables this antibody domain access to cryptic antigenic epitopes through unusually long and variable CDR3 loops (Greenber et al. (1995) supra; Ewert et al. (2002) Biochemistry 47:3628-2636; Nuttall et al. (2004) Proteins 55:187-197; Stanfield et al. (2004) Science 505:1770-1773; Streltsov et al. (2004) Proc Natl Acad Sci USA 101: 12444-12449; Streltsov et al. (2005) Protein Sci 74:2901-2909).

[0063] The terms "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. 55:3800; Sprinzl et al. (1977) Eur. J. Biochem. 57:579; Letsinger et al. (1986) Nucl. Acids Res. 74:3487; Sawai et al. (1 84) Chem. Lett. 805; Letsinger et al. (1988) J. Am. Chem. Soc. 770:4470 and Pauwels et al. (1986) Chemica Scripta 25: 1419), phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 79: 1437; US Patent No. 5,644,048) and phosphorodithioates (Briu et al. (1989) J. Chem. Soc. 777:2321), O- methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press).

[0064] Other analog 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. YS Sanghui and P Dan Cook; Mesmaeker et al. (1994) Bioorganic & Medicinal Chem. Lett. 4:395; Jeffs et al. (1994) J. Biomolecular NMR 34: 11; Tetrahedron (1996) Lett. 57:743) and non-ribose backbones, including those described in US Patent Nos. 5,235,033 and 5,034,506 and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. YS Sanghui and P Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (Jenkins et al. (1995) Chem. Soc. 7?ev. : 169-176). Several nucleic acid analogs are described in Rawls (1997) C & E News:35. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments. [0065] The term "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.

[0066] Again, the term "agent" may be replaced with "compound", "molecule", "medicament" and the like as listed above.

[0067] 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.

[0068] 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. [0069] 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. [0070] 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 a glypican or glypican functional homolog or an agonist thereof, which glypican inhibits signaling by an osteogenic BMP, to inhibit BMP-induced mineralization. [0071] 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. In an embodiment, the BMP is BMP2. [0072] 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. [0073] 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. [0074] In a further particular embodiment, a method is provided 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.

[0075] 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.

[0076] 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.

[0077] As indicated above, the BMP may be in homomultimeric or heteromultimeric form.

[0078] 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. An example is where a bone pathology results from or is exacerbated by deficient bone mineralization. In this case, for example, a GPC 1 or GPC3 antagonist is used to enhance BMP2 signaling. Also taught herein is the use of the glypican itself or an agonist thereof to dampen or otherwise reduce osteogenic BMP signaling. An example of the latter is where a bone pathology results from or is exacerbated by excess bone mineralization.

(0079] Also taught herein is 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).

[0080] The identification that particular glypicans inhibit BMP signaling further enables development of diagnostic protocols. 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.

[0081] In another embodiment, provided here is a method of screening for an agent which modulates the level or activity of the glypican. The method 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. In an embodiment, the control is a negative control cell contacted with the test agent at a lower concentration than the test cell. In an embodiment, the 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.

[0082] Taught herein, therefore, are therapeutic agents which interact with a gene or transcript which encodes the protein portion of a glypican or glypican protein or glypican- BMP complex. There are several steps commonly taken in the design of such therapeutic agents. First, the particular parts of glypican protein or gene critical for activity or expression are determined. In the case of a protein, for example, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. Alanine scans of proteins, for example, are commonly used to define such protein motifs. These parts or residues constituting the active region of the compound are known as its "pharmacophore". As indicated above, the terms "peptide", "polypeptide" or "protein" may be used interchangeably.

[0083] 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.

[0084] In a variant of this approach, the three-dimensional structure of the glypican protein is modeled. Modeling can be used to generate agents which interact with the linear sequence or a three-dimensional configuration.

[0085] 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. Alternatively, where 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. Further optimization or modification can then be carried out to arrive at one or more final agents for in vivo or clinical testing. [0086] The goal of rational drug design is to produce structural analogs of glypican which may act as antagonists, agonists, inhibitors or enhancers. (0087] 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.

[0088] 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.

[0089] In particular, a number of 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. (1992) Cell 65: 143-155; Wilkinson et al. (1992) Nucleic Acids Res 20:233-2239; Stratford-Perricaudet et al. (1990) Hum Gene Ther 7 :241- 256; Schneider et al (1998) Nat Genetics 75: 180-183), vaccinia virus (Moss (1992) Curr Top Microbiol Immunol 158: 5-38; Moss (1996) Proc Natl Acad Sci USA 95: 1 1341- 11348), adeno-associated virus (Muzyczka (1992) Curr Top Microbiol Immunol 158:91- 129; Ohi et al. (1990) Gene 59:279-282; Russell and Hirata (1998) Nat Genetics 75:323- 328), 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. (1996) Science 272:263-267), Sindbis and Semliki Forest virus (Berglund et al. (1993) Biotechnology :916-920) and retroviruses of avian (Bandyopadhyay and Temin (1984) Mol Cell Biol 4:749-754; Petropoulos et al. (1992) J Virol (56:3391-3397), murine (Miller (1992) Curr Top Microbiol Immunol 7JS.1 -24; Miller et al. (1985) Mol Cell Biol 5:431-437; Sorge et al. (1984) Mol Cell Biol 4: 1730-1737; Mann and Baltimore (1985) J Virol 54:401-407; Miller et al. (1988) J Virol 62:4337-4345) and human (Shimada et al. (1991) J Clin Invest 88: 1043- 1047; Helseth et al. ( 1990) J Virol 64:2416-2420; Page et al. (1990) J Virol 64:5270-52^"/ '6; Buchschacher and Panganiban ( 1982) J Virol 66:2731-2739) origin. (0090] 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. Alternatively, 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.

[0091] 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.

{0092] In one embodiment, 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. This is accomplished by providing oligonucleotides which specifically hybridize to or otherwise target one or more target nucleic acid molecules encoding the target gene product. Hence, the constructs may encode inter alia micro RNA, dsRNA, hairpin RNAs, RNAi, siRNA or DNA. As used herein, the term "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.

[0093] In another embodiment, a method is enabled herein 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. Pharmaceutically 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"). [0094] 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. One skilled in the art would appreciate that the choice of 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. [0095] 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.

[0096] For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, 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). Due to their ease in administration, 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. [0097] 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. Means of protecting compounds from digestion are well known in the art, see, e.g. Fix (1996) Pharm Res 75: 1760-1764; Samanen et al. (1996) J Pharm Pharmacol 48: 1 19-135; US Patent No. 5,391 ,377, describing lipid compositions for oral delivery of therapeutic agents. [0098] 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. In many cases, it will be preferable to include 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.

[0099] 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. Generally, 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. In the case of sterile powders for the preparation of sterile injectable solutions, 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.

[0100] For parenteral administration, the agent may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension. Illustrative of 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. When the agents are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.

[0101] For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used for delivering the agent. Such 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. For topical, transdermal administration, the agents are formulated into ointments, creams, salves, powders and gels. Transdermal delivery systems can also include patches.

[0102] For inhalation, 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. For example, the pharmaceutical formulation can be administered in the form of an aerosol or mist. For aerosol administration, the formulation can be supplied in finely divided form along with a surfactant and propellant. In another aspect, 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.

[0103] The agents contemplated herein can also be administered in sustained delivery or sustained release mechanisms, which can deliver the formulation internally. For example, 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).

[0104] In preparing pharmaceuticals contemplated herein, a variety of formulation modifications can be used and manipulated to alter pharmacokinetics and biodistribution. A number of methods for altering pharmacokinetics and biodistribution are known to one of ordinary skill in the art. Examples of such methods include protection of the compositions of the disclosure in vesicles composed of substances such as proteins, lipids (for example, liposomes), carbohydrates, or synthetic polymers. For a general discussion of pharmacokinetics, see, e.g., Remington (1990) supra.

[0105] In one aspect, 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. For example, 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. In cafculating the dosage regimen for a patient, 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.

[0107] In accordance with these methods, 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. [0108] As indicated above, the development of diagnostic and/or prognostic assays is 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.

[0109] Hence, taught herein is a method of diagnosing or predicting the development of a bone pathology in a subject, the method 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.

[0110] Generally, the levels are compared to normal control levels or statistically determined normal levels.

^• [0111] 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.

[0112] 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. WO 91/1 1465, 1991 and in Losman et al. (1990) Int. J. Cancer 46:310. [0113] The antibody should comprise at least a variable region domain. The 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. In general terms the variable (V) region domain may be any suitable arrangement of immunoglobulin heavy (VH) and/or light (VL) chain variable domains. Thus, for example, 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. Alternatively the 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). Where desired, however, 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).

[0114] The variable region domain may be any naturally occurring variable domain or an engineered version thereof. By engineered version is meant a variable region domain that has been created using recombinant DNA engineering techniques. Such 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. [0115] 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. Thus, for example, where a V_H domain is present in the variable region domain this may be linked to an immunoglobulin CH I domain or a fragment thereof. Similarly, a VL domain may be linked to a C domain or a fragment thereof. In this way for example, 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. [0116] Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") 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. [0117] 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).

[0118] Aspects taught herein are further described by the following non-limiting Examples. EXAMPLE 1

Inhibition of BMP2~induced signaling by GPCl and GPC3

[0119] 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.

EXAMPLE 2

Recombinant GPCl and GPC3 protein inhibit BMP2 induced signaling [0120] Figure 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.

EXAMPLE 3

Recombinant GPCl and GPC3 protein act together to inhibit BMP2 induced signaling

[0121] Figure 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.

EXAMPLE 4

GPCl and GPC3 inhibit BMP2-mediated mineralization [0122] Figure 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. Cells were cultured in minimal media (MM), osteogenic media, OM + lOOng/ml BMP2, OM + lOOng/ml BMP2 + GPCl (200ng/ml), OM + lOOng/ml BMP2 + GPC3 (200ng/ml), OM + lOOng/ml BMP2 + GPCl (lOOng/ml) + GPC3 (lOOng/ml) for up to 14 days, then stained with Alizarin Red to detect mineralization. Absorbance was quantified and normalized to cell number. Error bars represent mean + SD. N = 5; * - p<0.05 compared to D14 OM + BMP2; ▲ = p<0.05 compared to combined BMP2 + GPC 1 + GPC3 treatment at D 14.

[0123] It is proposed herein that a higher ratio of GPCl and/or GPC3 to BMP2 would have a more potent effect. EXAMPLE 5

Physical interaction between GPCl and BMP2 and GPC3 and BMP2

[0124] Figure 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.

EXAMPLE 6

Immunoblockade of GPC3

(0125] Figure 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, 25μg 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.

EXAMPLE 7

Immunoblockade of GPC1

[0126] Figure 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

(0127] Those skilled in the art will appreciate that aspects of the instant disclosure are susceptible to variations and. modifications other than those specifically described. It is to be understood that the aspects includes all such variations and modifications. Embodiments described herein also include all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

BIBLIOGRAPHY

Akimaru et al. ( 1995) Cytokines Mol Ther 1 : 197-210 Alving et al. (1995) Immunol Rev 145:5-31 Angew ( 1991 ) Chem. Intl. Ed. English 50:423 Bandyopadhyay and Temin (1984) Mol Cell Biol 4:149-154 Beaucage et al. (1993) Tetrahedron 49(10): \925 Berglund et al. ( 1993 ) Biotechnology U:9\ 6-920 Berkner et al. ( 1988) BioTechniques 6:616-629 Berkner (1992) Curr Top Microbiol Immunol 158:39-66 Bird et al. (1988) Science 242:424-426 Breakefield and Geller (1987) Mol Neurobiol 7:339-371 Brinkmann et al. (1993) Proc. Natl, Acad. Sci USA, 90:547-551 Briu et al. (1989) J. Am. Chem. Soc. 111 :2321 Buchschacher and Panganiban (1982) J Virol (56:2731-2739

Chapters 2 and 3, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. YS Sanghui and P Dan Cook Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. YS Sanghui and P Dan Cook

Coligan et al. (1991) Current Protocols in Immunology 7, John Wiley & Sons

Courtneay-Luck (1995) Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al (eds), Cambridge University Press: 166

Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA, 92:6097

Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press

Egleton and Davis ( 1997) Peptides 18: 1431 - 1439

Ewert et al. (2002) Biochemistry 47:3628-2636

Fink et al. (1992) Hum Gene Ther 5:1-19

Fink et al. (1996) Ann Rev Neurosci 79:265-287

Fix (1996) Pharm Res 75: 1760-1764

Freese et al. (1990) Biochem Pharmaco. 40:2189-2199

Gorziglia and Kapikian ( 1992) J Virol 12

Green et al. ( 1992) Immunochemical Protocols (Manson ed): l-5

Greenberg et al. (1995) Nature 574:168-173

He (2005) J Musculoskelet Neuronal Interact 5^:363-366 Helseth et al. (1990) J Virol (54:2416-2420

Huston et al. (1988) Proc. Nat. Acad Sci. USA, £5:5879-5883

Jeffs et al. ( 1994) J. Biomolecular NMR 34ΛΊ

Jenkins et al. ( 1995) Chem. Soc. Rev. : 169- 176

Johnson «?/ al. (1992) J ro/ 66:2952-2965

Jones et al. (1986) Native 527:522-525

Kohler and Milstein (l 975) Na/wre 256/ 495-499

Langer (1990) Sc/ewce 249: 1527-1533

Larrick a/. (1991) Methods: A Companion to Methods in Enzymology 2 : 106

Letsinger (1970) J. Org. Chem. 55:3800

Letsinger e/ a/. (1986) Nucl. Acids Res. 74:3487

Letsinger et al. (1988) J. Am. Chem. Soc. 770:4470

Letsinger et al. (1994) Nucleoside & Nucleotide 75: 1597

Losman et al. (1990) Int. J. Cancer 46:310

Madzak et al. (1992) J Gen Virol 75: 1533-1536 Mag et al. ( 1991 ) Nucleic A cids Res. J : 1437

Mann and Baltimore (1985) J Virol 54:401-407

Margolskee (1992) Curr Top Microbiol Immunol 158:67-95

Mesmaeker et al. (1994) Bioorganic & Medicinal Chem. Lett. 4:395

Midorikawa et al. (2003) Int. J. Cancer J03(4)A55-465

Miller et al. (1985) Mol Cell Biol 5:431-437

Miller et al. (1988) J Virol 62:4337-4345

Miller ( 1992) Curr Top Microbiol Immunol 158: 1 -24

Morrison et al (1984) Proc. Nat. Acad. Sci. USA, 57:6851 -6855

Moss (1992) Curr Top Microbiol Immunol 158: 5-38

Moss (1996) Proc Natl Acad Sci USA 93:1 1341-1 1348

Muenke and Wilkie (2000) Craniosynostosis Syndromes 3:61 17-6146

Muzyczka ( 1992) Curr Top Microbiol Immunol 158:91- 129

Naldini et al. (1996) Science 272:263-267

Nuttall et al. (2001) Mol Immunol 35:313-326

Nuttall et al. (2003) Eur J Biochem 270:3543-3554 Nuttall et al. (2004) Proteins 55: 187-197 Ohi et al. (1990) Gene 59:279-282 Page et al. (1990) J Virol 64:5270-5276. Patton ( 1998) Nat Biotech 16: 141- 143 Pauwels et al. ( 1986) Chemica Scripta 2(5: 1419 Petropoulos et al. (1992) J Virol 66:3391-3397

Picksley et al. (1995) DNA Cloning 2: Expression Systems, 2^nd Edition, Glover et al (eds), page 93 Oxford University Press

Putney and Burke (1998) Nat Biotech 16: 153-157

Quantin et al. ( 1992) Proc Natl Acad Sci USA 59:2581 -2584

Rawls (1997) C & ENews:35

Remington's Pharmaceutical Sciences 18^th Edition (1990) Mack Publishing Company, Easton, PA

Rosenfeld et al. ( 1992) Cell 68: 143- 155

Roux et al. (1998) Proc Natl Acad Sci USA 95: 11804-11809

Russell and Hirata (1998) Nat Genetics 75:323-328 Samanen et al. (1996) J Pharm Pharmacol ^.1 19-135

Sawai et al. (1984) Chem. Lett 805

Sayani and Chien (1996) Crit Rev Ther Drug Carrier Syst 73:85-184

Schneider et al. (1998) Nat Genetics 75: 180-183

Shimada et al. (1991) J Clin Invest 88: 1043-1047

Sorge et al. (1984) Mo! Cell Biol 4:1730-1737

Sprinzl et al. (1977) Eur. J. Biochem. 57:579

Stanfield et al. (2004) Science 305: 1770- 1773

Stratford-Perricaudet et al. ( 1990) Hum Gene Ther 1 :241 -256

Streltsov et al. (2004) Proc Natl Acad Sci USA 707: 12444-12449

Streltsov et al. (2005) Protein Sci 14:2901 -2909

Suntres and Shek (1994) J Pharm Pharmacol 4(5:23-28

Szoka and Papahadjopoulos (1980) Ann Rev Biophys Bioeng 9:467-508

Tetrahedron (1996) Lett. 37:743

Vutla et al. ( 1996) J Pharm Sci 55:5-8 Ward et al. (1995) Monoclonal Antibodies: Principles and Applications. Birch et al, Wiley-Liss, Inc.: 137

Wilkinson et al. (1992) Nucleic Acids Res 20:233-2239

- Williams et al. (1995) DNA Cloning 2: Expression Systems, 2^nd Ed, Oxford University Press

Woodle et al. (1992) Pharm Res 9:260-265 Zalipsky et al. ( 1995) Bioconjug Chem 6:705-708

Claims

CLAIMS:

1. A method. for modulating osteogenic bone morphogenetic protein (BMP) signaling in cells associated with bone formation, said method comprising administering to said cells, an effective amount of a glypican antagonist, which glypican inhibits the BMP signaling in order to promote BMP signaling or the glypican, glypican functional homolog or a glypican agonist in order to inhibit BMP signaling.

2. The method of Claim 1 wherein the cells are in vivo in a subject.

3. The method of Claim 1 wherein the cells are ex vivo in cell culture.

4. The method of Claim 1 wherein the glypican is selected from GPCl and GPC3.

5. The method of Claim 1 or 4 wherein the BMP is selected frorn BMP2, 4, 6, 7 and 9.

6. The method of any one of Claims 1 to 5 wherein a glypican 1 (GPCl) or glypican3 (GPC3) antagonist is used to promote BMP2 signaling.

7. The method of Claim 6 wherein the antagonist is an antibody specific for GPC l or GPC3 or a recombinant or synthetic or derivative of a GPCl or GPC3-specific antibody.

8. The method of Claim 7 wherein the GPCl or GPC3 antibody is humanized.

9. The method of Claim 6 wherein the antagonist is a small chemical molecule or a genetic molecule.

10. The method of Claim 2 wherein the subject is a human.

11. The method of Claim 1 wherein the BMP signaling is associated with bone osteogenesis.

12. The method of Claim 1 wherein the BMP signaling is associated with bone mineralization.

13. A method of promoting bone osteogenesis in a subject, said method comprising administering to said subject an effective amount of an antagonist of a glypican, which glypican inhibits osteogenic BMP signaling, for a time and under conditions to reduce glypican inhibition of a BMP.

14. The method of Claim 13 wherein the glypican is selected from GPCl and GPC3.

15. The method of Claim 13 or 14 wherein the osteogenic BMP is selected from BMP2, 4, 6, 7 and 9.

16. The method of Claim 14 or 15 wherein a GPCl or GPC3 antagonist is used to promote BMP2 signaling.

17. The method of Claim 13 wherein the method promotes bone mineralization.

18. The method of Claim 13 wherein the subject is a human.

19. A method of reducing excessive bone osteogenesis in a subject, said method comprising administering to said subject an effective amount of GPCl or GPC3, a GPCl or GPC3 functional homolog or a GPCl or GPC3 agonist for a time and under conditions sufficient to induce GPCl or GPC3 inhibition of BMP.

20. The method of Claim 19 wherein the glypican is selected from GPCl and GPC3.

21. The method of Claim 19 or 20 wherein the osteogenic BMP is selected from BMP2, 4, 6, 7 and 9.

22. The method of Claim 20 or 21 wherein GPCl or GPC3 or homolog or agonist thereof is used to induce inhibition of BMP2 signaling.

23. The method of Claim 19 wherein the method reduces excessive bone mineralization.

24. The method of Claim 1 wherein the subject is a human.

25. Use of a glypican antagonist in the manufacture of a medicament for the treatment or prophylaxis of a bode pathology in a subject.

26. Use of a glypican or homolog or agonist thereof in the manufacture of a medicament for the treatment or prophylaxis of a bone pathology in a subject.

27. Use of Claim 25 or 26 wherein the glypican is selected from GPCl and GPC3.

28. Use of Claim 25 or 26 or 17 wherein the subject is a human.