EP1385532A1 - Stimulation d'osteogenese utilisant des proteines de fusion de ligands rank - Google Patents

Stimulation d'osteogenese utilisant des proteines de fusion de ligands rank

Info

Publication number
EP1385532A1
EP1385532A1 EP02741650A EP02741650A EP1385532A1 EP 1385532 A1 EP1385532 A1 EP 1385532A1 EP 02741650 A EP02741650 A EP 02741650A EP 02741650 A EP02741650 A EP 02741650A EP 1385532 A1 EP1385532 A1 EP 1385532A1
Authority
EP
European Patent Office
Prior art keywords
bone
composition
rankl
bone formation
kinase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02741650A
Other languages
German (de)
English (en)
Other versions
EP1385532A4 (fr
Inventor
Jonathan Lam
F. Patrick Ross
Steven L. Teitelbaum
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Barnes Hospital
Original Assignee
Barnes Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Barnes Hospital filed Critical Barnes Hospital
Publication of EP1385532A1 publication Critical patent/EP1385532A1/fr
Publication of EP1385532A4 publication Critical patent/EP1385532A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/38—Drugs for disorders of the endocrine system of the suprarenal hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to methods for enhancing processes of bone formation by the administration of effective amounts of oligomeric complexes of one or more of RANKL, a RANKL fusion protein, analog, derivative, or mimic or osteogenic compounds capable of 1 ) enhancing activity of intracellular proteins in osteoblasts or osteoblast precursors, wherein said activity is indicative of bone formation, or 2) inactivating phosphatases in osteoblasts or osteoblast precursors, wherein said inactivation is indicative of bone formation.
  • the present invention further relates to treating, preventing or inhibiting bone loss or reduced bone formation caused by diseases such as osteoporosis. It further relates to enhancing fracture repair and promoting bone ingrowth into orthopedic implants or sites of bony fusion by facilitating bone formation via administration of oligomeric complexes or osteogenic compounds described herein.
  • the invention further provides compositions for stimulating bone formation.
  • bone loss Other conditions known to involve bone loss include juvenile osteoporosis, osteogenesis imperfecta, hypercalcemia, hyperparathyroidism, osteomalacia, osteohalisteresis, osteolytic bone disease, osteonecrosis, Paget's disease of bone, bone loss due to rheumatoid arthritis, inflammatory arthritis, osteomyelitis, corticosteroid treatment, metastatic bone diseases, periodontal bone loss, bone loss due to cancer, age-related loss of bone mass, and other forms of osteopenia. Additionally, new bone formation is needed in many situations, e.g., to facilitate bone repair or replacement for bone fractures, bone defects, plastic surgery, dental and other implantations and in other such contexts.
  • Bone is a dense, specialized form of connective tissue. Bone matrix is formed by osteoblast cells located at or near the surface of existing bone matrix. Bone is resorbed (eroded) by another cell type known as the osteoclast (a type of macrophage). These cells secrete acids, which dissolve bone minerals, and hydrolases, which digest its organic components. Thus, bone formation and remodeling is a dynamic process involving an ongoing interplay between the creation and erosion activities of osteoblasts and osteoclasts. Alberts, et al., Molecular Biology of the Cell, Garland Publishing, N.Y. (3rd ed. 1994), pp. 1182- 1186.
  • Present forms of bone loss therapy are primarily anti-resorptive, in that they inhibit bone resorption processes, rather than enhance bone formation.
  • agents which have been used or suggested for treatment of osteoporosis because of their claimed ability to inhibit bone resorption are estrogen, selective estrogen receptor modulators (SERM's), calcium, calcitriol, calcitonin (Sambrook, P. et al., N.Engl.J.Med. 328:1747-1753), alendronate (Saag, K. et al., N.Engl.J.Med. 339:292- 299J and other bisphosphonates. Luckman et al., J. Bone Min. Res. 13, 581 (1998).
  • anti-resorptives fail to correct the low bone formation rate frequently involved in net bone loss, and may have undesired effects relating to their impact on the inhibition of bone resorption/remodeling or other unwanted side effects.
  • RANK ligand also known as osteoprotegerin ligand (OPGL), TNF-related activation induced cytokine (TRANCE), and osteoclast differentiation factor (ODF)
  • OPGL osteoprotegerin ligand
  • TRANCE TNF-related activation induced cytokine
  • ODF osteoclast differentiation factor
  • RANKL The cell surface receptor for RANKL is RANK, Receptor Activator of Nuclear Factor (NF)-kappa B.
  • RANKL is a type-2 transmembrane protein with an intracellular domain of less than about 50 amino acids, a transmembrane domain of about 21 amino acids, and an extracellular domain of about 240 to 250 amino acids. RANKL exists naturally in transmembrane and soluble forms.
  • RANKL The deduced amino acid sequence for at least the murine, rat and human forms of RANKL and variants thereof are known. See e.g., Anderson, et al., U.S. Pat. No. 6,017,729, Boyle, U.S. Pat. No. 5,843,678, and Xu J. et al., J. Bone Min. Res. (2000/15:2178) which are incorporated herein by reference.
  • RANKL (OPGL) has been identified as a potent inducer of bone resorption and as a positive regulator of osteoclast development. Lacey et al., supra. In addition to its role as a factor in osteoclast differentiation and activation,
  • RANKL has been reported to induce human dendritic cell (DC) cluster formation. Anderson et al., supra and mammary epithelium development J.Fata et al., "The osteoclast differentiation factor osteoprotegerin ligand is essential for mammary gland development," Cell, 103:41-50 (2000). However, that RANKL could play a role in anabolic bone formation processes or could be used in methods to stimulate osteoblast proliferation or bone nodule mineralization was previously unknown and unexpected.
  • the objects of the present invention is the provision of methods and compositions which stimulate osteogenesis, including enhanced activity of osteoblasts, commitment of osteoblast precursors to the osteoblast phenotype and in vivo bone matrix deposition.
  • methods are provided for enhancing bone formation as well as for treating diseases and conditions of bone loss by increasing bone formation, whether or not bone resorption processes are otherwise affected.
  • the present invention is directed toward a method of enhancing bone formation.
  • the method calls for administering effective amounts of 1 ) oligomeric complexes of one or more of RANKL, a RANKL fusion protein, analog, derivative, or mimic, 2) osteogenic compounds capable of enhancing activity of intracellular proteins in osteoblasts or osteoblast precursors, wherein said activity is indicative of bone formation, or 3) osteogenic compounds capable of inactivating phosphatases in osteoblasts or osteoblast precursors, wherein said inactivation is indicative of bone formation
  • a method of treating a disease or condition manifested at least in part by the loss of bone mass comprises administering a pharmaceutical composition comprising a RANKL fusion protein or an analog, derivative or mimic thereof in an amount effective to promote bone formation.
  • a pharmaceutical composition comprising an osteogenic compound capable of enhancing activity of intracellular proteins in osteoblasts or osteoblast precursors, wherein said activity is indicative of bone formation may be used.
  • a pharmaceutical composition comprising an osteogenic compound capable of inactivating phosphatases in osteoblasts or osteoblast precursors, wherein said inactivation is indicative of bone formation may be employed. The loss of bone mass is thereby prevented, inhibited or counteracted.
  • compositions for stimulating bone formation include an effective amount of a RANKL fusion protein, oligomeric complex, or an analog, derivative or mimic thereof in a pharmaceutically acceptable carrier or excipient.
  • compositions which include effective amounts of osteogenic compounds in pharmaceutically acceptable carriers or excipients wherein said osteogenic compounds are capable of 1 ) enhancing activity of intracellular proteins in osteoblasts or osteoblast precursors, wherein said activity is indicative of bone formation, or 2) inactivating phosphatases in osteoblasts or osteoblast precursors, wherein said inactivation is indicative of bone formation.
  • intracelllular proteins are selected from IKB- ⁇ and IKB- ⁇ .
  • the intracellular proteins exhibiting prolonged activity comprise intracellular kinases, and more preferably such kinases are ERK1/2, IKK,
  • PI3 kinase Akt, JNK, and p38.
  • the kinases are N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-like kinase
  • Akt Akt
  • JNK Jun N-like kinase
  • p38 p38
  • the activity of one or more intracellular proteins constitutes phosphorylation of said protein(s).
  • the phosphorylated proteins include ERK1/2, IKK, PI3 kinase, Akt, JNK, and p38. More preferably, the phosphorylated kinases are ERK1/2.
  • the activity of one or more intracellular proteins can be detected for at least about 15-30 minutes following the incubation of the osteogenic compound with osteoblasts or osteoblast precursors.
  • the activity can be detected for 40 minutes, and more preferably it can be detected for at least 60 minutes following said incubation.
  • osteogenic compounds capable of inactivating one or more phosphatases in osteoblasts or osteoblast precursors, wherein said inactivation is indicative of bone formation may be used in the methods and compositions of the present invention.
  • said phosphatase is selected from the group consisting of ERK1-, ERK2-, IKK-, PI3 kinase-, Akt-, JNK-, and p38- specific phosphatases, and more preferably the phosphatese is specific for ERK1/2.
  • inactivation comprises phosphorylation of a phosphatase.
  • the preferred oligomeric complexes used in the methods and compositions described herein include oligomeric complexes of GST-RANKL, AP-RANKL, leucine zipper-RANKL, and RANKL derivative comprising the "flap" domain of TALL-1.
  • FIG. 1 is the structure and sequence of the RANKL murine cDNA and protein used to produce the GST-RANKL fusion proteins discussed in Examples 1 and 25 below.
  • FIG. 2 depicts a size-exclusion chromatograph of the GST-RANKL fusion protein under conditions replicating the physiological milieu. See Example 1.
  • FIG. 3 is a histological presentation of GST-RANKL stimulation of bone formation ex vivo in whole calvarial organ culture, as discussed in Example 2. Arrows mark parietal bone thickness.
  • FIG. 4 is a graphic depiction of the dose-dependent increase in calvarial thickness due to GST-RANKL stimulation of bone formation in vitro, as discussed in Example 2.
  • White bars indicate 1 dose exposure, whereas black bars indicate 2 dose exposure to GST-RANKL.
  • FIG. 5(a) is a histological presentation of GST-RANKL stimulation of bone formation in vivo in mice, shown at low power magnification, as discussed in Example 3.
  • FIG. 5(b) is a histological presentation of GST-RANKL stimulation of bone formation in vivo in mice, shown at high power magnification, as discussed in Example 3.
  • FIG. 5(c) depicts a dual-energy X-ray absorptiometry (DEXA) analysis of tibial metaphyses comparing bone mineral density of animals administered GST-RANKL or control vehicle in vivo, as discussed in Example 3.
  • Scale bar 1 mm.
  • FIG. 6 is a histological presentation of a mouse tibia at high magnification, demonstrating in vivo activation of osteoblasts in animals administered GST-RANKL as discussed in Example 4. Arrow in the left panel indicates activated osteoblasts, whereas the arrow in the right panel indicates flat bone lining cells.
  • FIG. 7 is a graphical depiction of the impact of controlled administration of
  • FIG. 8 is a histological presentation of GST-RANKL stimulation of mineralized bone nodule formation in marrow cells cultured ex vivo, as discussed in Example 6.
  • Red histochemical reaction product represents mineralizing colony forming units of osteoblasts.
  • FIG. 9 is a depiction of an in vivo double fluorochrome label incorporation into mineralizing bone, as discussed in Example 4.
  • MAR represents mineral apposition
  • BFR indicates bone formation
  • (ex) and (en) indicate exocranial and endocranial surfaces of calvaria, respectively.
  • FIG. 10 is an image of a Western blot depicting the rapid activation of the members of the MAPK pathway in murine osteoclast precursors following the treatment of cells with GST-RANKL.
  • the activity was measured at the time of GST- RANKL/RANK interaction (0 minutes) and 5, 15, and 30 minutes following the interaction.
  • the second, fourth, and sixth panels show the total levels of JNK, p38, and ERK respectively.
  • the first, third, and fifth panels depict the phosphorylated (activated) forms of JNK, p38, and ERK respectively.
  • FIG. 11 is an image of a Western blot depicting the activity of Akt in murine osteoclast precursors following the treatment of cells with GST-RANKL. The activation was monitored at the time of GST-RAN KL/RANK interaction, and 5 and 15 minutes following the interaction. The bottom panel depicts the levels of total Akt at specified time points, whereas the top panel depicts the phosphorylated forms of Akt.
  • FIG. 12 is an image of a Western blot depicting the prolonged activity of the kinases in MAPK pathway in murine osteoblasts following the GST-RANKL treatment of cells compared to the treatment with RANKL alone. The time points for which the phosphorylation was measured included 0 minutes (time of GST-RANKL or RANKL stimulation of cells), and 5, 10, 20, 30, and 60 minutes after GST-
  • pERK designates phosphorylated ERK
  • ERK designates the total amount of the same protein
  • pJNK designates phosphorylated JNK
  • JNK designates the total amount of JNK
  • pp38 designates phosphorylated p38
  • p38 designates the total amount of p38
  • pAkt designates phosphorylated Akt
  • Akt designates the total amount of the same protein.
  • the first panel from the top is p-lkB ⁇ , which designates phosphorylated IkB ⁇ , whereas IkB ⁇ designates the total amount of the same protein.
  • FIG. 13 is an image of a Western blot depicting the prolonged activity of ERK1/2 in murine osteoblast precursors following the treatment of cells with GST- RANKL.
  • the time points at which ERK1/2 activity was measured include 0, 5, 10, 20, 30, and 60 minutes following GST-RANKL/RANK interaction.
  • pERK designates phosphorylated ERK whereas ERK designates the total amount of the same protein.
  • FIG. 14 is a graphic presentation of alkaline phosphatase (AP) activity following GST-RANKL exposure.
  • AP alkaline phosphatase
  • FIG. 15 depicts GST-RANKL as oligomeric complexes, whereas cleaved RANKL (GST removed) does not exist in oligmeric forms, (a) shows that cleaved RANKL migrates as a single trimeric species (1 n), while GST-RANKL exists as a polydisperse mixture of non-covalently associated mono-trimeric (1 n) and oligomeric (2-1 OOn) units under dynamic equlibrium. (b) depicts possible oligomeric structures.
  • FIG. 15 depicts GST-RANKL as oligomeric complexes, whereas cleaved RANKL (GST removed) does not exist in oligmeric forms, (a) shows that cleaved RANKL migrates as a single trimeric species (1 n), while GST-RANKL exists as a polydisperse mixture of non-covalently associated mono-trimeric (1 n) and oligomeric (2-1 OOn) units under dynamic e
  • FIG. 16 is an image of an agarose gel depicting the expression of Type I collagen in response to GST-RANKL treatment. "+” indicates the treatment of primary osteoblasts with GST-RANKL, whereas "-" indicates the lack of such treatment. Osteoblasts were exposed to GST-RANKL for 1 , 2, 4, or 6-hour exposures at the beginning of each successive 48-hour treatment window. All culltures harvested between 8-48 hours were exposed to GST-RANKL for 6 hours, ⁇ -actin expression is used as a control for the experiment.
  • FIG. 18 is an image of an agarose gel depicting the expression of Cbfal in the marrow of mice treated with GST-RANKL or GST alone (marked as “control”).
  • the bottom panel is the experiment control, depicting the expression of HPRT (hypoxanthine phosphoribosyl transferase).
  • FIG. 19 is a graphic representation of osteoblast proliferation as measured by BrdU (5-bromo-2'-deoxyuridine) incorporation in response to GST-RANKL treatment.
  • FIG. 20(a) is an image of a Western blot showing that osteoblasts transduced with dominant-negative ERK fail to phosphorylate an ERK substrate, known as RSK.
  • DN-ERK represents dominant-negative ERK.
  • LacZ represents ⁇ -galactosidase.
  • FIG. 20(b) is an image of an agarose gel showing that osteoblasts transduced with dominant-negative ERK fail to upregulate the expression of type I collagen in response to GST-RANKL.
  • MAP kinase or “MAPK” are used interchangeably herein, and are abbreviations for mitogen activated protein kinase.
  • ERK1/2 refers to ERK1 and ERK2, which are abbreviations for extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2, respectively.
  • JNK is an abbreviation for c-jun N-terminal kinase.
  • p38 is a kinase of 38 kDa, which is a member of the MAPK family of kinases.
  • Akt is Akt serine threonine kinase.
  • 1KB is an abbreviation for IkappaB protein.
  • IKB- ⁇ is IkappaB ⁇
  • IKB- ⁇ is IkappaB ⁇ .
  • IKK is an abbreviation for IkappaB (1KB) kinase.
  • RSK is an abbreviation for p90 ribosomal S6 protein kinase.
  • RKL or “RANK ligand” are used interchangeably herein to indicate a ligand for RANK (Receptor Activator of NFKB).
  • AP alkaline phosphatase
  • GST is an abbreviation for glutathione-s-transferase.
  • HPRT is an abrreviation for hypoxanthine phosphoribosyl transferase.
  • Cbfal is an abbreviation for core binding factor 1.
  • LacZ is an abbreviation for ⁇ -galactosidase.
  • Ostogenic potential or “osteogenic activity” are used interchangeably herein to refer to any compound that is able to enhance bone formation, as determined from bone formation assays.
  • “BrdU” is an abbreviation for 5-bromo-2'-deoxyuridine.
  • TALL-1 is an abbreviation for a protein "TNF-and APOL-related leukocyte expressed ligand 1".
  • an effective amount is meant an amount of the substance in question which produces a statistically significant effect.
  • an “effective amount” for therapeutic uses is the amount of the composition comprising an active compound herein required to provide a clinically significant increase in healing rates in fracture repair; reversal or inhibition of bone loss in osteoporosis; prevention or delay of onset of osteoporosis; stimulation and/or augmentation of bone formation in fracture non-unions and distraction osteogenesis; increase and/or acceleration of bone growth into prosthetic devices; repair or prevention of dental defects; or treatment or inhibition of other bone loss conditions, diseases or defects, including but not limited to those discussed herein above.
  • Such effective amounts will be determined using routine optimization techniques and are dependent on the particular condition to be treated, the condition of the patient, the route of administration, the formulation, and the judgment of the practitioner and other factors evident to those skilled in the art.
  • the dosage required for the compounds of the invention (for example, in osteoporosis where an increase in bone formation is desired) is manifested as that which induces a statistically significant difference in bone mass between treatment and control groups. This difference in bone mass may be seen, for example, as at least 1-2%, or any clinically significant increase in bone mass in the treatment group.
  • Other measurements of clinically significant increases in healing may include, for example, an assay for the N-terminal propeptide of Type I collagen, tests for breaking strength and tension, breaking strength and torsion, 4-point bending, increased connectivity in bone biopsies and other biomechanical tests well known to those skilled in the art.
  • General guidance for treatment regimens is obtained from the experiments carried out in animal models of the disease of interest.
  • treatment includes both prophylaxis and therapy.
  • the compounds of the invention may be administered to a subject already suffering from loss of bone mass or to prevent or inhibit the occurrence of such condition.
  • oligomeric complexes of RANKL fusion proteins can be administered in an amount and manner such that they stimulate a net increase in the numbers of activated osteoblasts and enhance the anabolic processes of bone formation.
  • Such discovery provides the basis for methods useful to facilitate bone replacement or repair, as well as for treating diseases or conditions involving loss of bone mass by stimulating anabolic processes of bone formation.
  • RANKL RANKL
  • its fragments, variants, analogs, mimics, fusion products and oligomeric complexes of such compounds, wherein said oligomeric complexes are capable of promoting bone formation as taught herein are within the ability of a person of ordinary skill in the art and are contemplated as being within the scope of this invention.
  • Boyle, supra provides a detailed discussion of the synthesis of various forms of RANKL therein (called "osteoprotegerin binding protein”), and discloses, e.g., murine and human variants, recombinant forms of RANKL, RANKL fragments, analogs, mimics and derivatives of RANKL, and fusion-proteins thereof.
  • RANKL derivatives or analogs of RANKL which have been modified post- translationally (such as glycosylated proteins), as well as polypeptides which are encoded by nucleic acids shown to hybridize to part or all of the polypeptide coding regions of RANKL cDNA under conditions of high stringency.
  • the murine RANKL nucleic acid and amino acid sequences are provided herein as SEQ ID NO. 1 and SEQ ID NO. 2, respectively (see Fig. 1 ).
  • SEQ ID NO. 1 The murine RANKL nucleic acid and amino acid sequences are provided herein as SEQ ID NO. 1 and SEQ ID NO. 2, respectively (see Fig. 1 ).
  • SEQ ID NO. 1 amino acid sequences from other species have been identified and are available at http://www.ncbi.nlm.nih.gov/.
  • RANKL nucleic acid and amino acid sequences have, for instance, the following accession numbers: AF019047 and AAB86811.
  • Rat RANKL nucleic acid and amino acid sequences have, for example, these accession numbers: NM_057149 and NP_476490. Accordingly, any of the RANKL molecules may be used in the methods of the present invention, and are thus contemplated within the scope of the present invention.
  • RANKL and related molecules can be synthesized by using nucleic acid molecules which encode the peptides of this invention in an appropriate expression vector which include the encoding nucleotide sequences using procedures well known in the art.
  • DNA molecules may be prepared, and subsequently analyzed, e.g., using automated DNA sequencing and the well-known codon-amino acid relationship of the genetic code.
  • Such a DNA molecule also may be obtained as genomic DNA or as cDNA using oligonucleotide probes and conventional hybridization methodologies.
  • DNA molecules may be incorporated into expression vectors, including plasmids, which are adapted for the expression of the DNA and production of the polypeptide in a suitable host such as bacterium, e.g., Escherichia coli, yeast cell, insect cell or mammalian cell. See, e.g., Examples 1 and 25.
  • bacterium e.g., Escherichia coli, yeast cell, insect cell or mammalian cell. See, e.g., Examples 1 and 25.
  • oligomers of GST-RANKL results in enhanced anabolic processes of bone formation.
  • size exclusion chromatography indicates that RANKL fusion proteins are capable of existing as oligomeric complexes under physiologic conditions. Oligomers of GST-RANKL are believed to be formed as a result of RANKL's and GST's tendencies to trimerize and dimerize, respectively. Accordingly, other fusion partners besides GST may be used to form oligomeric complexes comprising RANKL. Preferred fusion partners include alkaline phosphatase and leucine zippers, however any other proteins with a tendency to form oligomeric structures are contemplated within the scope of the present invention.
  • RANKL fusion partners are added to the N-terminal of RANKL. Formation of GST-RANKL used to form oligomeric complexes is described in Examples 1 and 25. Furthermore, it is within the skill of the art to generate other forms of RANKL oligomers by well known techniques. For example, one could construct RANKL oligomers using alternative proteins or polypeptides that have an intrinsic tendency to self-associate and/or form higher-order complexes. One could also create such oligomers by chemical modification or by synthesizing a polymeric form of RANKL in which many copies are linked together, e.g., similar to a chain of pearls. Such alternative embodiments are also within the scope of this invention.
  • AP Alkaline phosphatase
  • AP Alkaline phosphatase
  • GST Alkaline phosphatase
  • APs form a large family of enzymes that are common to all organisms. Humans possess four isoforms of AP, three of which are tissue-specific and one which is non-specific and can be found in bone, liver, and kidney. The three tissue-specific APs include: placental AP (PLAP), germ cell AP (GCAP), and intestinal AP.
  • PLAP placental AP
  • GCAP germ cell AP
  • the construction of an amino-terminal AP-RANKL may be performed similarly to the construction of GST-RANKL fusion protein.
  • alkaline phosphatases examples include but are not limited to human placental AP-1 , human placental AP-2, human placental AP precursor, mouse secreted AP, mouse embryonic AP precursor, and mouse embryonic AP with the corresponding accession numbers: AAA51710, AAA51707, AAC97139, AAL17657, P24823, and AAA37531.
  • human placental alkaline phosphatase is employed, however other APs, isolated either from humans or from other mammalian species such as Mus musculus may be used.
  • the use of many different alkaline phosphatases is believed to be feasible due to the ability of all APs to dimerize.
  • a cDNA encoding a desired isoform of AP can be isolated from a cDNA library and spliced upstream (at amino terminal) of a RANKL cDNA in a suitable expression vector, such as, e.g., pcDNA 3.1 , using appropriate restriction endonucleases, such that the resulting DNA sequence is in frame, with no intervening stop codons.
  • a suitable expression vector such as, e.g., pcDNA 3.1
  • the expression vector, comprising the nucleotide sequence encoding AP-RANKL can then be introduced into host cells of choice by any of several trasfection or transduction techniques known in the art. See also Example 17.
  • a RANKL fusion protein may comprise a peptide with the ability to oligomerize, such as a leucine zipper domain.
  • Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, 1988). Leucine zipper domain is a term used to refer to a conserved peptide domain present in these (and other) proteins, which is responsible for dimerization of the proteins.
  • the leucine zipper domain comprises a repetitive heptad repeat, with four or five leucine residues interspersed with other amino acids. Examples of leucine zipper domains are those found in the yeast transcription factor GCN4 and a heat-stable DNA-binding protein found in rat liver (C/EBP; Landschulz et al., Science 243:1681 , 1989).
  • Leucine zipper domains are known to fold as short, parallel coiled coils. (O'Shea et al., Science 254:539; 1991 ) The general architecture of the parallel coiled coil has been well characterized, with a "knobs-into-holes" packing as proposed by Crick in 1953 (Acta Crystallogr. 6:689).
  • the dimer formed by a leucine zipper domain is stabilized by the heptad repeat, designated (abcdefg) n according to the notation of McLachlan and Stewart (J. Mol. Biol.
  • residues a and d are generally hydrophobic residues, with d being a leucine, which line up on the same face of a helix.
  • Oppositely-charged residues commonly occur at positions g and e.
  • leucine zipper domains Peptides containing these substitutions are still referred to as leucine zipper domains.
  • leucine zippers capable of dimerizing proteins are used as RANKL fusion partners. Construction of a fusion RANKL-leucine zipper fusion protein may be performed in a similar manner as for GST-RANKL and AP- RANKL. See Example 18. In addition to bacteria, other suitable expression systems such as mammalian cells and insect cells may be used. One of ordinary skill in the art can easily make necessary adjustments in order to express a leucine zipper-RANKL fusion protein.
  • a RANKL derivative may be used to form oligomeric complexes. It has recently been discovered that a newly found TNF ligand family member TALL-1 (also known as BAFF, THANK, BLyS, and zTNF4) possesses the ability to oligomerize under physiological conditions (Liu et al., Cell, 108:383-394, 2002). Liu et al. have shown that the "flap" region, named so due to the length of the loop that forms the flap and allows it to extend from the molecule, mediates trimer-trimer ineractions and subsequent cluster formation.
  • TALL-1 also known as BAFF, THANK, BLyS, and zTNF4
  • This flap region is unique to TALL-1 among TNF family members and is created by a surface DE loop (the loop that connects the strands D and E of TALL-1 ) that is longer than any DE loop of other TNF family proteins, which have been discovered so far.
  • the oligmerization is thought to occur through a noncovalent interaction of the long DE loop with surrounding TALL-1 molecules, thereby resulting in the formation of large clusters.
  • RANKL and TALL-1 are both TNF ligand family members and possess similar ⁇ -strand core structure, in accordance with the invention, RANKL is mutated to create a mutant RANKL molecule that oligomerizes spontaneously at physiological conditions.
  • modification of RANKL is designed so that its DE loop (amino acids 245-249 containing the amino acid sequence SIKIP) is substituted with the DE loop of TALL-1 (amino acid sequence KVHVFGDEL).
  • TALL-1 amino acid sequence KVHVFGDEL.
  • the following amino acid changes may be made throughout the RANKL molecule: 168T-I, 187Y ⁇ L, 194K ⁇ F, 212F- ⁇ , 252H-V, 279F-I, and 283R ⁇ E. See Example 20.
  • the mutations can be introduced into RANKL by PCR-driven site-directed mutagenesis, using, for example, the QuickChange Multi-Site Directed Mutagenesis Kit (available from Stratagene).
  • the QuickChange Multi-Site Directed Mutagenesis Kit available from Stratagene.
  • One of ordinary skill in the art can make said mutations and test the structure and function of the mutated RANKL without undue experimentation.
  • In vitro or in vivo assays can be used to determine the efficacy of oligomeric
  • osteoblast-like cells can be used. Suitable osteoblast-like cells include, but are not limited to, primary marrow stromal cells, primary osteoblasts, ST-2 cells, C1 cells, ROS cells, and MC3T3-E1 cells. Many of the cell lines are available from American Type Culture Collection, Rockville, Md., and can be maintained in standard specified growth media.
  • oligomeric complexes can be tested by cultu ng the cells with a range of concentrations of compounds and assessing markers or indicia of bone formation such as osteoblast activation, bone matrix deposition, calvarial thickness and bone nodule formation. See Example 2 below.
  • markers or indicia of bone formation such as osteoblast activation, bone matrix deposition, calvarial thickness and bone nodule formation.
  • osteoblast proliferation, expression of Collagen type I and/or expression of Cbfal may be used to assess bone formation. See Example 14 below.
  • the cells were plated at 5000/cm 2 in plastic 25 cm 2 culture flasks in ⁇ -MEM supplemented with 5% fetal bovine serum, 26 mM NaHCO 3 , 2 mM glutamine, 100 u/ml penicillin, and 100 ⁇ g/ml streptomycin, and grown in humidified 5% CO 2 /95% air at 37°C. Cells were passaged every 3-4 days after releasing with 0.002% pronase E in PBS. The cells in treatment groups were grown for 24 hours, then incubated with BMP-2 (50 ng/ml) dissolved in PBS containing 4 mM HCI and 0.1 % bovine serum albumin (BSA) at 37 ° C for 24 and 48 hours.
  • BMP-2 50 ng/ml
  • BSA bovine serum albumin
  • Control groups received equal volumes of vehicles only. Exemplary conditions for treatment of osteoblast cells or precursors with oligomers, such as GST-RANKL, are described below. Osteoblast precursor cells are incubated in the presence of vehicle, GST (a negative control), or increasing concentrations of purified oligomeric GST-RANKL (e.g. concentrations ranging from 1 ng/ml to 100ng/ml). Bone morphogenetic protein (BMP)-2 is administered as a positive control. Test compositions are administered for a period of 12 hours only at the initiation of the culture or once at initiation and once three days later, again for a duration of 12 hours.
  • BMP bone morphogenetic protein
  • oligomeric RANKL compositions which enhance bone formation according to applicants methods may be evaluated in various animal models. See Examples 3-6 and descriptions below.
  • a commonly used assay is a neonatal mouse calvaria assay. Briefly, four days after birth, the front and parietal bones of ICR Swiss white mouse pups are removed by microdissection and split along the sagittal suture. The bones are then incubated in a specified medium, wherein the medium contains either test or control compounds. Following the incubation, the bones are removed from the media, and fixed in 10% buffered formalin for 24-48 hours, decalcified in 14% EDTA for 1 week, processed through graded alcohols, and embedded in paraffin wax. Three micron sections of the calvaria are prepared and assessed using histomorphometric analysis of bone formation or bone resorption. Bone changes are measured on sections cut 200 microns apart. Osteoblasts and osteoclasts are identified by their distinctive morphology.
  • the effect of compounds on murine calvarial bone growth can also be tested in vivo.
  • male ICR Swiss white mice, aged 4-6 weeks are employed, using 4-5 mice per group. Briefly, the test compound or the appropriate control is injected into subcutaneous tissue over the right calvaria of normal mice. The mice are sacrificed on day 14, and bone growth is measured by histomorphometric means. Bone samples are cleaned from adjacent tissues and fixed in 10% buffered formalin for 24-48 hours, decalcified in 14% EDTA for 1-3 weeks, processed through graded alcohols, and embedded in paraffin wax.
  • Three to five micron sections of the calvaria are prepared, and representative sections are selected for histomorphometric assessment of the effects of bone formation and bone resorption. Sections are measured by using a camera lucida attachment to trace directly the microscopic image onto a digitizing plate. Bone changes are measured on sections cut 200 microns apart, over 4 adjacent 1X1 mm fields on both the injected and noninjected sides of calvaria. New bone is identified by its characteristic tinctorial features, and osteoclasts and osteoblasts are identified by their distinctive morphology. Histomorphometry software (OsteoMeasure, Osteometrix, Inc., Atlanta) can be used to process digitized input to determine cell counts and measure areas or perimeters.
  • Additional in vivo assays include dosing assays in intact animals, and dosing assays in acute ovariectomized (OVX) animals (prevention model), and assays in chronic OVX animals (treatment model).
  • Prototypical dosing in intact animals may be accomplished by, for example, subcutaneous, intraperitoneal, transepithelial, or intravenous administration, and may be performed by injection, or other delivery techniques.
  • the time period for administration of test compound may vary (for instance, 28 days as well as 35 days may be appropriate).
  • in vivo transepithelial or subcutaneous dosing assays may be performed as described below.
  • test compound PBS, and vehicle are administered subcutaneously once per day for 35 days. All animals are injected calcein nine days and two days before sacrifice (to ensure proper labeling of newly formed bone). Weekly body weights are determined. At the end of 35 days, the animals are weighed and bled by orbital or cardiac puncture.
  • Serum calcium, phosphate, osteocalcin, and CBCs are determined. Both leg bones (femur and tibia) and lumbar vertabrae are removed, cleaned of adhering soft tissue, and stored in 70% ethanol or 10% formalin for evaluation, as performed by peripheral quantitative computed tomography (pQCT; Ferretti, J, Bone, 17: 353S-364S, 1995), dual energy X-ray absorptiometry (DEXA; Laval-Jeantet A. et al., Calcif Tissue Intl, 56:14-18, 1995, and Casez J. et al., Bone and Mineral, 26:61-68, 1994) and/or histomorphometry. The effect of test compounds on bone remodeling can thus be evaluated.
  • pQCT Ferretti, J, Bone, 17: 353S-364S, 1995
  • DEXA dual energy X-ray absorptiometry
  • the effect of test compounds on bone remodeling can thus be evaluated.
  • Test compounds can also be assayed in acute ovariectomized animals. Such assays may also include an estrogen-treated group as a control. An example of the test in these animals is briefly described below.
  • a typical study 80 three-month-old female Sprague-Dawley rats are weight-matched and divided into eight groups, with ten animals in each group. This includes a baseline control group of animals sacrificed at the initiation of the study; three control groups (sham OVX and vehicle only, OVX and vehicle only, and OVX and PBS only); and a control OVX group that is administered a compound known to enhance bone mass. Three dosage levels of the test compound are administered to remaining groups of OVX animals.
  • test compound, positive control compound, PBS or vehicle alone is administered transepithelially or subcutaneously once per day for 35 days.
  • test compounds can be formulated in implantable pellets that are implanted for 35 days, or may be administered transepithelially, such as by nasal administration. All animals are injected with calcein at intervals determined empirically, including but not limited to nine days and two days before sacrifice. Weekly body weights are determined. At the end of the 35-day cycle, the animals blood and tissues are processed as described above.
  • Test compounds may also be assayed in chronic OVX animals. Briefly, 80 to 100 six month old female, Sprague-Dawley rats are subjected to sham surgery (sham OVX), or ovariectomy (OVX) at the beginning of the experiment, and 10 animals are sacrificed at the same time to serve as baseline controls. Body weights are monitored weekly. After approximately six weeks or more of bone depletion, 10 sham OVX and 10 OVX rats are randomly selected for sacrifice as depletion period controls. Of the remaining animals, 10 sham OVX and 10 OVX rats are used as placebo-treated controls. The remaining animals are treated with 3 to5 doses of test compound for a period of 35 days.
  • a group of OVX rats can be treated with a known anabolic agent in this model, such as PTH (Kimmel et al., Endocrinology, 132: 1577-1584, 1993).
  • PTH Kinmel et al., Endocrinology, 132: 1577-1584, 1993.
  • the animals are sacrificed and femurs, tibiae, and lumbar vertebrael to 4 are excised and collected.
  • the proximal left and right tibiae are used for pQCT measurements, cancellous bone mineral density (BMD), and histology, while the midshaft of each tibiae is subjected to cortical BMD or histology.
  • the femurs are prepared for pQCT scanning of the midshaft prior to biomechanical testing.
  • LV lumbar vertebrae
  • LV2 are processed for BMD (pQCT may also be performed)
  • LV3 are prepared for undecalcified bone histology
  • oligomeric RANKL and its receptor RANK on osteoblasts or osteoblast precursors results in prolonged intracellular activity of intracellular proteins.
  • Mouse osteoblasts when treated with GST-RANKL in vitro manifested activation, as characterized by the activation of NFKB and ERK intracellular signal pathways.
  • the time course of intracellular protein activity, especially ERK activity is different from that observed in osteoclast precursors, which also express RANK on the surface.
  • osteoclast precursors ERK activity peaks 5-15 minutes after RANK/GST-RANKL interaction, and returns to basal levels after 15-30 minutes.
  • osteoblasts and osteoblast precursor cells also exhibit prolonged activity of kinases such as IKK, PI3 kinase, Akt, p38 and JNK.
  • This osteoblast-related activity contrasts with GST-RANKL interaction with RANK on osteoclasts, which results in short-lived activity of MAP kinases and bone resorption. While not being bound to a particular theory, it therefore appears that the prolonged activity of kinases observed in osteoblasts following oligomeric GST-RANKL stimulation plays a role in the anabolic bone processes.
  • TNF family cytokine-induced intracellular signaling is attenuated by internalization of the receptor-ligand complex (see, e.g., Higuchi, M and Aggarwal, B.B., J. Immunol., 152:3550-3558, 1994).
  • Applicants therefore believe that oligomeric complexes comprising RANKL are not internalized as promptly as RANKL trimers, thus allowing for a longer interaction with the receptor and prolonged intracellular signaling. See Fig. 16 and Example 13.
  • osteogenic compounds capable of enhancing activity of one or more intracellular proteins in osteoblasts or osteoblast precursors, wherein such activity is indicative of bone formation, may be used in the methods of the present invention.
  • Activated intracellular proteins include but are not limited to kinases.
  • the kinases comprise ERK1/2, JNK, PI3 kinase, IKK, Akt, and p38, and even more preferably, the kinases are ERK1/2.
  • Other intracellular proteins include IKB- ⁇ and IKB- ⁇ .
  • the activity comprises phosphorylation of one or more intracellular proteins, and more preferably of kinases.
  • MAP kinase family full activation requires dual phosphorylation on tyrosine and threonine residues separated by a glutamate residue (known as TEY motif, where T is threonine, E is glutamic acid, and Y is tyrosine) by a single upstream kinase known as MAP kinase kinase (MKK).
  • MKK MAP kinase kinase
  • Any of the assays available in the art for determining whether a kinase has been phosphorylated may be used.
  • such assays include Western blots or kinase assays.
  • a Western blot can be generally performed as follows. Once the cell lysates are generated, the intracellular proteins are separated on the basis of size by utilizing SDS-PAGE (sodium dodecyl sulfate- ⁇ olyacrylamide gel electrophoresis). The separated proteins are transferred by electroblotting to a suitable membrane (such as nitrocellulose or polyvinylidene flouride) to which they adhere. The membrane is washed to reduce non-specific signals, and then probed with an antibody which recognizes only the specific amino acid which has been phosphorylated as a result of RANK signaling.
  • SDS-PAGE sodium dodecyl sulfate- ⁇ olyacrylamide gel electrophoresis
  • a second antibody which recognizes the first antibody (bound to specifically-phosphorylated proteins on the membrane) and contains a reporter moiety is applied to the membrane.
  • the addition of a developing agent, which interacts with a reporter moiety on the second antibody results in visualization of the bands.
  • a kinase assay for example for ERK1/2, can be performed by utilizing a known substrate for this kinase such as p90 ribosomal S6 protein kinase (RSK).
  • a known substrate for this kinase such as p90 ribosomal S6 protein kinase (RSK).
  • RSK ribosomal S6 protein kinase
  • treated osteoblasts are washed in ice-cold PBS, e.g., three times, and extracted with lysis buffer in order to obtain cell lysates.
  • Supernatants obtained after microcentifugation of cell lysates are incubated with goat anti-RSK2 antibody (1 :200) together with protein G-Sepharose at 4°C overnight.
  • the beads are collected by microcentrifugation, washed twice with lysis buffer, followed by kinase buffer.
  • RSK2 phosphotransferase activity in the beads is measured by
  • an additional assay that can be applied to determine activation of osteoblasts is an electrophoretic mobility gel shift assay (EMSA).
  • EMSA electrophoretic mobility gel shift assay
  • Nuclei of treated osteoblasts are isolated and their extracts generated.
  • the nuclear proteins are then incubated with a specific oligonucleotide probe that has been labeled with 32 P orthophosphate.
  • the putative protein- DNA complexes are separated on a PAGE gel (no SDS present), which is dried and exposed to an X-ray film.
  • a band will be visible on the developed film.
  • appropriate controls are run in parallel with the experimental sample(s) in order to ensure that the band is specific for activated osteoblasts.
  • the activation in osteoblasts can be detected up to at least 60 minutes following the incubation of said cells with oligomers, such as GST-RANKL. In osteoblast precursor cells, the activation peaks after 5-10 minutes, and can be detected for up to at least 60 minutes.
  • the activity of one or more intracellular proteins may be detected for at least about 30 minutes after the incubation of the osteogenic compound with osteoblasts or osteoblast precursors.
  • the activity is detected for at least about 40 minutes, and more preferably for at least about 60 minutes after said incubation.
  • the intracellular proteins whose activity is detected for at least about 30 minutes are kinases, and more preferably, the kinases are ERK1/2.
  • a compound that activates osteoblasts and/or stimulates differentiation of osteoblast precursors can enhance anabolic bone processes, such compound can be tested in a bone formation assay, wherein an increase in bone mass over the increase in background bone mass designates a compound as having osteogenic activity.
  • bone formation assays there are multiple bone formation assays that can be used successfully to screen potential osteogenic compounds of this invention.
  • cell-based assays for osteoblast differentiation and function based on measuring collagen levels and alkaline phosphatase activity may be used.
  • these assays are well known in the art and easily performed by a skilled artisan.
  • multiple in vitro and in vivo bone formation assays have been described in above sections. It should be noted that in vitro assays may be performed with either osteoblasts or osteoblast precursors since both cell types exhibit prolonged activity of the same kinases following stimulation with anabolic forms of RANKL, such as GST-RANKL.
  • Neogenesis automated ligand identification system
  • data analysis software allow for a highly specific identification of a ligand structure based on the exact mass of the ligand.
  • One skilled in the art may also perform mass spectrometry experiments to determine the identity of the compound.
  • osteogenic compounds capable of inactivating one or more phosphatases in osteoblasts or osteoblast precursors, wherein said inactivation is indicative of bone formation may be used in the methods of the present invention.
  • the phosphatases inhibit the kinases involved in osteogenesis, including p38, ERKs, JNK, IKK, and Akt. More preferably, the phosphatases are MAPK specific or Akt specific, and even more preferably they are ERK1/2 specific. While not being bound to a particular theory, this method is feasible for this purpose due to the fact that a kinase activity is tightly regulated by its corresponding phosphatase.
  • the phosphatase is known as the mitogen activated protein kinase phosphatase-3 (MKP-3).
  • MKP-3 mitogen activated protein kinase phosphatase-3
  • This phosphatase belongs to a family of dual specificity phosphatases, which are responsible for the removal of phosphate groups from the threonine and tyrosine residues on their corresponding kinases (Camps et al., FASEB J., 14, pp.6-16, 1999).
  • the prompt removal of phosphate groups by phosphatases ensures that kinase activation is short-lived and that the level of phosphorylation is low in a resting cell.
  • osteoblasts/osteoblast precursors In order for the phosphatase to be active and remove phosphate groups, it also needs to be phosphorylated. Therefore, inhibition of phosphatase activity results in activation or prolongation of ERK1/2 activity.
  • One method of determining the ability of an osteogenic compound to inactivate phosphatases in osteoblasts/osteoblast precursors involves initially activating osteoblasts/osteoblast precursors with a substance known to activate these cells, such as GST-RANKL or BMP-2 (bone morphogenetic protein 2). This leads to activation of phosphatases, at which point osteoblasts/osteoblast precursors are treated with a test compound and cell lysates are obtained.
  • test compound dephosphorylate (inactivate) phosphatase(s) is determined by performing Western blots or kinase assays. See above. For additional details on assessing phosphatase activity, see Muda et al., J Biol Chem., 273:9323-9329, 1998, and Camps et al., Science 280:1262-1265, 1998. If the compound is determined to possess phosphatase inhibitory activity, it can further be tested in one of the bone formation assays to determine its osteogenic activity. These assays were also described above.
  • a method of preventing or inhibiting bone loss or of enhancing bone formation is provided by administering 1 ) oligomeric complexes of one or more of RANKL, a RANKL fusion protein, analog, derivative, or mimic, 2) osteogenic compounds capable of enhancing activity of intracellular proteins in osteoblasts or osteoblast precursors, wherein said activity is indicative of bone formation, or 3) osteogenic compounds capable of inactivating intracellular proteins in osteoblasts or osteoblast precursors, wherein said inactivation is indicative of bone formation.
  • the bone forming compositions of the present invention may be utilized by providing an effective amount of such compositions to a patient in need thereof.
  • compositions are used to treat conditions selected from the group consisting of: osteoporosis, juvenile osteoporosis, osteogenesis imperfecta, hypercalcemia, hyperparathyroidism, osteomalacia, osteohalisteresis, osteolytic bone disease, osteonecrosis, Paget's disease, rheumatoid arthritis, inflammatory arthritis, osteomyelitis, corticosteroid treatment, periodontal disease, skeletal metastasis, cancer, age-related bone loss, osteopenia, and degenerate joint disease.
  • the compounds of the invention can be formulated as pharmaceutical or veterinary compositions.
  • a summary of such techniques is found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA.
  • the administration of RANKL-comprising oligomers or osteogenic compounds of the present invention may be pharmacokinetically and pharmacodynamically controlled by calibrating various parameters of administration, including the frequency, dosage, duration mode and route of administration.
  • bone mass formation is achieved by administering anabolic compositions such as an oligomeric complex of one or more of RANKL, a RANKL fusion protein, analog, derivative or mimic in a non-continuous, intermittent manner, such as by daily injection and/or ingestion.
  • anabolic compositions such as an oligomeric complex of one or more of RANKL, a RANKL fusion protein, analog, derivative or mimic in a non-continuous, intermittent manner, such as by daily injection and/or ingestion.
  • any osteogenic compound as described herein may be administered intermittently to achieve the same affect.
  • Variations in the dosage, duration and mode of administration may also be manipulated to produce the activity required.
  • the dosage of the compounds of the invention is typically 0.01-100mg/kg. However, dosage levels are highly dependent on the nature of the disease or situation, the condition of the patient, the judgment of the practitioner, and the frequency and mode of administration. If the oral route is employed, the absorption of the substance will be a factor effecting bioava
  • the appropriate dosage of the substance should suitably be assessed by performing animal model tests, wherein the effective dose level (e.g. ED 50 ) and the toxic dose level (e.g. TD 50 ) as well as the lethal dose level (e.g. LD 50 or LD 10 ) are established in suitable and acceptable animal models. Further, if a substance has proven efficient in such animal tests, controlled clinical trials should be performed.
  • effective dose level e.g. ED 50
  • TD 50 toxic dose level
  • LD 50 or LD 10 lethal dose level
  • compositions of the invention may be used alone or in combination with other compositions for the treatment of bone loss.
  • Such compositions include anti-resorptives such as a bisphosphonate, a calcitonin, a calcitriol, an estrogen, SERM's and a calcium source, or a supplemental bone formation agent like parathyroid hormone or its derivative, a bone morphogenetic protein, osteogenin, NaF, or a statin. See U.S. Patent No. 6,080,779 incorporated herein by reference.
  • the compounds will be formulated into suitable compositions.
  • Formulations may be prepared in a manner suitable for systemic administration or for topical or local administration.
  • Systemic formulations include, but are not limited to those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection) or may be prepared for transdermal, transmucosal, or oral administration.
  • the formulation will generally include a diluent as well as, in some cases, adjuvants, buffers, preservatives and the like.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • the compounds can be administered also in liposomal compositions or as microemulsions. Suitable forms include syrups, capsules, tablets, as is understood in the art.
  • formulations can be prepared in conventional forms as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions.
  • Suitable excipients include, for example, water, saline, dextrose, glycerol and the like.
  • Such compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, and so forth.
  • RANKL-comprising oligomers and osteogenic compounds described herein also may be administered locally to sites in patients, both human and other vertebrates, such as domestic animals, rodents and livestock, where bone formation and growth are desired using a variety of techniques known to those skilled in the art.
  • these may include sprays, lotions, gels or other vehicles such as alcohols, polyglycols, esters, oils and silicones.
  • Such local applications include, for example, at a site of a bone fracture or defect to repair or replace damaged bone.
  • oligomeric complexes and osteogenic compounds of the present invention may be administered e.g., in a suitable carrier, at a junction of an autograft, allograft or prosthesis and native bone to assist in binding of the graft or prosthesis to the native bone.
  • compositions include, but are not limited to, physiological saline, Ringer's, tocopherol, phosphate solution or buffer, buffered saline, and other carriers known in the art.
  • Pharmaceutical compositions may also include stabilizers, anti-oxidants, colorants, and diluents.
  • Pharmaceutically acceptable carriers and additives are chosen such that side effects from the pharmaceutical compound are minimized and the performance of the compound is not canceled or inhibited to such an extent that treatment is ineffective.
  • RANKL as a GST-RANKL fusion protein.
  • cDNA encoding murine RANKL residues 158-316 was cloned into pGEX-4T-1 (Amersham; GenBank Accession No. U 13853 - see National Library of Medicine listing at http://ncbi.nlm.nih.gov under nucleic acids.) downstream of glutathione S- transferase using the Sail and Notl restriction endonucleases.
  • the isolated protein was then subjected to ion exchange chromatography, eluted with a salt gradient ranging from 0-500mM NaCI, and dialyzed against physiologic salt and pH.
  • Purified GST-RANKL was then assayed for endotoxin contamination by limulus amoebocyte lysate assay, and quantitated for bioactivity by an in vitro osteoclastogenesis readout.
  • GST-RANKL forms large oligomeric complexes, as demonstrated by size exclusion chromatography. See FIG. 2. The majority of the protein, as determined by the area under the curve in FIG. 2, exists as oligomeric complexes of GST-RANKL.
  • calvarial thickness was determined histomorphometrically and compared among the various control and experimental groups to assess bone formation. Briefly, calvarial bones were removed from the incubation medium, fixed in 10% neutral buffered formalin for 12 hours, decalcified in 14% EDTA for 3 days, dehydrated through graded alcohols, and embedded in paraffin for histological sectioning. Calvaria were sectioned coronally through the central portion of the parietal bone, perpendicular to the sagittal suture. Representative coronal sections of comparable anatomic position were subjected to histomorphometric assessment (OsteoMeasure, Osteometrics Inc., Atlanta, GA) of calvarial thickness. See FIG. 3. GST-RANKL induced a dose-dependent increase in cavarial thickness when administered 1X or 2X. See FIG. 4. At the highest doses tested (100 ng/ml) calvarial thickness had doubled.
  • mice In vivo stimulation of bone formation in mice.
  • Mice, C3H/HeN (Harlan, Indianapolis, IN) were administered 100 micrograms GST (control) or 100 micrograms GST-RANKL as obtained in Example 1 , subcutaneously, once a day for nine days. Histological examination of tibia reveals a marked increase in bone mass and a net increase in the numbers of activated osteoblasts in GST-RAN KL-treated as compared to control mice. See FIGS. 5(a) and 5(b), taken at low power and high power magnification, respectively. The figures revealed a marked increase in cortical thickness and augmentation of the trabecular architecture of the primary spongiosa, relative to control animals receiving GST.
  • Dual-energy X-ray absorptiometry (DEXA) analysis of GST or GST-RANKL administered mice was also conducted using standard procedures. Results (see FIG. 5(c) show a significant increase in bone mineral density of GST-RANKL compared to control.
  • GST-RANKL versus GST control, was accomplished by intraperitoneal administration of 20 mg/kg calcein in 2% NaHC0 3 seven and two days before euthanasia to allow incorporation of two fluorescent labels into mineralizing bone matrix. Following dissection, calvaria were fixed in 70% EtOH and embedded in polymethyl methacrylate for histological sectioning. Shown in FIG. 9 are fluorescent micrographs of coronal sections of the parietal bone taken mid-way between the coronal and lambdoidal sutures, with the external surface of the calvarium oriented upwards on the figure and the internal surface oriented downwards. The amount of bone synthesized during the five day period is that encompassed within the two sets of parallel fluorescent bands. While the magnitude of bone formation in control animals receiving only GST is insufficient to produce distinctly separated double labels, there is clear deposition of bone during the five days between the first and second labels in GST-RANKL-treated animals.
  • GST-RANKL stimulates osteoblast proliferation without substantially affecting osteoclastogenesis.
  • Purified GST-RANKL fusion product was administered subcutaneously to mice C3H/HeN (Harlan, Indianapolis, IN), in increasing dosages of 5, 50, 500, 1 ,500, 5,000 ⁇ g/kg, once a day, for 7 days.
  • GST in moles equivalent to the highest dosage of RANKL served as a negative control.
  • the mice were sacrificed and long bones were fixed, decalcified and stained for tartrate resistant acid phosphatase (TRAP) activity.
  • TRAP activity is a specific phenotypic marker of the osteoclast in the context of bone.
  • the number of activated osteoblasts and osteoclasts, per mm trabecular bone surface was histomorphometrically quantitated.
  • GST-RANKL administered in an intermittent fashion resulted in a dose-dependent increase in activated osteoblast, but not osteoclast number.
  • GST had no noticeable impact on either osteoblasts or osteoclasts.
  • Marrow cells derived from GST-RANKL treated mice generated substantially more mineralized bone nodules than did their GST administered counterparts (See FIG. 8).
  • BMMs bone marrow macrophages
  • Cells were lysed in the buffer containing 20 mM Tris (pH 7.5), 150 mM NaCI, 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100, 2.5 mM sodium pyrophoshate, 1 mM ⁇ -glycerophosphate, 1 mM Na 3 P0 4 , 1 mM NaF, and 1X protease inhibitor cocktail.
  • Fifty ⁇ g of cell lysates were boiled in the presence of SDS sample buffer (0.5 M Tris-HCI, pH 6.8, 10% w/v SDS, 10% glycerol, 0.05% w/v bromphenol blue) for 5 minutes and separated on SDS-PAGE, using 8% gels.
  • Proteins were transferred to nitrocellulose membranes using a semi- dry blotter (Bio-Rad, Richmond, CA) and incubated in blocking solution (5% non-fat dry milk in tris-buffered saline containing 0.1 % Tween 20) for 1 hour to reduce nonspecific binding. Membranes were then exposed to primary antibodies overnight at 4°C, washed three times, and incubated with secondary goat anti-mouse or rabbit IgG horseradish peroxidase-conjugated antibody for 1 hour. Membranes were washed extensively, and enhanced chemiluminiscence detection assay was performed following the manufacturer's directions (Amersham).
  • Osteoclast precursors were isolated, maintained, and manipulated as described in
  • Example 7 Immnublotting protocol was also the same as in Example 7, except that a primary antibody was specific for phospho-Akt, obtained from Cell Signaling.
  • Fig. 11 shows that there was a detectable phosphorylation of Akt at the time of GST-RANKL stimulation, indicating rapid activation of this protein.
  • Akt is a substrate for PI3 kinase, and in its active state is involved in anti-apoptotic signaling.
  • Akt activity increased with time, i.e. the number of phosphorylated Akt molecules in osteoclast precursors increased with time.
  • the activity of Akt was greater at 5 minutes than at 0 minutes, and it peaked at 15 minutes following GST-RANKL stimulation.
  • Example 9 GST-RANKL-induced activity of MAP kinases is prolonged in murine osteoblasts.
  • Primary osteoblasts were isolated from neonatal murine calvaria by sequential enzymatic digestion. Briefly, calvaria were minced and incubated at room temperature for 20 minutes with gentle shaking in an enzymatic solution containing 0.1 % collagenase, 0.05% trypsin, and 4 mM NA 2 EDTA in calcium- and magnesium- free phosphate buffered saline (PBS). This procedure was repeated to yield a total of six digests. The cells isolated from the last four to six digests were cultured in MEM containing 15% FBS, 50 ⁇ M ascorbic acid, and 10 mM ⁇ -glycerophosphate.
  • Cells were maintained at 37"C in a humidified atmosphere containing 6% C0 2 , with daily replenishment of media and cytokines. Following cytokine treatment at the indicated times and dosages, cells were lysed in RIPA buffer containing 10 mM Tris-HCI pH 7.4, 150 mM NaCI, 1 % Nonidet P-40, 0.2% sodium deoxycholate, and 1 mM EDTA, with 1 mM Na 3 P0 4 , 1 mM NaF, and 1X protease inhibitor cocktail added immediately prior to use. Protein concentration was quantitated and standardized by Micro BCA Protein Assay (Pierce). Lysates were denatured by heat in Laemmli buffer, resolved by SDS- PAGE, and transferred onto nitrocellulose.
  • ERK, JNK, p38, Akt, and IkB ⁇ were determined using primary and secondary antibodies according to the manufacturer's established protocols, with conventional chemiluminiscent detection. Membranes were stripped between hybridizations in PBS containing 10 ⁇ M ⁇ -mercaptoethanol and 2% SDS. The results of the immunoblot assay measuring the activity of MAP kinases following GST-RANKL or equimolar RANKL stimulation are shown in Fig. 12. GST- RANKL stimulation was performed as described in Example 7. The kinases whose phosphorylation was measured include ERK, JNK, p38, and Akt.
  • osteoclast precursors the amount of total protein did not significantly change in the cell at any time points.
  • all of the kinases tested exhibited prolonged activity in osteoblasts. Both ERKs were activated by 5 minutes after GST-RANKL stimulation, and their activity could be detected at 60 minutes following the stimulation. The activity of JNK, p38, and Akt was detectable at the time of GST- RANKL stimulation, and could be detected for at least 60 minutes following the stimulation.
  • phosphorylation of IkB ⁇ was detected 10 minutes after the stimulation and it increased until the end of the assay (60 minutes), indicating increased translocation of NFkB into the nucleus.
  • ERK activity in osteoblast precursors was prolonged and it increased with time. Whereas in osteoblasts the activity was prolonged but did not change significantly over time, ERK activity in osteoblast precursors was first detected at 10 minutes following GST-RANKL stimulation, and it increased up to 60 minutes following the activation, which was the length of time for which the assay was performed.
  • Example 11 AP activity following GST-RANKL exposure in osteoblasts.
  • Primary calvarial osteoblasts were cultured in MEM containing 15% FBS, 50 ⁇ M ascorbic acid, and 10 mM ⁇ -glycerophosphate. Cells were maintained at 37"C, with daily replenishment of media and cytokines.
  • Osteoblast alkaline phosphatase (AP) activity was quantitated by addition of a colorimetric substrate, 5.5 mM p-nitrophenyl phosphate. The cells were then exposed to GST-RANKL, administered in different regimens.
  • GST-RANKL was subjected to proteolysis to isolate the cleaved RANKL fragment from its GST fusion partner. Briefly, GST- RANKL was incubated with the type-14 human rhinovirus 3C protease (Amersham Pharmacia Biotech) for 4 hours at 4°C in 50 mM Tris-HCI, pH 7.0, 150 mM NaCI, 10 mM EDTA, and 1 mM DTT. Uncleaved fusion protein and GST-tagged protease were removed by passage over a glutathione affinity matrix.
  • Elution volumes were calibrated to molecular weight using the following standards: ribonuclease A (13,700), chymotrypsinogen A (25,000), ovalbumin (43,000), bovine serum albumin (67,000), aldolase (158,000), catalase (232,000), ferritin (440,000), thyroglobulin (669,000), and blue dextran 2000 (2,000,000).
  • Fractions containing protein from different elution volumes were subjected to Western analysis using a monoclonal anti-GST primary antibody. As FIG.
  • Example 14 Expression of Type I collagen and Cbfal in response to GST-RANKL.
  • mice were administered 5 ⁇ g/kg GST-RANKL or GST alone as a control by subcutaneous injection and euthanized one hour later.
  • primary osteoblasts were exposed to 100 ng/ml GST-RANKL or GST alone as a control.
  • RNA was isolated with the RNeasy Total RNA System (Qiagen) and digested with deoxyribonuclease to eliminate genomic DNA.
  • RNA was subsequently isolated from total RNA with the Oligotex mRNA Purification System (Qiagen) and analyzed with the Platinum Quantitative RT-PCR Thermoscript One-Step System (Life Technologies). Briefly, 1 ⁇ g mRNA was reverse-transcribed to cDNA using murine gene-specific oligonucleotide primers designed to span exon-intron boundaries: Cbfal sense 5'-
  • Type I collagen synthesized by osteoblasts, is the major organic component of bone.
  • primary osteoblasts gradually upregulate collagen expression as they differentiate in culture.
  • Intermittent GST-RANKL exposure accelerates this process, inducing robust collagen expression within 12 hours of initial exposure to it.
  • Cbfal is the master transcription factor for osteoblastogenesis, and its absence results in a complete lack of osteoblasts and bone formation in mice (see, e.g., Otto et al., Cell 89, pp.765-771 , 1997, and Komori et al., Cell 89, pp. 755- 764, 1997).
  • expression of Cbfal is enhanced in the marrow within one hour of systemic GST-RANKL administration relative to the expression of control animals receiving GST alone.
  • GST-RANKL stimulates osteoblast proliferation.
  • the proliferation rate of osteoblasts in vitro was assessed by incorporation of 5-bromo-2'-deoxyuridine (brdU) into DNA. Briefly, cells were cultured in the presence of 10 ⁇ M BrdU for 48 hours, in the presence or absence of 100 ng/ml GST-RANKL, or a molar equivalent of GST alone as control. BrdU incorporation was quantitated by ELISA (Amersham Pharmacia Biotech) using a peroxidase-labelled anti-BrdU antibody. Spectrophotometric measurement was performed at 450 nm following addition of the colorimetric substrate 3,3'-5,5'-tetramethylbenzidine.
  • ERK activation is involved in anabolic effects of GST-RANKL.
  • a kinase- defective ERK1 cDNA (see Robbins et al., J. Biol. Chem., 268, pp.5097-5106, 1993) used in this experiment was a result of mutating alanine nucleotides at positions 211 and 212 to cytosine and guanine, respectively, resulting in replacement of lysine 71 with arginine (Erk1 K71 R).
  • ERK1 K71 R functions in a dominant-negative fashion to block both ERK1 and ERK2 activities (see Li et al., Immunol., 96, pp.524-528, 1999).
  • the ERK1 K71 R cDNA was cloned into the Ncol and BamHI restriction endonuclease sites of the SFG retroviral vector as described previously (see Ory et al., Proc. Natl. Acad. Sci. USA, 93, pp.11400-11406, 1996).
  • VSV vesicular stomatitis virus
  • the SFG-ERK1 K71 R retroviral vector was transfected into a 293GPG packaginig cell line that expresses Mul V gag-pol and VSV-G glycoprotein under tetracycline regulation.
  • Conditioned medium was harvested following tetracycline withdrawal from days 3 to 7, and found to contain a viral titer ⁇ 5X10 6 colony forming units/ml. Before transduction, the medium was filtered through a 0.45 ⁇ m membrane, and hexadimethrine bromide (polybrene) was added to a concentration of 8 ⁇ g/ml. As a negative control, a retrovirus carrying a LacZ cDNA was generated in the same fashion.
  • FIG. 20(a) shows that osteoblasts transduced with dominant-negative ERK failed to phosphorylate RSK, a known downstream ERK substrate in response to a treatment with GST-RANKL.
  • Fig. 20(b) shows that osteoblasts transduced with dominant-negative ERK failed to upregulate expression of type I collagen in response to GST-RANKL.
  • cDNA encoding murine RANKL residues 158-316 is cloned into the appropriate vector using the appropriate restriction endonucleases.
  • a cDNA encoding the human alkaline phosphatase 1 is isolated from a cDNA library and spliced upstream (at amino terminal) of a RANKL cDNA in a suitable mammalian expression vector, such as, e.g., pcDNA3.1 , using appropriate restriction endonucleases, such that the resulting DNA sequence is in frame, with no intervening stop codons.
  • the resulting vector is transduced into a mammalian cell line, suce as, e.g., CHO cells by standard methods.
  • Purified AP-RANKL is then assayed for endotoxin contamination by limulus amoebocyte lysate assay, and quantitated for bioactivity by an in vitro osteoclastogenesis readout.
  • Human AP 1 is a secreted protein, and as a result, AP fusion protein is secreted into the media.
  • the media is affinity purified to isolate AP-RANKL.
  • the empirical mass of the AP-RANKL fusion protein is determined by mass spectrometry.
  • the ability of AP-RANKL to form oligomeric complexes is checked by size exclusion chromatography.
  • Example 18 Expression of RANKL as a GCN4-RANKL fusion protein.
  • cDNA encoding murine RANKL residues 158-316 is cloned into the appropriate vector using the appropriate restriction endonucleases.
  • a DNA sequence encoding the GCN4 peptide is spliced upstream (at amino terminal) of a RANKL cDNA in a suitable expression vector, such as, e.g., pGEX-6P-1 (Accession No. U78872), using appropriate restriction endonucleases, such that the resulting DNA sequence is in frame, with no intervening stop codons.
  • the empirical mass of the GCN4-RANKL fusion protein is determined by mass spectrometry.
  • the ability of GCN4-RANKL to form oligomeric complexes is checked by size exclusion chromatography.
  • RANKL derivative comprising the TALL-1 flap region.
  • Murine RANKL containing residues 158-316 is mutated so that its DE loop (amino acids 245-249 containing the amino acid sequence SIKIP) is substituted with the DE loop of TALL-1 (amino acid sequence KVHVFGDEL).
  • the mutations can be introduced into RANKL by PCR-driven site-directed mutagenesis, using the QuickChange Multi- Site Directed Mutagenesis Kit (available from Stratagene).
  • the mutated RANKL is cloned into the appropriate vector, such as, e.g., pGEX-6P-1 (Accession No.
  • the isolated protein is then subjected to ion exchange chromatography, eluted with a salt gradient ranging from 0-500mM NaCI, and dialyzed against physiologic salt and pH.
  • Purified RANKL derivative is then assayed for endotoxin contamination by limulus amoebocyte lysate assay, and quantitated for bioactivity by an in vitro osteoclastogenesis readout.
  • the empirical mass of the mutant RANKL is determined by mass spectrometry.
  • the ability of mutated RANKL to form oligomeric complexes is checked by size exclusion chromatography.
  • RANKL derivative comprising the TALL-1 flap region and additional amino acid changes.
  • Murine RANKL containing residues 158-316 is mutated so that its DE loop (amino acids 245-249 containing the amino acid sequence SIKIP) is substituted with the DE loop of TALL-1 (amino acid sequence KVHVFGDEL).
  • the following amino acid changes are made throughout the RANKL molecule to increase the similarity with the TALL-1 structure: 168T-I, 187Y ⁇ L, 194K-F, 212F-Y, 252H-V, 279F-I, and 283R-E.
  • the mutations can be introduced into RANKL by PCR-driven site-directed mutagenesis, using the QuickChange Multi- Site Directed Mutagenesis Kit (available from Stratagene).
  • the mutated RANKL is cloned into the appropriate vector, such as, e.g., pGEX-6P-1 using the appropriate restriction endonucleases such that the resulting DNA sequence is in frame, with no intervening stop codons.
  • the appropriate vector such as, e.g., pGEX-6P-1
  • the appropriate restriction endonucleases such that the resulting DNA sequence is in frame, with no intervening stop codons.
  • IPTG-mediated (0.05mM) induction of protein expression in BL21 (DE3) Escherischia coli (Invitrogen) cells are triturated into a lysis buffer comprising 150mM NaCI, 20mM Tris-HCI pH 8.0, and 1 mM EDTA.
  • Lysates are incubated with glutathione sepharose (Amersham) for affinity purification of the mutated RANKL protein, followed by excessive washing with buffer comprising 150mM NaCI and 20mM Tris-HCI pH 8.0. Following competitive elution (10mM reduced glutathione) from the affinity column, The isolated protein is then subjected to ion exchange chromatography, eluted with a salt gradient ranging from 0-500mM NaCI, and dialyzed against physiologic salt and pH. Purified RANKL derivative is then assayed for endotoxin contamination by limulus amoebocyte lysate assay, and quantitated for bioactivity by an in vitro osteoclastogenesis readout. The empirical mass of the mutant RANKL is determined by mass spectrometry. The ability of mutated RANKL to form oligomeric complexes is checked by size exclusion chromatography.
  • Neo-natal mouse calvariae are placed in organ culture in the presence of vehicle, AP (a negative control), or increasing concentrations of purified AP-RANKL.
  • Bone morphogenetic protein (BMP)-2 is administered as a positive control.
  • Test compositions are administered for a period of 12 hours only at the initiation of the culture (1X) or once at initiation and once three days later, again for a duration of 12 hours (2X). After seven days, calvarial thickness is determined histomorphometrically and compared among the various control and experimental groups to assess bone formation.
  • Dual-energy X-ray absorptiometry (DEXA) analysis of AP or AP-RANKL administered mice is also conducted using standard procedures to assess the change in bone mineral density in AP-RANKL mice compared to AP-treated mice.
  • DEXA Dual-energy X-ray absorptiometry
  • mice In vivo stimulation of bone formation in mice. Mice, C3H/HeN (Harlan, Indianapolis, IN) are administered 100 micrograms GCN4 (control) or 100 micrograms GCN4-RANKL subcutaneously, once a day for nine days. Histological examination of tibia is then performed to assess the increase in bone mass and a net increase in the numbers of activated osteoblasts in GCN4-RANKL-treated as compared to control mice.
  • Dual-energy X-ray absorptiometry (DEXA) analysis of GCN4 or GCN4- RANKL administered mice is also conducted using standard procedures to assess the change in bone mineral density in GCN4-RANKL mice compared to GCN4- treated mice.
  • DEXA Dual-energy X-ray absorptiometry
  • Example 25 Expression of RANKL as a GST-RANKL fusion protein.
  • cDNA encoding murine RANKL residues 158-316 was cloned into pGEX-6p-1 (Amersham; GenBank Accession No. U78872 - see National Library of Medicine listing at http://ncbi.nlm.nih.gov under nucleic acids.) downstream of glutathione S- transferase using the Sail and Notl restriction endonucleases.
  • the isolated protein was then subjected to ion exchange chromatography, eluted with a salt gradient ranging from 0- ⁇ OOmM NaCI, and dialyzed against physiologic salt and pH.
  • Purified GST-RANKL was then assayed for endotoxin contamination by limulus amoebocyte lysate assay, and quantitated for bioactivity by an in vitro osteoclastogenesis readout.
  • GST-RANKL formed large oligomeric complexes, as demonstrated by size exclusion chromatography (data not shown). The majority of the protein existed as oligomeric complexes of GST-RANKL (data not shown).
  • mice Twenty, six week old C67BL/6 mice were randomly assigned to two experimental groups. Group 1 mice (10) received 100ug injection of GST- RANKL in the intramedullary cavity of the right femur. Group 2 mice (10) received an equimolar volume injection of GST vehicle in the intramedullary cavity of the right femur.
  • mice were anesthetized with a Ketamine/Xylazine cocktail (100mg/kg ketamine and 10mg/kg xylazine IP) and placed in left lateral recumbancy.
  • the major trochanter and lateral femoral condyle of the right femur were identified and the intramedullary injection site was equidistant between these landmarks.
  • the injections were made with 29 gauge needles on tuberculin syringes.
  • Ketamine/Xylazine cocktail 100mg/kg ketamine and 10mg/kg xylazine IP
  • DEXA dual energy x-ray absorptiometry
  • the DEXA analysis showed a significant difference in total bone mineral density (TBMD) between GST-RANKL-treated group and the control group ( see Table 1 ). No significant difference was seen in either GST-RANKL or control group when comparing bone mineral density of the right and left femurs (see Table 2). There was no significant difference in skeletal density when comparing plain radiographs of both groups.
  • TBMD total bone mineral density

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Rheumatology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Diabetes (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Urology & Nephrology (AREA)
  • General Engineering & Computer Science (AREA)
  • Endocrinology (AREA)
  • Oncology (AREA)
  • Pain & Pain Management (AREA)

Abstract

L'invention concerne un procédé d'amélioration de la formation osseuse, consistant à administrer une quantité efficace 1) d'un complexe oligomérique d'un ou plusieurs éléments parmi RANKL, une protéine de fusion RANKL ou un analogue, un dérivé ou un mimétique de cette dernière, 2) d'un composé ostéogénique capable d'améliorer l'activité d'une ou de plusieurs protéines intracellulaires dans des ostéoblastes ou des précurseurs d'ostéoblastes, ladite activité étant indicative d'une formation osseuse, ou 3) d'un composé ostéogénique capable d'inactiver une ou plusieurs phosphatases dans des ostéoblastes ou des précurseurs d'ostéoblastes, ladite inactivation étant indicative d'une formation osseuse. Le procédé peut également être utilisé pour traiter une maladie ou une condition se manifestant au moins en partie par la perte de masse osseuse, en administrant à un patient une composition pharmaceutique renfermant un complexe oligomérique ou un composé ostéogénique décrit ci-dessus.
EP02741650A 2001-03-22 2002-03-22 Stimulation d'osteogenese utilisant des proteines de fusion de ligands rank Withdrawn EP1385532A4 (fr)

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
US27785501P 2001-03-22 2001-03-22
US277855P 2001-03-22
US31116301P 2001-08-09 2001-08-09
US311163P 2001-08-09
US32887601P 2001-10-12 2001-10-12
US32923101P 2001-10-12 2001-10-12
US329231P 2001-10-12
US328876P 2001-10-12
US32939301P 2001-10-15 2001-10-15
US329393P 2001-10-15
PCT/US2002/009271 WO2002080955A1 (fr) 2001-03-22 2002-03-22 Stimulation d'osteogenese utilisant des proteines de fusion de ligands rank

Publications (2)

Publication Number Publication Date
EP1385532A1 true EP1385532A1 (fr) 2004-02-04
EP1385532A4 EP1385532A4 (fr) 2004-12-15

Family

ID=27540621

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02741650A Withdrawn EP1385532A4 (fr) 2001-03-22 2002-03-22 Stimulation d'osteogenese utilisant des proteines de fusion de ligands rank

Country Status (5)

Country Link
EP (1) EP1385532A4 (fr)
JP (1) JP2004526748A (fr)
CA (1) CA2441538A1 (fr)
EA (1) EA200301041A1 (fr)
WO (1) WO2002080955A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6884598B2 (en) 2000-09-22 2005-04-26 Immunex Corporation Screening assays for agonists and antagonists of receptor activator of NF-κB
SI1732575T1 (sl) * 2004-02-26 2011-05-31 Osteologix As Spojine, ki vsebujejo stroncij, za uporabo pri preprečevanju ali zdravljenju nekrotičnih kostnih stanj
EP1736482A1 (fr) * 2005-06-20 2006-12-27 INSERM (Institut National de la Santé et de la Recherche Medicale) Ligand recombinant trimeric de 4-1BB
EP2020445B1 (fr) 2006-05-12 2013-01-02 Keio University Détection d'une maladie inflammatoire et composition destinée à la prévention ou au traitement d'une maladie inflammatoire
CA2666415C (fr) * 2006-10-11 2012-11-27 Oriental Yeast Co., Ltd. Reactif contenant une proteine fusionnee de rankl soluble avec un marqueur d'epitope
WO2008044379A1 (fr) * 2006-10-11 2008-04-17 Oriental Yeast Co., Ltd. Modèle animal de perte osseuse
WO2008044797A1 (fr) * 2006-10-11 2008-04-17 Oriental Yeast Co., Ltd. Animal modèle d'ostéopénie
WO2010048610A2 (fr) * 2008-10-24 2010-04-29 Osteotech, Inc. Compositions et procédés pour favoriser la formation osseuse
CN114391506A (zh) * 2021-12-24 2022-04-26 华中科技大学同济医学院附属协和医院 一种恢复节律促进骨组织生长发育的运动方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999058674A2 (fr) * 1998-05-14 1999-11-18 Immunex Corporation Procede pour inhiber l'activite osteoclastique
WO2000015807A1 (fr) * 1998-09-15 2000-03-23 M & E Biotech A/S Procede de regulation negative de l'activite des ligands d'osteoprotegerine
WO2001042298A1 (fr) * 1999-12-09 2001-06-14 Kornbluth Richard S Formes multimeres de ligands de la superfamille tnf

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69738841D1 (de) * 1996-12-23 2008-08-28 Immunex Corp Ligand für rezeptor aktivator of nf-kappa b, ligand ist mitglied der tnf superfamilie

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999058674A2 (fr) * 1998-05-14 1999-11-18 Immunex Corporation Procede pour inhiber l'activite osteoclastique
WO2000015807A1 (fr) * 1998-09-15 2000-03-23 M & E Biotech A/S Procede de regulation negative de l'activite des ligands d'osteoprotegerine
WO2001042298A1 (fr) * 1999-12-09 2001-06-14 Kornbluth Richard S Formes multimeres de ligands de la superfamille tnf

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KONG Y-Y ET AL: "Osteoprotegerin ligand: a regulator of immune responses and bone physiology" IMMUNOLOGY TODAY, ELSEVIER PUBLICATIONS, CAMBRIDGE, GB, vol. 21, no. 10, 1 October 2000 (2000-10-01), pages 495-502, XP004217459 ISSN: 0167-5699 *
LACEY D L ET AL: "Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation" CELL, CELL PRESS, CAMBRIDGE, NA, US, vol. 93, no. 2, 17 April 1998 (1998-04-17), pages 165-176, XP002117567 ISSN: 0092-8674 *
LAM J ET AL: "RANK ligand stimulates anabolic bone formation" September 2001 (2001-09), JOURNAL OF BONE AND MINERAL RESEARCH, VOL. 16, NR. SUPPL. 1, PAGE(S) S150 , TWENTY-THIRD ANNUAL MEETING OF THE AMERICAN SOCIETY FOR BONE AND MINERAL RESEARCH; PHOENIX, ARIZONA, USA; OCTOBER 12-16, 2001 , XP009038403 ISSN: 0884-0431 * abstract * *
See also references of WO02080955A1 *

Also Published As

Publication number Publication date
WO2002080955A1 (fr) 2002-10-17
EP1385532A4 (fr) 2004-12-15
CA2441538A1 (fr) 2002-10-17
EA200301041A1 (ru) 2004-08-26
JP2004526748A (ja) 2004-09-02

Similar Documents

Publication Publication Date Title
Missbach et al. A novel inhibitor of the tyrosine kinase Src suppresses phosphorylation of its major cellular substrates and reduces bone resorption in vitro and in rodent models in vivo
Kneissel et al. Everolimus suppresses cancellous bone loss, bone resorption, and cathepsin K expression by osteoclasts
US9234036B2 (en) Modulation of activity of proneurotrophins
Li et al. Role of endoplasmic reticulum stress in disuse osteoporosis
Hale et al. CDK/ERK-mediated phosphorylation of the human influenza A virus NS1 protein at threonine-215
EP1477180A1 (fr) Inhibiteurs de l'activité de proteasome pour favoriser la croissance osseuse et la pousse des cheveux
US20030013651A1 (en) Stimulation of osteogenesis using rank ligand fusion proteins
Shi et al. Role of tyrosine phosphorylation in thrombin-induced endothelial cell contraction and barrier function
Jie et al. Phosphorylation of phosvitin plays a crucial effects on the protein-induced differentiation and mineralization of osteoblastic MC3T3-E1 cells
Guo et al. Desalted duck egg white peptides promoted osteogenesis via wnt/β‐catenin signal pathway
EP1385532A1 (fr) Stimulation d'osteogenese utilisant des proteines de fusion de ligands rank
Zhang et al. Calcitonin induces dephosphorylation of Pyk2 and phosphorylation of focal adhesion kinase in osteoclasts
Stolzenberger et al. Specific inhibition of interleukin‐4‐dependent Stat6 activation by an intracellularly delivered peptide
Xia et al. PC-PLC is involved in osteoclastogenesis induced by TNF-α through upregulating IP3R1 expression
Kawai et al. Prevention of skeletal muscle atrophy in vitro using anti-ubiquitination oligopeptide carried by atelocollagen
US9381245B2 (en) Methods for inhibiting osteolysis
Kang et al. A Vitronectin-Derived Peptide Restores Ovariectomy-Induced Bone Loss by Dual Regulation of Bone Remodeling
AU2002314726A1 (en) Stimulation of osteogenesis using rank ligand fusion proteins
US6498139B1 (en) Remedies for diseases caused by insulin resistance
MXPA03008571A (es) Estimulacion de osteogenesis usando proteinas de fusion de ligando rank.
AU763488B2 (en) Methods for maintaining or restoring tissue-appropriate phenotype of soft tissue cells
US20030092603A1 (en) Identification of specific modulators of bone formation
WO2003065972A2 (fr) Methodes de criblage de composes osteogenes
WO2016100431A1 (fr) Procédés de traitement d'arthrite inflammatoire
US20030082638A1 (en) Methods for screening osteogenic compounds

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20031022

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

A4 Supplementary search report drawn up and despatched

Effective date: 20041028

RIC1 Information provided on ipc code assigned before grant

Ipc: 7A 61P 19/08 B

Ipc: 7A 61K 38/16 B

Ipc: 7C 07K 14/00 B

Ipc: 7A 61K 38/00 A

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20050228