EP0917469A1 - Procedes permettant d'induire la formation osseuse - Google Patents

Procedes permettant d'induire la formation osseuse

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Publication number
EP0917469A1
EP0917469A1 EP97922438A EP97922438A EP0917469A1 EP 0917469 A1 EP0917469 A1 EP 0917469A1 EP 97922438 A EP97922438 A EP 97922438A EP 97922438 A EP97922438 A EP 97922438A EP 0917469 A1 EP0917469 A1 EP 0917469A1
Authority
EP
European Patent Office
Prior art keywords
bone
cells
mammal
protein
leu
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Withdrawn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
EP97922438A
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German (de)
English (en)
Inventor
Diane M. Durnam
Joseph L. Kuijper
David S. Weigle
Chung C. Liu
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.)
University of Washington
Zymogenetics Inc
Original Assignee
University of Washington
Zymogenetics Inc
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Filing date
Publication date
Application filed by University of Washington, Zymogenetics Inc filed Critical University of Washington
Publication of EP0917469A1 publication Critical patent/EP0917469A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • 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
    • A61K38/22—Hormones
    • A61K38/2264—Obesity-gene products, e.g. leptin
    • 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
    • A61P19/08—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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/355—Leptin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/07—Coculture with; Conditioned medium produced by endocrine cells
    • C12N2502/076—Coculture with; Conditioned medium produced by endocrine cells pituitary cells

Definitions

  • Bone remodeling is the dynamic process by which tissue mass and skeletal architecture are maintained. The process is a balance between bone resorption and bone formation, with two cell types thought to be the major players. These cells are the osteoblast and osteoclast .
  • Osteoblasts synthesize and deposit matrix to become new bone.
  • the activities of osteoblasts and osteoclasts are regulated by many factors, systemic and local, including growth factors.
  • Osteoporosis is a disease that afflicts many elderly people, particularly post-menopausal women. In the U.S. alone, there are approximately 1.5 million fractures annually that are attributed to osteoporosis. The impact of these fractures on the quality of the patient's life is immense. Of people identified with hip fractures due to osteoporosis, 10-20% will die and 50% of sufferers will no longer be able to walk independently (Riggs et al . , Bone 17-.505S- 511S, 1995) .
  • It is an object of the present invention to provide methods for stimulating a cell population containing marrow mesenchymal cells comprising: exposing the cell population to an amount of ob protein effective for expansion of osteogenic cells.
  • the population is enriched for marrow mesenchymal cells.
  • the hematopoietic cells have been removed.
  • the osteogenic cells are primarily osteoblasts.
  • the method further comprises the step of combining the cell population with an anti-resorptive agent.
  • the method further comprises infusing the cell population into a mammal .
  • It is a further object of the present invention to provide methods for stimulating a cell population containing marrow mesenchymal cells comprising: exposing the cell population to a biological fluid obtained from an ob protein-treated animal.
  • the biological fluid is serum.
  • the biological fluid has been fractionated to enrich for molecules effective for expansion of osteogenic cells.
  • the cells are pituitary or hypothalamic cells.
  • It is a further object of the present invention to provide methods for promoting bone repair or healing in a mammal comprising: administering to the mammal an amount of ob protein effective to provide a clinically significant increase in the bone repair or bone healing rate.
  • the mammal is an adolescent or pre-adolescent . It is a further object of the present invention to provide methods for stimulating active bone growth in mammal having a fracture in a bone growth plate T S97/06892
  • It is a further object of the present invention to provide methods for inducing bone formation in mammal having an oncological resection of a bone comprising: administering to the mammal an amount of ob protein effective to induce bone formation at the site of the resection.
  • ob/ob mice Inbred mice that are homozygous for an inactivating mutation at the ob (obese) locus. ob/ob mice are hyperphagic and hypometabolic, and are believed to be deficient in production of circulating satiety factor.
  • ob As used herein, ob or ob denotes nucleic acid. This designation is distinct from the rodent mutant phenotype designations defined above (i.e., ob/ob mice) , which are used in the format "*/*", but not in the singular "*" .
  • ob As used herein, ob denotes protein.
  • stem cell As used herein, the term “stem cell” refers to a pluripotent cell that has the potential to differentiate into multiple cell types and that is not limited to a fixed number of mitotic divisions.
  • precursor cell As used herein, the term
  • osteogenic cells refers to a cell that is committed to a differentiation pathway, but generally does not express markers or function as a mature, fully differentiated cell. osteogenic cells: As used herein, the term
  • osteoogenic cells includes osteoblasts and osteoblast precursor cells.
  • fibroblasts or reticular cells As used herein, this terms refers to cells of the soft connective tissue network; such cells are found throughout the marrow and spaces in the bone.
  • adipocytes As used herein, the term
  • adipocytes refers cells distinguished by the presence of their lipid inclusions and expression of specific gene products.
  • the present invention is based on the discovery that ob protein, when administered to mammals, produces a dramatic increase in osteogenic cells or their bone- forming activity.
  • the sequence encoding mouse and human ob protein is known (Friedman et al . , WO 96/05309) .
  • the human and mouse ob DNA and protein sequences are shown in SEQ ID NOS:l, 2, 3 and 4, respectively.
  • SEQ ID NOS. 1, 2, 3 and 4 correspond to a single allele of the human and mouse ob gene, and that allelic variation is expected to exist.
  • an allelic variant of the human and mouse ob gene does not have a Gin residue at position 49 present .
  • DNA sequence shown in SEQ ID NOS: 1 and 3, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the methods of the present invention, as are ob proteins which are allelic variants of SEQ ID NO: 2 or 4.
  • the present invention contemplates use of DNA molecules encoding ob proteins, wherein the DNA molecules are generally at least 60%, preferably at least 80%, and may be 90-95% or more identical in sequence to SEQ ID NO: 1 or
  • the present invention also contemplates use of isolated proteins that are substantially homologous to the proteins of SEQ ID NO: 2 or 4 and their species homologs.
  • isolated is meant a protein which is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated protein is substantially free of other proteins, particularly other proteins of animal origin. It is preferred to provide the proteins in a highly purified form, i.e., greater than 95% pure, more preferably greater than 99% pure.
  • substantially homologous is used herein to denote proteins having 50%, preferably at least 80%, sequence identity to the sequence shown in SEQ ID NO: 2 or 4 or their species homologs. Such proteins will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO: 2 or 4 or their species homologs.
  • Substantially homologous proteins are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding or activity of the protein; small deletions, typically of one to about 45 amino acids; and small amino- or carboxyl-terminal or internal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain. See, in general Ford et al . , Protein Expression and Purification 2 : 95- 107, 1991, which is incorporated herein by reference.
  • ob protein of the present invention can be joined to other bioactive molecules, particularly targeting moieties or anti-resorptive agents, to provide multi-functional molecules.
  • Such hybrid ob protein molecules may be formed by chemical conjugation or polynucleotide fusion or a combination thereof, and may further comprise a linker
  • polypeptide linker e.g., a polypeptide linker
  • the methods of the present invention include the use of fragments of these proteins and isolated polynucleotide molecules encoding the fragments.
  • ob protein fragments of at least 10 amino acids in length that exhibit bone-forming or osteogenic cell stimulatory activity, and polynucleotide molecules of at least 30 nucleotides in length encoding such polypeptides.
  • Polypeptides of this type are identified by known biological screening and assay methods, and can be produced by proteolytically digesting the intact ob protein or by synthesizing small, overlapping polypeptides or polynucleotides (and expressing the latter) . The resultant polypeptide fragments are then tested for bone- forming activity, or for an activity or effect associated with enhanced bone formation. Larger polypeptides up to the size of the entire mature ob protein are also useful herein.
  • ob protein has similarity to the helical cytokine structure class . Additional evidence of the possible relatedness of ob protein to the helical cytokine structure class was provided by the two forms of ob cDNA (+Gln and -Gin at position 49) . These two forms may be caused by slippage at a splice acceptor sequence, indicating the location of a exon-intron boundary (Zhang et al . , Nature 372 :425-32 , 1994) .
  • the location of the exon-intron boundary of ob was determined to be coincident with an exon-intron boundary in interleukin-3 and GM-CSF, and is within two residues of exon-intron boundaries in interleukin-2 and interleukin-6.
  • OB-R ob protein receptor
  • OB-R is a membrane- spanning receptor that is closely related to the gpl30 signal-transducing component of the IL-6, G-CSF and LIF receptors.
  • the extracellular domain of OB-R shows many similarities to the class I cytokine family.
  • a substantially homologous ob protein or ob protein fragment is capable of folding into a four alpha-helix bundle and capable of self-assembly.
  • amino acid residues that impart the four alpha-helical bundle character of the ob protein molecule are preferably maintained with little or no amino acid substitutions, additions or deletions. However, the remaining amino acid residues may tolerate more extensive amino acid substitutions, additions or deletions with retention of desired biological function.
  • Essential amino acids in the ob polypeptides useful herein can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244 , 1081-85, 1989) .
  • the DNA molecule shown in SEQ ID NO:1 was cloned by polymerase chain reaction (PCR; see Mullis et al . , U.S. Patent No. 4,683,195; Mullis, U.S. Patent No. 4,683,202) using a probe designed from the sequence of the mouse obese gene (Zhang et al . , Nature 372 :425-432, 1994) and cDNA from a human adipose tissue cDNA library (obtained from Clontech, Palo Alto, CA) . By this method, the DNA molecule shown in SEQ ID NO: 1 was isolated.
  • PCR polymerase chain reaction
  • Bone is a tissue and, therefore, contains a composite of heterogeneous cell populations. Some of the cell types found in bone include osteoblasts, osteoclasts, chondrocytes and osteocytes . Examples of other tissues include bone marrow, skin, skeletal and cardiac muscle, pancreas, brain and liver. Tissues usually consist of a mixture of tissue-specific cell types, as well as cell types that are found in many tissues, such as fibroblasts. It is within the bone marrow that many of the earliest progenitor cells are found, and these are generally described as either hematopoietic stem cells or mesenchymal stem cells.
  • Human hematopoietic stem cells are identified as CD34 + , and differentiate to become either myeloid cells (including erythrocytes, megakaryocytes, macrophages and neutrophils) or lymphoid cells (T cells and B cells) .
  • myeloid cells including erythrocytes, megakaryocytes, macrophages and neutrophils
  • lymphoid cells T cells and B cells
  • mesenchymal lineage includes stem cells and precursor cells for osteoblasts, chondrocytes, adipocytes, fibroblasts and reticular cells. Markers for mesenchymal stem cells have not been well identified (Owen et al. , J. of Cell Sci.
  • mesenchymal cells are histologically distinct from hematopoietic cells.
  • Stromal cells are generally considered the subpopulation of bone marrow cells that are adherent when cultured in vitro and are included in the mesenchymal lineage cells.
  • Differentiation is the process of maturation of cells. It is a progressive and dynamic process, beginning with pluripotent stem cells and ending with terminally differentiated cells that progress no further down the cell lineage pathway. A cell's function, phenotype and growth characteristics are affected by the cell's degree of differentiation. As stem cells or progenitor cells begin the process of differentiation, they generally migrate to selected tissues and/or organs, where they appear to undergo additional differentiation.
  • a cell's differentiated state can be identified using a set of differentiation markers that is specific to a particular cell type. Differentiation markers are transiently exhibited at various stages of cell lineage development. Pluripotent stem cells can regenerate without commitment to a lineage, and express a set of differentiation markers that are lost when commitment to a cell lineage is made. Precursor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress down the cell lineage pathway toward maturation. Differentiation markers that are expressed exclusively by mature cells are usually associated with functional properties, such as cell products, enzymes to produce cell products and receptors.
  • Mature osteoclasts express a set of differentiation markers that is selected from the group consisting of tartrate-resistant acid phosphatase (TRAP) and calcitonin receptor (Suda et al . , Endocrine Rev. 12:66-80, 1992) .
  • Identification of osteoblasts is primarily phenotypic, and includes expression of alkaline phosphatase (Manduca et al . , J. Bone Min. Res. £:281, 1993) ; type 1 collagen synthesis (Kurihara et al. , Endocrinol . 118 (3) : 940-947, 1986) ; production of osteocalcin (Rodan et al . , Crit . Rev. Eucar. Gene Expr. 1:85-98, 1991)) ; and responsiveness to parathyroid hormone (Aubin et al . , J. Cell Biol. 92:452-461, 1982) .
  • TRIP tartrate-resistant acid phosphat
  • cardiac myosin isozyme expression a cardiac specific pattern of creatine kinase isozyme expression (Yaffe et al . , Develop. Biol. 15_:33-50, 1967 and Richler et al . , Develop. Biol. 2_3:l-22, 1970) and insulin and insulin-like growth factor receptors I
  • Progenitor or precursor cells can be stimulated to differentiate, thereby providing differentiated cells. Differentiation is induced by exposing the undifferentiated stem cells or precursor cells to factors that can be supplied by surrounding cells (paracrine regulation) , by the cell itself (autocrine regulation) or by distant hormone-secreting cells (endocrine regulation) . In addition, it has been demonstrated that certain factors can be added exogenously and will stimulate differentiation of cells. For example, osteoclasts are stimulated to differentiate by exposure to vitamin D and dexamethasone. Osteoblasts are induced to differentiate by exposure to retinoic acid, TGF-b or bone morphogenic proteins (BMP) .
  • BMP bone morphogenic proteins
  • ob protein is a potent stimulator that drives osteogenic cells to become activated osteoblasts.
  • Ob protein has been previously implicated as a regulator of appetite (Friedman et al., WO 96/05309) and fertility (Chehab et al . , Nature Genetics 12:318-320, 1995) , but was previously not known to be a bone-stimulating factor.
  • the receptor for ob protein has been reported to be found in brain, kidney and lung, but not in bone (Tartaglia et al . , Cell 85:1263- 1271, 1995) .
  • test samples containing cells of the mesenchymal lineage are incubated in the presence of ob protein and in the absence of ob protein (control sample) . The test samples are then scored for cell proliferation and differentiation by visual examination and/or staining. Such test samples may be in vi tro cultured cells or may be obtained from an in vivo model system.
  • ob protein may stimulate the growth of isolated osteoblasts, as determined by [ 3 H] thymidine incorporation or [ 35 S] - labeling of proteins.
  • exposure of mesenchymal lineage cells to ob protein may modulate the biological activity of the cells (such as up-regulation or activation of bone-stimulatory molecules) , such that bone formation (i.e., in vivo) or a bone-forming molecule (i.e., in vi tro) is induced.
  • ob protein may enhance recruitment or migration of bone-forming cells to the proper bone-forming tissues and tissue sites.
  • ob protein on bone formation or osteoblast activation may be either direct or indirect. It has been reported that there is an inverse relationship between the differentiation of adipocytes and osteogenic cells in vi tro (Beresford et al . , J. Cell Sci. 102 :34l- 351, 1992) , and correlations between increased volume of marrow adipose tissue and osteoporosis have been established (Meunier et al . , Clin. Orthop. Rel. Res. £0:147-154, 1971; Burkhardt et al . , Bone £: 157-164, 1987 and Minaire et al. , Calcif. Tiss . Int.
  • Bone marrow adipocytes are known to secrete factors that are required for the maturation of other cell types found in the bone marrow (Gimble, The New Biologist 1:304-312, 1990) . Therefore, changes in levels of growth and differentiation factors due to shifts within the cell population in the bone marrow microenvironment would be expected to alter the osteoblast maturation process.
  • ob protein The effects of ob protein are believed to be both central and peripheral (Yu et al . , Proc. Natl. Acad. Sci . USA £4:1023-1028, 1997) .
  • endocrine or CNS tissues or cell types that have been exposed to ob protein and then cultured may contain additional factors that contribute to the osteogenic effects.
  • culturing of CNS cells, such as pituitary or hypothalamic cells, after exposure to ob protein may enhance the osteogenic effects.
  • PTH provides a paradigm for bone forming agents. At specific dose levels in ovariectomized mice, PTH increases osteoblast number and size (Lui et al . , J. Bone Mineral Res. ⁇ >:973-981, 1990) . This increase in osteoblast size and number corresponds with increases in cancellous bone formation.
  • Estrogen is an anti-resorptive agent and is used for treatment of osteoporosis.
  • Adipocytes have been shown to produce estrogen (Heiss et al . , J. Clin. Endocrinol. Metabolism 80:1591-1596, 1995) , and may be a primary source of estrogen in post-menopausal women.
  • Low body fat level is considered a risk factor for osteoporosis.
  • marrow fat has been shown to be increased in a variety of osteoporotic patients. When the bone loss stopped, the marrow fat levels decreased (Martin et al . , Bone 12:123-131, 1991) .
  • a composition comprising ob protein is used as a therapeutic agent to enhance osteoblast-mediated bone formation.
  • the methods of the invention may be applied to promote the repair of bone defects and deficiencies, such as those occuring in closed, open and non-union fractures; to promote bone healing in plastic surgery; to stimulate bone ingrowth into non-cemented prosthetic joints and dental implants; in the treatment of peridontal disease and defects; to increase bone formation during distraction osteogenesis; and in treatment of other skeletal disorders that may be treated by stimulation of osteoblastic activity, such as osteoporosis and arthritis.
  • De novo bone formation provided by the methods of the present invention would have use in repair of congenital, trauma- induced, oncologic resection of bone or healing bone following radiation-induced osteonecrosis (Hart et al, Cancer £7:2580-2585, 1976) .
  • the methods of the present invention may also find use in plastic surgery.
  • Ob polypeptides have been shown to augment growth hormone releasing hormone (GHRH) stimulation of growth hormone from cultured pituitary cells.
  • GHRH growth hormone releasing hormone
  • Many of the peripheral actions of growth hormone, including de novo bone formation, are mediated by IGF-I.
  • the methods of the present invention result in increased levels of IGF-I, consistant with a putative pathway in which ob protein exerts an effect on bone.
  • compositions of the present invention may be administered systemically or locally.
  • ob proteins are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, intranasal or transdermal) or enteral (e.g., oral or rectal) delivery according to conventional methods.
  • parenteral e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, intranasal or transdermal
  • enteral e.g., oral or rectal
  • Intravenous administration will be by a series of injections or by continuous infusion over an extended period.
  • Administration by injection or other routes of discretely spaced administration will generally be performed at intervals ranging from weekly to once to three times daily. Treatment will continue until the desired outcome is achieved.
  • pharmaceutical formulations will include an ob protein in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water, borate- buffered saline containing trace metals or the like.
  • a pharmaceutically acceptable vehicle such as saline, buffered saline, 5% dextrose in water, borate- buffered saline containing trace metals or the like.
  • Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, lubricants, fillers, stabilizers (for example, soluble receptors) , etc.
  • Methods of formulation are well known in the art and are disclosed, for example, in Remington's Pharmaceutical Sciences, Gennaro, ed. , Mack Publishing Co., Easton PA, 1990, which is incorporated herein by reference.
  • compositions for use within the present invention can be in the form of sterile, non-pyrogenic liquid solutions or suspensions, coated capsules, suppositories, lyophilized powders, transdermal patches or other forms known in the art.
  • Local administration may be by injection at the site of injury or defect or by insertion or attachment of a solid carrier at the site, or by direct, topical application of a viscous liquid. Delivery of ob protein to wound sites may be enhanced by the use of controlled-release compositions such as those described in pending U.S. Patent Application No. 07/871,246 (corresponding to WIPO publication WO 93/20859 which is incorporated herein by reference in it ⁇ entirety) . Films of this type are particularly useful as coatings for prosthetic devices and surgical implants.
  • the films may, for example, be wrapped around the outer surfaces of surgical screws, rods, pins, plates and the like. Implantable devices of this type are routinely used in orthopedic surgery.
  • the films can also be used to coat bone filling materials, such as hydroxyapatite blocks, demineralized bone matrix plugs, collagen matrices and the like.
  • a film or device as described herein i ⁇ applied to the bone at the fracture site. Application is generally by implantation into the bone or attachment to the surface using standard surgical procedures.
  • the biodegradable films and matrices may include other active or inert components.
  • agents that promote tissue growth or infiltration include bone morphogenic proteins (U.S. Patent No. 4,761,471; PCT Publication WO 90/11366) , osteogenin (Sampath et al . , Proc. Natl. Acad. Sci. USA 84 -. 7109-7113, 1987) and NaF (Tencer et al . , J. Biomed. Mat . Res. 23 : 571-589, 1989) are preferred.
  • Alternative methods for delivery of ob protein include use of ALZET osmotic minipumps (Alza Corp. Palo Alto, CA) ; sustained release matrix materials such as those disclosed in Wang et al . (WO 90/11366) ; electrically charged dextran beads a ⁇ disclosed in Bao et al . (WO 92/03125) ; collagen-based delivery systems, for example, as disclosed in Ksander et al . (Ann. Surg. 211 (3) :288-294, 1990) ; methylcellulose gel systems as disclosed in Beck et al. (J. Bone Min. Res. 6 (11) :1257-1265, 1991) and alginate-based sy ⁇ tems as disclosed in Edelman et al .
  • Targeting molecule refers to a molecule that binds to the tissue of interest.
  • bone-targeting molecules include tetracyclines; calcein; bisphosphonates,- polyaspartic acid; polyglutamic acid; aminophosphosugars,- peptides known to be associated with the mineral phase of bone such as osteonectin, bone ⁇ ialoprotein and osteopontin; bone specific antibodies; proteins with bone mineral binding domains and the like. See, for example, the disclosures of Bentz et al . (EP 0512844) and Murakami et al. (EP 0341961) .
  • the methods of the present invention provide the use of ob protein for treating bone loss in conjunction with HRT and other antiresorptive agents, such as bisphosphonates and calcitonin.
  • an "effective amount" of a composition is that amount which produces a statistically significant effect .
  • an "effective amount” is the amount of the composition comprising ob protein required to provide a clinically significant increase in healing rates in fracture repair, reversal of bone loss in osteoporo ⁇ is, stimulation and/or augmentation of bone formation in fracture non-unions and distraction osteogenesis, increase and/or acceleration of bone growth into prosthetic devices and repair of dental defects.
  • Such effective amounts will depend, in part, on the particular condition to be treated and other factors evident to those skilled in the art. For example in osteoporosis, increase in bone formation is manifested as a statistically significant difference in bone mass between treatment and control groups. This difference in bone mas ⁇ may be seen, for example, as a 5-20% or more increase in bone mass in the treatment group.
  • Other measurements of clinically significant increases in healing may include, for example, tests for breaking strength and tension, breaking strength and torsion, 4- point bending, increased connectivity in bone biopsies and other biomechanical test ⁇ well known to those skilled in the art.
  • General guidance for treatment regimens is obtained from experiments carried out in animal models of the disease of interest. In vi tro, the preferred range for ob protein concentration is about 1 pg/ml to 100 ng/ml, preferably 50 pg/ml to 5 ng/ml .
  • Therapeutic doses will generally be in the range of 10 ⁇ g/kg to 100 mg/kg of patient weight per day, preferably 0.1-10 mg/kg per day, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose i ⁇ within the level of ordinary skill in the art. In general, sustained release compositions will be formulated to provide doses in the higher ends of the stated ranges. Doses will be adjusted to the release rate. Initial dose determinations can be made by extrapolation from animal models according to standard principles, taking into consideration route of administration and pharmacokinetic factors such as rate of absorption, distribution, biotransformation, bioavailability and rate of excretion.
  • compositions described above are administered over a period ranging from a day to 6 months or more, depending on the condition to be treated.
  • doses will be adminstered from 5 times a day to once a month, and preferably from once a day to once a month until healing is substantially complete or bone mass increase is achieved.
  • the actual treatment regimen will depend upon such factors as the age and general condition of the patient, the condition to be treated, and the route of delivery. Determination of treatment regimen is within the level of ordinary skill in the art.
  • mice Ten-week-old C57BL/6J ob/ob female mice (Jackson Labs, Bar Harbor, ME) were treated with vehicle or ob protein (50 ⁇ g twice per day i.p., total 100 ⁇ g/day/animal) for 14 days. All mice were pair-fed throughout the experiment . To achieve matched food intake between the ob-treated and vehicle-treated animals, the pairs were initially matched for age and body weight. The pair mate for ob treatment was injected a fed and 24 hours later vehicle-treated animals were injected and food intake was restricted to the amount consumed by the ob- treated pair mate. After 14 days, the animals were sacrificed and the tibias were removed.
  • Tibial bone samples were fixed in 10% neutral buffered formalin, decalcified in 5% formic acid with 10% sodium citrate, washed in tap water, dehydrated in a series of 70%-100% ethanol, and embedded in glycol methacrylate.
  • the proximal end of the tibia (about 5 mm long) was cut frontally at 5 ⁇ m, stained for tartrate-resistant acid phosphatase (TRAP) activity, and counter-stained with methyl green and thionin for identification of bone cells.
  • TRIP tartrate-resistant acid phosphatase
  • Osteoblasts were identified by central negative (clear) Golgi area, eccentric nucleus, and the strong basophilic counter- ⁇ tain of methyl green and thionin, while osteoclasts by TRAP stain, multinucleation, and non-uniform shape. The following bone parameters were evaluated for histomorphometric changes.
  • Growth plate activity width measured every 50-100 ⁇ m at 42 X magnification to determine the growth plate activity.
  • Endocortical osteoblast size measured using all the osteoblasts counted in the vehicle-treated mice or 50 osteoblasts randomly selected in the ob-treated mice at 424 X magnification.
  • Number of endosteal osteoblasts measured along the endosteal surface of cancellous bone in the metaphysis at 212 X magnification in a zone area 0.62- 3.10 mm distal to the growth plate.
  • Number of endosteal osteoclasts measured simultaneously when endosteal osteoblast counts were taken.
  • BV/TV Percentage of cancellous bone
  • Ob protein treatment significantly increased growth plate activity as measured by increased growth plate width.
  • Osteobla ⁇ t-mediated bone mineralization following expo ⁇ ure of osteoblasts to ob protein was analyzed using the following in vi tro assay: 24-well plates (American Scientific Products,
  • Osteoblast cells were plated so as to be 90-100% confluent by the next day.
  • the cells used were derived from p53 knockout mice (see WO 96/07733) and plated at approximately 5 x IO 4 cells/well for cell lines 2-45 and CCC-4, and at 1 x IO 5 cells/well for cell line 2-29 using growth medium (Table 1) at 1-2 ml/well and vehicle or serial dilutions of ob protein that ranged 50 ng/ml to 5 pg/ml .
  • alpha-MEM JRH Biosciences, Lenexa, KS
  • fetal calf serum Hyclone, Logan, UT
  • L-glutamine (Hazelton, Lenexa, KS) lx PSN (5 mg/ml penicillin, 5 mg/ml streptomycin,
  • Beta-Glycerophosphate (Sigma, St. Louis, MO) was added to a final concentration of from 2 to 10 mM.
  • the cells were fed every 2-4 days with medium containing beta-glycerophosphate, L-ascorbic acid and ob protein or vehicle.
  • the solution was buffered by mixing 250 ul of the 0.5 N HCI dissolved mineralized layer and adding 600 ul 2 M Tris, pH 7.4, 600 ul Nova 7/9/10 urine diluent (Nova Biomedical, Waltham, MA) and 50 ul 2N- NaOH. Total calcium was measured using the Nova 7/7+7 Electrolyte analyzer (Nova Biomedical) .
  • ob protein The effects of ob protein on bone growth were tested in ten-week-old CD-I male mice (Jackson Labs) by injecting ob protein into the subcutaneous tissue over the calvarium of the mice. Doses ranged from 0.001-5.0 mg/mouse, given three times daily for five days.
  • mice After 14 days, the mice were sacrificed and calvarial bone growth was measured by histomorphometry.
  • the ovariectomized rat is accepted as an animal model of human post-menopausal osteoporosis.
  • female normal rats are either sham- operated or surgically ovariectomized. Seven days after surgery, treatment is begun, administering either vehicle, ob protein or estrogen (160 ⁇ g/kg, subcutaneously) .
  • single doses of tetracycline or demeclocycline are administered to assess bone formation and mineralization. Tibias and lumbar vertebrae are removed, fixed, processed and analyzed as described in Example 1.
  • mice Thirteen-week-old genetically obese mice (ob/ob,
  • Ten- ⁇ m sections were mounted without staining for evaluation of calcein labels, while 5- ⁇ m sections were stained for tartrate-resistant acid phosphatase (TRAP) activity and counter-stained with methyl green and thionin for identification of bone cells.
  • TRAP tartrate-resistant acid phosphatase
  • Osteoblasts were identified by central negative (clear) Golgi area, eccentric nucleus, and the strong basophilic stain, while osteoclasts by TRAP stain, multinucleation, and non- uniform shape.
  • Endocortical bone was evaluated at both the anterior and the posterior region within 3.7 mm below the growth plate at 90X magnification.
  • bone- forming activity can differ significantly at various sites within the bone, with greater activity at the posterior than the anterior region of the bone site evaluated. The evaluations were as follows:
  • bone formation rate (BFR/BS, ⁇ m3/ ⁇ m2/day) : The amount of new bone formed per unit bone surface per day (bone surface referent) .
  • bone cells are usually less responsive compared with the other two bone sites (e.g., do not respond to short-term estrogen deficiency or treatment) .
  • Osteoblasts were active following leptin treatment in ob/ob mice as shown previously. To confirm this unusual finding, dynamic bone changes were further evaluated. The following bone parameters were evaluated from the entire endosteal surface of the cancellous bone at 180X magnification:
  • Leptin treatment of ob/ob mice significantly increased the tibial endocortical bone formation at both the anterior and the posterior sites as indicated by increased % single- and/or double-labeled surfaces, mineral apposition rate, and bone formation rate, confirming the earlier findings of increased osteoblast activity by leptin treatment.
  • leptin treatment resulted in significant increases in % single-labeled surface, % double labeled surface and bone formation rate. 7.
  • the stimulation of bone formation by leptin parallels with the increased serum IGF-I levels following leptin treatment and may indicate its potential role in mediating leptin effects on osteoblasts in ob/ob mice.
  • a combined treatment with an anti- resorptive agent could potentially augment the anabolic effect of leptin on bone.
  • CAA GCT GTG CCC ATC CAA AAA GTC CAA GAT GAC ACC AAA ACC CTC ATC 150 Gin Ala Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He 20 25 30 35
  • AGC AGG CTG CAG GGG TCT CTG CAG GAC ATG CTG TGG CAG CTG GAC CTC 534 Ser Arg Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu 150 155 160

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Abstract

Procédés permettant d'induire la formation osseuse à l'aide de la protéine ab. On peut utiliser ces procédés pour traiter l'ostéoporose, réparer des fractures, réparer des défauts dentaires, la résection due à l'encogène et l'élongation de la plaque de croissance/os long. On peut également utiliser ces procédés pour pratiquer la thérapie ex vivo et la réintroduction dans un mammifère.
EP97922438A 1996-04-19 1997-04-18 Procedes permettant d'induire la formation osseuse Withdrawn EP0917469A1 (fr)

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EP0950417A3 (fr) * 1998-02-23 2000-02-23 Pfizer Products Inc. Traitement d' anomalies du squelette
AU3147699A (en) * 1998-03-27 1999-10-18 Andre Rosenthal Nucleic acid molecules which code proteins influencing bone development
AU3863699A (en) * 1998-04-20 1999-11-08 Mayo Foundation For Medical Education And Research Treatment of osteoporosis with leptin
AU767068B2 (en) * 1999-06-11 2003-10-30 Baylor College Of Medicine Methods and compositions for control of bone formation via modulation of leptin activity
WO2000076552A1 (fr) * 1999-06-11 2000-12-21 Baylor College Of Medicine Procedes et compositions pour la regulation de l'osteoformation par modulation de l'activite de la leptine
AU781936C (en) * 1999-08-03 2006-02-16 University Of Melbourne, The A method of treatment and agents useful for same
JP2003526677A (ja) * 2000-03-14 2003-09-09 ザ・ユニバーシテイ・オブ・ウエスタン・オンタリオ 骨形成に影響を与える組成物および方法
AU2015267006B2 (en) * 2014-05-30 2020-04-09 The Research Foundation For The State University Of New York Compositions and methods to promote bone formation

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US6309853B1 (en) * 1994-08-17 2001-10-30 The Rockfeller University Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof
EP0956862A1 (fr) * 1995-11-22 1999-11-17 Amgen Inc. Procédés permettant d'accroitre la masse tissulaire maigre à l'aide de compositions à base de proteine ob

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