JP2000509377A - Methods for inducing bone formation - Google Patents

Abstract

  (57) [Summary] Disclosed are methods for inducing osteogenesis using ob proteins. The method can be used for treating osteoporosis, repairing fractures, repairing tooth defects, repairing resections due to tumor formation, and expanding growth plates / long bones. In addition, the method can be used for ex vivo treatment and can be re-injected into vertebrates.

Description

DETAILED DESCRIPTION OF THE INVENTION Methods for inducing bone formation Background of the Invention   Bone remodeling is a dynamic process in which tissue mass and skeletal structure are maintained. The process Is the equilibrium between bone resorption and bone formation, and two cell types It is a layer. These cells are osteoblasts and osteoclasts. oclast). Osteoblasts synthesize a matrix that becomes new bone and deposit it. Stack. The activity of osteoblasts and osteoclasts depends on a number of factors: systemic and local. Regulated by sex factors, such as growth factors.   The interaction between local and systemic factors has not been fully elucidated, but Some hormones such as parathyroid hormone (PTH), vitamin D and calcium There is evidence that tonin can be mediated by local factors found in bone. On the bone Some of the growth factors that have been identified are IFG-I, IGF-II, TGF-β₁, TGF −β_Two, BFGF, aFGF, PDGF, and types of bone morphogenetic proteins (Baylin k etc.,J.Bone Mineral Res . 8 (Supp. 2): S565-S572, 1993).   If bone resorption exceeds bone formation, a final fracture of the bone will result, and the bone The tendency to break is increased. Reduced bone formation is related to age and certain pathological conditions. Are linked. Osteoporosis is a disease that afflicts many older people, especially postmenopausal women It is. In the United States alone, about 1.5 million fractures attributable to osteoporosis Present every year. The impact of those fractures on the essence of a patient's life is immunity. Of those identified with osteoporotic hip fractures, 10-20% have died and 50% will no longer be able to walk alone (Riggs Such.,Bone 17: 505S-511S, 1995). Health management system in the United States The associated costs for $ 5 to $ 10 billion annually, excluding long-term management costs Estimated.   Currently available treatments for the prevention of osteoporosis include salmon calcitonin, estr Hormone replacement therapy (HRT) with gen and / or progesterone and Is limited to the bisphosphonate compound of Worldwide about salmon calcitonin The market exceeds $ 500 million a year. However, patients did not respond to salmon calcitonin. And estrogen therapy are known to evolve Correlate with the increased risk of palatal and possibly breast cancer. Bisphosphonates are Related to gastrointestinal problems. Therefore, the need for more effective treatment for bone loss Survive. Summary of the Invention   Large amounts of ob protein in bone marrow mesenchymal cells (marro w. a cell population comprising exposing a cell population comprising It is an object of the present invention to provide a method for stimulating a group. In one aspect In which the population is enriched for bone marrow mesenchymal cells. In one aspect Hematopoietic cells have been removed. In another embodiment, the osteogenic cells are predominantly Osteoblasts. In one embodiment, the method comprises combining an anti-absorbent with a cell population. Further comprising the step of combining. In another embodiment, the method comprises a cell Further comprising injecting the population into the mammal.   A cell population containing bone marrow mesenchymal cells was obtained from an ob protein-treated animal. A method for stimulating said cell population comprising exposing to a physical fluid. It is a further object of the present invention to provide It is. In another embodiment, the biological fluid is serum. Another one In an embodiment, the biological fluid is one that is effective for expanding osteogenic cells. Sorted out to enrich the child.   Endocrine or CNS cells or tissues exposed to ob proteins Expose the cell population, including bone marrow mesenchymal cells, to the conditioned culture medium. It is an object of the present invention to provide a method for stimulating said cell population, comprising: For further purposes. In another embodiment, the cells are pituitary or thalamic The lower cell.   Effective for providing a clinically significant increase in bone repair or bone healing rate Administering to the mammal a large amount of the ob protein, the bone in the mammal. It is a further object of the present invention to provide a method for promoting repair or healing. You.   To provide a clinically significant increase in bone growth into prosthetic devices or tooth implants Administering to the mammal an effective amount of an ob protein. Provided is a method for stimulating bone growth into an inserted prosthetic device or tooth implant Is a further object of the present invention.   Large amounts of ob effective to confer a clinically significant increase in bone growth plate width Treating bone loss in a mammal comprising administering the protein to the mammal It is a further object of the present invention to provide a method for doing so. Another aspect In, the mammal is an adolescent or pre-adolescent mammal.   A large amount of ob protein is injected into mammals to repair bone length in anterior fractures. Activating bone in a mammal having a bone growth plate fracture, comprising providing It is a further object of the present invention to provide a method for stimulating proliferation.   Feeding large amounts of ob protein effective to induce bone formation at site of bone resection Bone shape in mammals with oncological resection of bone, comprising administering to bones It is a further object of the present invention to provide a method for inducing growth. Detailed description of the invention   Before describing the present invention in detail, it is necessary to define certain terms as used herein. Is useful:   ob / Ob mouse: Homozygous for inactivating mutations at the ob (obese) locus A syngeneic mouse. ob / ob mice are hyperphagic and hypometabolic and have circulatory satiation. It appears to be a defect in the production of full factors.   ob: As used herein, ob or ob indicates a nucleic acid. This name Is used in the form “* / *”, but not alone “*”. Of the rodent mutant phenotype (ie, ob / ob mice).   ob: Ob as used herein indicates a protein. ob protein Is also known as "leptin".   Stem cells(stem cell): As used herein, the term “stem cell” Has the potential to differentiate into multiple cell types, and is restricted to a fixed number of mitosis Refer to pluripotent cells that are not.   Precursor cells(Precurcor cell): As used herein, the term "precursor cell" A vesicle "is conferred on the differentiation pathway, but generally does not express a marker and Refers to cells that do not function as mature, well-differentiated cells.   Osteogenic cells(osteogenic cell): As used herein, the term “osteogenic cell” “Vesicle” means osteoblast or osteoblast precursor cell cell).Fibroblasts or reticulocytes(Reticulan cell): used in this specification When used, the term refers to cells of the soft connective tissue network; And through the space in the bone.   Fat cells(adirocyte): As used herein, the term “adipocyte” Cells differentiated by the presence of their lipid inclusions and the expression of specific gene products Means   The present invention relates to a method for treating osteoclasts or their cells when the ob protein is administered to a mammal. Based on a finding that results in a dramatic increase in osteogenic activity of osteoporosis.   Sequences encoding mouse and human ob proteins are known (Friedman et al. , WO 96/05309). The human and mouse abDNA and protein sequences are These are shown in column numbers 1, 2, 3, and 4. One of skill in the art will recognize SEQ ID NOs: Corresponds to a single allele of the human and mouse ob genes, and It will be appreciated that the presence of allelic variation is expected. For example, humans and And murine ob gene allelic variants do not have a Gln residue at position 49 .   O encoded by allelic variants of the DNA sequences shown in SEQ ID NOs: 1 and 3 b proteins, such as those containing silent mutations and sudden Those proteins whose mutations result in an amino acid sequence change are shown in SEQ ID NO: 2 or 4 Are within the methods of the invention, such as the ob protein, which is an allelic variant of . The present invention also relates to the use of a DNA molecule encoding the ob protein, wherein said DNA molecule is The molecule has a sequence in relation to SEQ ID NO: 1 or 3 or an allelic variant thereof. Generally at least 60%, preferably at least 80%, and 90-95% or The above is the same.   The present invention also provides for a single homologous to the protein of SEQ ID NO: 2 or 4 It also relates to the use of isolated proteins and their species homologs. "Isolated" Are found under conditions other than their natural environment, e.g., apart from blood and animal tissues. Means the protein to be used. In a preferred form, the isolated protein The quality is substantially free of other proteins, especially other proteins of animal origin. High In purified form, i.e., at least 95% pure, more preferably at least 99% pure. It is preferred to supply the protein. The term “substantially homologous” refers to the sequence number No. 2 or 4, or 50%, preferably In order to indicate proteins having at least 80% sequence identity, used. Such a protein is more preferably SEQ ID NO: 2 or 4, or Have at least 90% identity to their species homologs, and most preferably 95% % Or more. A substantially homologous protein is one or Is characterized as having multiple amino acid substitutions, deletions or additions. It These changes are preferably of a minor nature, i.e., protein folding. Conservative amino acid substitutions that do not significantly affect activity or activity; small deletions, typical Has a deletion of 1 to about 45 amino acids; and a few amino- or carboxyl-terminals. Terminal or internal extensions, e.g., amino-terminal methionine residue extensions, up to about 20-25 A small linker peptide extension of the residue, or a small extension to facilitate purification, For example, those that extend the poly-histidine tract, antigenic epitope or binding domain. You. Generally, Ford etc.,Protein Expression and Purification 2： 95-107 , 1991 (incorporated herein by reference). Furthermore, the ob of the present invention Proteins (or polypeptide fragments thereof) supply multifunctional molecules Other bioactive molecules, especially targets Or an anti-absorbent. Such hybrid ob protein content The child is formed by chemical conjugation or polynucleotide fusion, or a combination thereof. And a linker between said component proteins or (poly) peptides (eg, Polypeptide linker).   In addition to the ob proteins disclosed above, the methods of the present invention And an isolated polynucleotide encoding said fragment Include the use of the molecule. At least 10 cells exhibiting osteogenic or osteogenic cell stimulating activity An amino acid-long ob protein fragment, and such a polypeptide Of particular interest are polypeptide molecules at least 30 nucleotides in length that encode The subject of. This type of polypeptide can be used for known biological screening and The intact ob protein identified by the assay method and intact By digestion or by small overlapping polypeptides or Can be produced by synthesizing a polynucleotide (and expressing the latter) . The resulting polypeptide fragment can then be used to enhance or enhance osteogenic activity. Tested for an activity or effect associated with the bone formation performed. Full mature ob tampa Larger polypeptides up to the size of the protein are also useful in the present invention. You.   Pattern recognition for generation of secondary structures (Cohen et al.,Biochem . twenty five: 266-75, 1 986) and neural networks (Kneller et al.,J . Mol. Biol.214: 171-82, 1990) Software The analysis of the amino acid sequence shown in SEQ ID NO: 2 and the formation of the structural model using It was predicted that the protein would fold into four α-helix bundles. This helic Prediction of protein structure is based on multiple alignment with other helical cytokines (mulitiple aliynment) (Buzan,Immunology T oday 11: 350-54, 1990; Gribskov et al.,PNAS USA 84: 4355-58, 1987). That Multiple matches indicate that the ob protein has similarities to helical cytokine structural types That was shown. Ob protein possible for its helical cytokine structure type Further evidence of relevance is due to the two forms of the ob cDNA (+ Gln and -Gln at position 49). Provided. The two forms are pulled by a shift in the splice receptor sequence. Which indicates the location of the exon-intron boundary (Zhang et al.,Nature 372: 425-32, 1994). For multiple matches, the position of the exon-intron boundary of ob The alignment is consistent with the exon-intron boundaries in interleukin-3 and GM-CSF. And inter-leukin-2 and inter-leukin It resides within two residues of the exon-intron boundary at -6.   This prediction is supported by a recent report describing the ob protein receptor (OB-R; Tartaglia and others.,Cell 83: 1263-71, 1995). OB-R is IL-6, G-CSF and L Membrane proteins closely related to the gp130 signal-transduction component of the IF receptor It is a pan receptor. The extracellular domain of OB-R is Show many similarities.   In a preferred embodiment, the substantially homologous ob protein or ob protein Can be folded into four α-helix bundles and self-assembled. Can be Obtaining four α-helix bundle characteristics of ob protein molecules The amino acid residue preferably has most or no amino acid substitutions, additions or deletions. Maintained without loss. However, the remaining amino acid residues are To a wider range of amino acid substitutions, additions or deletions, with retention of biological function There is.   Essential amino acids in the ob polypeptides useful herein are Methods known in the art, such as site-directed mutagenesis or Nin-scanning mutagenesis can be identified (Cunningham and Wells,Science 244, 1081-85, 1989). In the latter technique, a single alanine mutation is identified. Introduced at every residue in the offspring, and the resulting mutant molecule Amino acids that are critical for the activity of the protein polypeptide or fragment To identify residues, the biological activity (ie, osteoblast-activating activity or Is tested for bone forming activity). The site of ligand-receptor interaction In addition, nuclear magnetic resonance, crystallography or photoaffinity labeling may be used. It can also be determined by analysis of the crystal structure, as determined by such techniques. For example, de Vos.Science 255: 306-12, 1992; Smith et al.,J . Mol. Bio l .224: 899-904, 1992; Wlodaver and others.,FEBS Letl. 309: See 59-64, 1992 thing.   Multiple amino acid substitutions are known in mutagenesis and screening methods, BA Reidhaar-Olson and Sauer (Science 241: 53-57, 1988) or Bowie and Sauer (Proc . Natl. Acad. Sci. USA 86: 2152-56, 1989). Can be performed and tested. In short, their authors are Proteins that function by randomizing multiple positions in the protein simultaneously And then determine the spectrum of acceptable substitutions at each position. Disclosed are methods for sequencing mutated proteins. It These methods allow for the rapid determination of the importance of individual amino acid residues in the protein of interest. And can be applied to proteins of unknown structure.   The DNA molecule shown in SEQ ID NO: 1 is a mouse Obese gene (Zhang et al.,Nature 37 Two : 425-432, 1994), and human adipose tissue cDNA Designed from cDNA from library (obtained from Clontech, Polo Alto, CA) Polymerase chain reaction (PCR; Mallis et al., US patent) No. 4,683,195; see Mallis, U.S. Pat. No. 4,683,202). Has been implemented. According to this method, the DNA molecule shown in SEQ ID NO: 1 was identified. .   Analysis of the amino acid sequence shown in SEQ ID NO: 2 indicates that the protein has 21 amino acids. It was shown to include the amino terminal signal peptide of the acid residue. Therefore, mature tamper The protein started at amino acid residue 22 (Val) of SEQ ID NO: 2.   Bone is tissue and, therefore, comprises a composite of a heterogeneous cell population. Found in bone Some of the cell types involved include osteoblasts, osteoclasts, chondrocytes and osteocytes . Examples of other tissues include bone marrow, skin, skeleton and myocardium, pancreas, brain and liver. Tissues are usually tissue-specific cell types, and cell types found in many tissues, such as Consists of a mixture of fibroblasts. It is in the bone marrow where many of the early precursor cells are found And they are commonly found in hematopoietic stem cells or mesenchymal stem cells. tem cell). Human hematopoietic stem cells, CD34⁺Identified as And myeloid cells (eg, red blood cells, megakaryocytes, macrophages, and Differentiate to become either neutrophils) or lymphocyte cells (T cells or B cells). Of particular interest herein are cells of the mesenchymal lineage. Yes, and for osteoblasts, chondrocytes, adipocytes, fibroblasts and reticulocytes Includes stem cells and precursor cells (Owen et al.,Ciba Fdn . Symp.136: 42-46, 198 8). Markers for mesenchymal stem cells are poorly identified (Owen et al., J . of Cell Sci .87: 731-738, 1987), so that identification is usually in precursor and mature cells It takes place in stages. Mesenchymal cells, as defined herein, are hematopoietic cells Is histologically different. Stromal cells generally adhere when cultured in vitro. Is considered as a subpopulation of bone marrow cells and is included in mesenchymal cells.   Differentiation is the process of cell maturation. It is a progressive and dynamic process, Starting from pluripotent stem cells, and not progressing further down the cell line Terminated by the dead cells. Cell function, phenotype and growth characteristics depend on the degree of cell differentiation. More affected. When stem cells or progenitor cells initiate the process of differentiation, they Generally, they move to selected tissues and / or organs where they are It seems to be subject to change.   Typically, the differentiated state of a cell is a set that is specific for a particular cell type. Can be identified using differentiating markers. Differentiation markers are used at various stages of cell line growth. Shown transiently on the floor. Pluripotent stem cells are connected to lineages. It can be regenerated without commitment and has been linked to cell lineages. Capable of expressing a set of differentiation markers that are lost when the killing is performed You. Progenitor cells are expressed as the cells progress down the mature cell lineage Express a set of differentiation markers that can or cannot be continued You. Differentiation markers that are exclusively expressed by mature cells usually have functional properties, such as For example, it relates to a cell product, an enzyme that produces the cell product, and a receptor. Mature Are osteoclasts tartrate-resistant acid phosphatase (TRAP) and calcitonin receptor? Express a set of differentiation markers selected from the group consisting of (Suda et al.,Endocrine R ev. 13: 66-80, 1992). The identification of osteoclasts is mainly phenotypic and Phosphatase expression (Manduca et al.,J . Bone Min. Res.8: 281, 1993); Thailand Collagen synthesis (Kurihara et al.,Endocrinol.118 (3): 940-947, 1986); Steocal Generation of thin (Rodan et al.,Crit . Rev. Eucar. Gene Expr.1: 85-98, 1991); And responsiveness to parathyroid hormone (Aubin et al.,J . Cell Bio1.92: 452-461, 1 982).   Other differentiation markers for specific cell types include: for cardiomyocytes As for the expression of cardiac myosin isozyme and creatine kinase isozyme, Cardiac specific patterns (Yaffe et al.,Develop . Biol.15: 33-50, 1967 and Richler Such.,Develop . Biol.twenty three: 1-22, 1970), and insulin and insulin-like Growth factor receptor I (Wolleben et al.,Am . J. Physiol.252: E673-E678, 1987); bone For skeletal muscle cells, myosin isozyme expression and creatine kinase eye Muscle-specific patterns of sozyme expression (I and II) (Yaffe et al.,Develop . Biol.1 1 : 300-317, 1965; Yaffe et al.,Develop . Biol.5: 33-50, 1967; Richler, etc. .,DeveloP . Biol. twenty three: 1-22, 1970); For chondrocytes, aggrecan (aggrec an) (Doege etc.,J . Biol. Chem.266: 894-902, 1991) and collagen type II B (Sandell et al.,J . Cell Biol.114: 1307-1319,1991); For megakaryocytes , Mpl receptor (Souyri et al.,Cell63: 1137-1147, 1990) and acetylcholine (Ravid Such.,J . Cell. Biol. one two Three: 1545-1553, 1993); for pancreatic b-cells, Shurin (Powers etc.,Diabetes 39: 406-414, 1990); for pancreatic a-cells Are glucagon and glucagon-like polypeptides (such as Lacy.Diabetes 16: 35, 1 967; Gotoh et al.,Transplantation 40: 437-438, 1985: Hamaguchi and others.,Diabe tes 40: 842-849, 1991); for pancreatic d-cells, somatostatin (Wiliiams) Etc., Somatostatin and Pancreatic Polypeptide inInternational Textbook of Diabetes Mellitus , Albert; et al., Eds., 1992); 4. Fat protein lipase, AP2, PPARr (Butt erwith,Pharmac . Ther. 61: 399-411, 1994), triglycerides and perilipin ( perilipin) (Greenberg et al.,J . Biol. Chem.266(17): 11341-11346,1991; Gre enberg et al.,Proc . Natl. Acad. Sci.90 (24): 12035-12039, 1993); For monocyte cells, including osteoporosis and osteoclast precursors, Ly-6C and Mac-1 (McCormack etc.,J . Immunol.151: 6389-6398, 1993; Gordon et al.,Current Up in in Immunol .Four(25): 25-32, 1992) and non-specific esterases (NSE; Yam et al.). ,Amer . J. Clin. Path.55: 283, 1971); and for hepatocytes, albumin; Liver-specific glucokinase, liver-specific pyruvate kinase, and glycogen Hepatic isozymes of synthase (Miller et al.,J . Biol. Chem.261: 785-790, 1986; Magnuson,Diabetes 39: 523-527, 1990).   Progenitor cells or progenitor cells are stimulated to differentiate and Supplies the differentiated cells. Differentiation is an undifferentiated stem cell or progenitor Somatic cells are mediated by surrounding cells (paracrine regulation) and by the cells themselves (autoc Supply (line regulation) or by distant hormone-secreting cells (endocrine regulation) Induced by exposure to the factors mentioned. In addition, certain factors are extrinsic Could be added and would stimulate the differentiation of cells. For example, osteoclasts Differentiation is stimulated by exposure to Tamine D and dexamethasone. Osteoclasts Differentiation is induced by exposure to formic acid, TGF-b or bone morphogenetic protein (BMP) .   In the present invention, the ob protein induces osteogenic cells to become activated osteoblasts. It is disclosed that the stimulus has the ability to perform. ob protein is used for appetite (Friedma n et al., WO 96/05309) and fertility (Chehab et al.,Nature Genetics 12: 318-320 , 1995), but have previously been meant as modulators of bone-stimulating factors. What is this Was not known for. Receptors for ob proteins found in brain, kidney and lung But not found in bone (Tartagia Such.,Cell 83: 1263-1271, 1995).   When ob / ob mice are treated with leptin, various effects can be attributed to the mesenchymal lineage. Vesicles (eg, stem cells, stromal cells, osteoclast precursor cells, and mature osteoclasts) Is observed. Stimulation of proliferation and / or differentiation of mesenchymal cells can be performed using standard assays. As determined more, results in a large number of mature osteoblasts. Typically, mesenchymal strains Test samples containing cells in the presence and absence of ob protein (Control sample). Next, the test sample is And / or by staining for cell proliferation and differentiation. Such test services The sample can be a cell cultured in vitro, or from an in vivo model system. can get. In addition, exposure to the ob protein will increase the size of the resulting mature osteoblasts. Can be increased. On the other hand, the ob protein is^ThreeH] J Incorporation of midine or [³⁵S] Isolated osteoblasts as determined by labeling It can stimulate cell proliferation. In addition, mesenchymal cells exposed to ob protein Dew can be either osteogenic (ie, in vivo) or osteogenic molecules (ie, in vitro). Is induced so that the biological activity of cells (eg, up-regulation of bone stimulating molecules) Regulation or activation). In particular, in vivo environments In addition, ob protein recruits osteogenic cells to appropriate osteogenic tissues and tissue sites. Or movement can be enhanced.   The effect of the ob protein on bone formation or osteoblast activation may be direct or indirect. It can be any of the targets. Inverse relationship between in vitro differentiation of adipocytes and osteogenic cells Exists (such as Berestord.,J . Cell. Sci.102: 341-351,1992), and bone marrow fat With increased volume of fatty tissue A correlation between osteoporosis has been established (Meunier et al.,Clin . Orthop. Rel. Res .80: 147-154, 1971: Barkhardt etc.,Bone 8: 157-164, 1987 and Minaire And so on.Calcif . Tiss. Int.36: 338-340, 1984). These observations are combined with the present invention. In turn, the ob protein induces normal adipocyte-osteoblast precursors to the osteogenic phenotype Suggest that it may play a role in the mesenchymal cell differentiation pathway.   Bone marrow adipocytes are factors required for the maturation of other cell types found in the bone marrow It is known to secrete offspring (Gimble,The New Biologist 2: 304-312, 1 990). Thus, growth and differentiation factors due to migration within the cell population in the bone marrow microenvironment Is predicted to alter the osteoblast maturation process.   The effects of ob proteins appear to be both central and peripheral (Yu et al.,Proc . Natl. Acad. Sci. USA 94: 1023-1028, 1997). Therefore, the ob protein And then cultured, endocrine, or CNS tissue or The cell type may include additional factors that contribute to the osteogenic effect. In particular, ob ta Following protein exposure, culture of CNS cells, such as pituitary or hypothalamic cells, Will enhance the osteogenic effect.   PTH provides a paradigm for osteogenic agents. Ovarian dissected mouse At certain dose levels, PTH increases osteoblast number and size (Lui et al.,J . Bone Mineral Res .5: 973-981, 1990). This increase in osteoblast size and number is Corresponds to increased bone formation.   Estrogens are anti-absorbents and can be used for the treatment of osteoporosis You. Adipocytes have been shown to produce estrogen (Heiss et al.,J . C lin. Endocrinol. Metabolism 80: 1591-1596,1995), and for postmenopausal women Of estrogen in Can be a source. Low body fat levels appear to be a risk factor for osteoporosis. I However, bone marrow fat has been shown to be elevated in various osteoporosis patients. Was. When bone loss ceases, bone marrow fat levels fall (Martin et al.,Bone 12 : 123-131, 1991). Thus, peripheral adipocytes, bone marrow adipocytes, and bone formation and loss The relationship between is complex and requires further elucidation.   In one embodiment of the invention, the composition comprising the ob protein is an osteoblast. Used as a therapeutic to enhance vesicle-mediated bone formation. The method of the present invention comprises: Bone defects and defects, such as those that occur in closed, open and non-union fractures To promote bone restoration; to promote bone healing in plastic surgery; cementing To stimulate bone growth into unprosthetic interstitial and dental implants: periodontal disease and To treat bone defects and defects; to increase bone formation during distraction bone formation; and to increase osteoblast activity. Suitable for the treatment of other skeletal disorders that can be treated by stimulation, such as osteoporosis and arthritis Can be used. The new bone formation provided by the method of the present invention can Bone following wound-induced repair of oncological resection or radiation-induced osteonecrosis (Hart, etc.)Cancer 37: 2580-2585, 1976). The method of the present invention Can also be used in plastic surgery.   Growth hormone is found in bone growth in the presence of excess glucocorticoid Has been shown to reduce (and possibly eliminate) some pathological phenotypes (Altman et al.,Calcif . Tissue Int.51: 298-304, 1992). ob polypeptide Growth hormone release hormone (GHRH) from cultured pituitary cells ) It has been shown to increase irritation. Growth hormone, including new bone formation Many peripheral effects of are mediated by IGF-I. The method of the present invention Quality is consistent with putative pathways exerting effects on bone Results in increased levels of IGF-I.   The compositions of the present invention may be administered systemically or locally. For systemic use In accordance with conventional methods, ob protein is administered parenterally (eg, intravenously, subcutaneously). Intramuscular, intraperitoneal, intranasal or transdermal) or enteral (eg oral or rectal) Formulated for. Intravenous administration can be a series of injections or a continuous Will be done by injection. Injections or other separately spaced doses Administration by the route is generally performed at intervals ranging from once a week to 1 to 3 times daily. Will. Processing will continue until the desired result is achieved. Generally, a doctor The drug formulation may be a pharmaceutically acceptable vehicle such as a salt solution, buffer solution, water, % Dextrose, a borate buffer solution containing trace metals, or the like. Will contain the ob protein. The formulation further comprises one or more excipients, preservatives, Lysing agents, buffers, albumin to prevent protein loss on vial surfaces , Lubricants, fillers, stabilizers (eg, soluble receptors), and the like. Formulation methods are well known in the art and include, for example,Reminaton's Pharmaceutical Sciences , Gennaro, ed., Mack Publishing Co., Easton PA, 1 990, which is incorporated herein by reference. Used within the present invention Pharmaceutical compositions for sterile, non-pyrogenic liquid solutions or suspensions, Capsules, suppositories, lyophilized powders, transdermal patches, or Can exist in other forms. Local administration should be performed at the site of injury or defect. Injection, or insertion or binding of a solid carrier at said site, or This can be done by direct local application of an ionic liquid.   Delivery of the ob protein to the wound site may be controlled by a controlled open composition, such as U.S. Patent Application Serial No. 07 / 871,246 (general) (Corresponding to WIPO publication WO 93/20859), which is incorporated herein by reference) It can be enhanced by the use of a composition. This type of film is used for prosthetic devices and surgical Particularly useful as a coating for implants. This film is used for surgical screws, rods, pins, The rate and the like can be wrapped around the outer surface. This type of port Possible devices are commonly used in corrective surgery. The film also contains bone filling material, For example, hydroxyapatite block, demineralized bone matrix plastic Can also be used to coat collagen, collagen matrices and the like. Generally, the films or devices described herein are applied to bone at the site of a fracture. It is. Application is generally to implantation into bone or its surface using standard surgical methods. This is done by binding to   In addition to the above copolymers and carriers, biodegradable films and matrices May contain other active or inactive ingredients. Promotes tissue growth or invasion Those agents are of particular interest. Promotes bone growth or inhibits bone resorption Agents such as bone morphogenetic proteins (US Pat. No. 4,761,471; PCT Publication WO 9) 0/11366), osteogenin (Sampath et al.,Proc . Natl. Acad. Sci. USA 84: 7109-7113, 1987), and NaF (Tencer et al.,J . Biomed. Mat. Res .twenty three: 571-589, 1989).   Another method for supplying ob proteins is the ALZET osmotic minipump (Alza Corp. . Palo Alto, CA); sustained release matrix materials such as Wang. (WO 90/113 66); Bao et al. (WO 92/03125) Dextran beads charged; for example, Ksander, etc.Ann . Surg.211 (3): 288- (294, 1990); a collagen-based delivery system as disclosed in Beck et al.J . B one Min. Res .6 (11): 1257-1265, 1991) Methylcellulose gel system; and Edelman et al.Biomaterials,12: 619-626 , 1991) involves the use of alginate-based systems. Bone Other methods well-known in the art for long-term local supply at A porous coated metal prosthesis that can be impregnated, having a therapeutic composition incorporated therein. Solid plastic rods and porous hydroxy as growth factor carriers Including apatite / tricalcium phosphate ceramics (Toriumi et al.,Laryngos cope 101: 395-404, 1991).   Delivery of the systemically administered composition of the present invention conjugates the ob protein to the target molecule Can be enhanced. "Target molecule" is a molecule that binds to the tissue of interest Means For example, bone targeting molecules include tetracycline; calcein; Phosphonate; polyaspartic acid; polyglutamic acid; aminophosphoric acid; Mineral phases such as osteonectin, bone sialoprotein and osteopontin Peptides known to be associated with; bone-specific antibodies; Proteins, and the like. For example, Bentz etc. (EP05128 44) and the disclosure of Murakami et al. (EP0341961).   The method of the present invention relates to HRT and other anti-absorbents such as bisphosphonates and Provides the use of the ob protein to treat bone loss, along with cytotonin.   In the present invention, an “effective amount” of a composition is an amount that produces a statistically significant effect. It is. ob protein is used to stimulate osteoblast proliferation in vitro When compared to cells grown in the absence of the ob protein,^ThreeH-thymidine It can produce at least a 50% increase in proliferation as measured by integration. Generally desired. For therapeutic use, an “effective amount” refers to the cure in fracture repair. Clinically significant increase in healing rate, reversal of bone loss in osteoporosis, non-union fracture And / or augmentation of bone formation in bone formation and distraction osteogenesis, bone growth into prosthetic devices Ob tan needed to provide and / or enhance and / or promote tooth restoration The amount of the composition comprising the protein. Such an effective amount will It will depend in part on the particular conditions and other factors which will be apparent to those skilled in the art. And For example, in osteoporosis, increased osteogenesis is the quality of bone quality between treatment and control groups. It is evident as a statistically significant difference in the amount. This difference in bone mass For example, it can be seen as a 5-20% or more increase in bone mass in the treatment group You. Other measures of clinically significant increase in healing include, for example, strength and tension Fracture, strength and torsional fracture, four-point bending, increased connectivity in bone biopsy, and And tests for other biomechanical tests well known to those skilled in the art. it can. A general guide to treatment methods can be found in animal models of the disease of interest. Obtained from experiments performed.   The preferred range for ob protein concentration in vitro is from about 1 pg / ml to 100 ng / ml, preferably 50 pg / ml to 5 ng / ml.   Therapeutic doses generally range from 10 μg / kg to 100 mg / kg of patient weight / day, preferably Is present in the range of 1-10 mg / kg / day, and the exact dose depends on the condition to be treated. Clinicians according to accepted standards, taking into account the nature and rigor, patient constitution, etc. Determination of the six doses to be determined is within the level of ordinary skill in the art. In general, The efficacy composition will be formulated to deliver a dose at the upper end of the stated range. Would. The dose will be adjusted to the release rate. Determination of initial dose depends on route of administration And pharmacokinetic factors such as absorption, distribution, rate of bioconversion, bioavailability and By estimating from animal models according to standard principles, taking into account the rate of Can be done. For example, Goodman and Gilman, eds.,The Pharmacological Basis of Therapeutics, Fif th Edition, MacMillan Publishing Co., Inc., New York, 1975.   The compositions may range from one day to six months or more, depending on the condition to be treated. Administered over a period of time. Generally, the dose will be from 5 degrees per day to once a day, and Preferably once a day to once a month, healing is substantially complete, or on bone mass It will be administered until elevation is achieved. The actual treatment depends on the patient's age and Will depend on factors such as general conditions, conditions to be treated, and supply routes . Determination of treatment is within the level of ordinary skill in the art. An exampleExample 1 Analysis of mouse tibia A. Female mouse   10 week old C57BL / 6J ob / ob female mice (Jackson Labs, Bar Harbor, ME) With vehicle or ob protein (50 μg i.p. twice daily, 100 μg total) / Day / animal) for 14 days. All animals are paired throughout the experiment. Paid. ob-harmonized diet between treated and vehicle treated animals To achieve food intake, pairs were first matched for age and weight. ob Inject food into pairmates for treatment and vehicle treated 24 hours later Inject food into animals and consume food intake by ob-treated pairmates Limited to amount. After 14 days, the animals were killed and the tibia was removed. Tibia sump Is fixed in 10% neutral buffered formalin and 10% sodium citrate is added. Demineralized in 5% formic acid containing, washed with tap water, a series of 70% to 100% ethanol Dehydrate in ethanol and fix in glycol methacrylate Was. Proximal resection of the proximal end of the tibia (approximately 5 mm long) at 5 μm, tartrate-resistant Stain for acid phosphatase (TRAP) activity, and screen for bone cell identification. Counterstained with chill green and thionine.   Osteoblasts were collected in the central negative (clear) Golgi area, ectopic nuclei, and methyl green Identified by strong basophilic counterstaining of thiol and thionin, and TRAP staining of osteoclasts , Multinucleation, and non-uniform shape. For the following bone parameters, the tissue type Evaluated for measurement changes;   1) Growth plate activity: 50 times at 42 × magnification to determine growth plate activity Width measured every 100 μm.   2) Number of intracortical osteoblasts: Measured along one side of the intracortical surface at a magnification of 212x. You.   3) Intracortical osteoclast size: counted in vehicle-treated mice All osteoblasts, or 50 randomly selected in ob-treated mice Of osteoblasts at 424-fold magnification.   4) Number of endosteal osteoblasts: 0.62 to 3.10 mm distal to growth plate It is measured along the endosteal surface of the reticular bone at the metaphysis at a magnification of 212x.   5) Number of endosteal osteoclasts: measured simultaneously when endosteal osteoblasts are counted Is done.   6) Reticulated bone% (bone volume / tissue volume, BV / TV): Reticulated per control tissue area The same pair calculated from the bone area and where the endosteal osteoblast and osteoclast counts are taken It is measured in the illumination area.   7) Epiphyseal osteoblast number: osteoblasts are also found in this metabolically inactive bone site. To the epiphysis to determine if It is measured from the endosteal surface of the reticular bone.   Analysis of the data (mean ± SEM, n = 5 / group) showed that:   1. ob protein processing as measured by increased growth plate width Growth plate activity was significantly enhanced.   2. ob protein treatment is about 32 times greater than levels in control group The cortex at the metaphysis and epiphyseal, with the most dramatic changes occurring at the endocortical surface Osteoblast numbers at all bone sites tested, including inner and endosteal surfaces Significantly increased. The results show that ob protein stimulated osteoblast activity Suggests.   3. ob protein treatment does not significantly alter the number of endosteal osteoclasts, Indicates that the income level did not change.   4. The percentage of reticular bone volume was not significantly changed by the ob protein, which was The osteogenic effect of the protein is continuous, and similar experiments with PTH show As indicated, it suggests that it takes a long time to find hardened bone.B. Male mouse     The same experiment performed with C57BL / 6J ob / ob male mice shows similar results. And the mechanism underlying the observed osteoblast activation and bone morphology It suggests that no logen is mediated.Example 2. Calcification   Osteoblast-mediated bone mineralization following exposure of osteoblasts to the ob protein leads to subsequent in vivo Were analyzed using a microassay:   Rack 24-well plates (American Scientific Products, Chicago, IL) Tail Collagen Type 1 (Collaborative Research, Inc., Bedford, MA) Mix 300 μl of 1 mg / ml rat tail collagen with 14.7 ml of 0.02 N acetic acid By coating. Previous The mixture was added at 2 ml per well and incubated for 1 hour at room temperature .   After the incubation, the wells are rinsed twice with 2.5 ml of PBS and dried. For 1 hour in a hood. Immediately after drying, store the plate at 4 ° C. Was.   Osteoblasts were plated to 90-100% confluency by the next day. Cells used are derived from P53 knockout mice (see WO 96/07733) And about 5 × 10 5 for cell lines 2-45 and CCC-4^FourCells / well 1 × 10 for cell line 2-29^FourCells / well, at 1-2 ml / well Growth medium and vehicle or a series of ob proteins ranging from 50 ng / ml to 5 pg / ml Using a diluted solution of                                   Table 1 Growth medium α-MEM (JRH Biosciences, Lenexa, KS), 15% fetal bovine serum (Hyclone, Logan, UT), (1 mM) sodium pyruvate (Irvine, Santa Ana, CA), (0.29 mg / ml) L-glutamine (Hazelton, Lenexa, KS), 1 × PSN (5 mg / ml penicillin, 5 mg / ml streptomycin,   10 mg / ml neomycin) (Gibco BRL, Gaithersburg, MD).   β-glycerophosphate (Sigma, St. Louis, MO) was added and a 2-10 mM minimum was added. The final concentration was reached. Magnesium L-ascorbic acid phosphate n-hydrate (Wak o Pure Chemical Industries, Ltd., Osaka, Japan) and add 50 μg / ml The final concentration was reached.   Cells are treated with β-glycerophosphate, L-ascorbic acid and ob protein or Was supplied every 2-4 days with the medium containing the vehicle.   Prior to calcification measurement, the wells were rinsed with PBS and the calcified layer was Add 300 μl / well N HCl and incubate overnight at room temperature And dissolved.   After the incubation, the solution was quenched with a 250 μl 0.5N HCl-dissolved calcified layer. and 600 μl of 2 M Tris (pH 7.4), 600 μl of Nova 7/9/10 Add urea dilution solution (Nova Biomedical, Waltham, MA) and 50 μl of 2N NaOH Buffered. Total calcium, Nova7 / 7 + 7Electorolyte The measurement was performed using an analyzer (Nova Biomedical).   The results show that calcification is more than 500 pg / ml ob from vehicle treated cells for 1-6 days. Increased in cells treated with the protein.Example 3 Calvarial assay   The effect of ob protein on bone growth was determined by subcutaneous tissue on the calvaria of mice. By injection of protein, 10-week-old CD-1 male mice (Jackson Labs) Tested. Doses range from 0.001 to 5.0 mg / mouse, 3 days a day for 5 days. Degree given.   After 14 days, the mice were sacrificed and calvarial bone growth was measured by histomorphometry. Was. The parameters measured are as described in Example 1. The result is negative And no significant calcification was found in the calvaria.Example 4. Ovariectomized rat assay   Ovariectomized rats were accepted as an animal model for human postmenopausal osteoporosis. Associated with estrogen deficiency similar to estrogen deficiency found in postmenopausal women Administration of ob protein to skeletal tissue in animal models of acute bone loss To evaluate the effect of ovaries on female normal rats, sham surgery or ovarian surgery To remove. hand 7 days after surgery, treatment is started and vehicle, ob protein or estrogen (160 μl) g / kg, subcutaneous). Before killing the animal, Administers a single dose of demeclocycline and evaluates bone formation and calcification. shin Bone and lumbar vertebrae were removed, fixed, processed and dissected as described in Example 1. Analyze.Example 5 Histomorphometric study of leptin-treated ob / ob mice   13-week-old genetically obese mice (ob / ob, C57BL / 6J) Divided into groups and processed as follows:     PBS 28 days (n = 4)     Vehicle 14 days (14 days on and 14 days off) (n = 6)     Leptin 14 days (14 days on and 14 days off) (n = 7)     Vehicle 28 days (n = 6)     Leptin 28 days (n = 6)   Both vehicles (0.007 mM borate in PBS) and leptin were administered twice daily i.p. Injected (leptin 50 μg 2 × / day, total 100 μg). Mice have food and water Freely given. Calcein injection (15 mg / kg body weight) was used to evaluate mechanical bone changes. Give 9 and 2 days before killing to label newly formed bone for value Was. All animals were killed at the end of day 28. Tibial sample in 70% ethanol Fixed and fixed in methyl methacrylate without decalcification. Tibia (about 5 mm length) were cut laterally in the sagittal direction at 5 and 10 μm. 10μm break The strips were fixed without staining for evaluation of the calcein label and 5 μm sections Was stained for tartrate-resistant acid phosphatase (TRAP) activity, and Were counterstained with methyl green and thionine for identification. Osteoblasts, Identified by central negative (clear) Golgi area, ectopic nuclei and strong basophil staining And TRAP staining, multinucleation and non-uniform shape of osteoclasts Identified by status.   The following bone sites were evaluated for histomorphometric changes: A. Intracortical bone   Intracortical bone at 90x magnification in anterior and posterior regions within 3.7mm below the growth plate evaluated. In general, osteogenic activity is significantly different at various sites within bone, The activity was higher in the posterior region than in the anterior region of the evaluated bone site.   The evaluation was as follows:   1)% single labeled surface (sLS): bone surface with single calcein label Percentage of.   2)% double labeled surface (dLS): of bone surface with double calcein label percentage.   3) Mineral deposition rate (MAR, μm / d): deposited on intracortical bone surface per day The width of the hardened bone.   4) Bone formation rate (BFR / BS, μm^Three/ Μm^Two/ Day): amount of new bone formed / Unit bone surface / day (for bone surface). B. Metaphyseal   Changes in bone cell activity following leptin treatment occur in the reticular bone (primary cavernous body) immediately below And the growth plate (secondary corpus cavernosum) are further different, Conversion was assessed at both sites.   The next mechanical bone change is preceded by 180 × magnification and 0.62 mm distal to the growth plate. The following were evaluated in the secondary cavernous body:   1)% single labeled surface   2)% double labeled surface   3) Mineral deposition rate   4) Bone formation rate (BFR / BS)   5) Number of endosteal osteoblasts (# osteoblasts / mm around reticular bone)   6) Number of endosteal osteoclasts (# osteoclasts / mm around reticular bone)   7)% reticulated bone volume (bone weight / tissue volume, BV / TV)   In the primary corpus cavernosum, only the last three bone parameters are scaled by a factor of 360. And within 0.3 mm of the growth plate. C. Epiphysis   At this bone site, the bone cells usually respond poorly compared to the other two bone sites. No (eg, do not respond to short-term estrogen deficiency or treatment). However The osteoblasts were subjected to subsequent leptin treatment in ob / ob mice as previously indicated. It was active against. To confirm this rare finding, perform a mechanical bone change. Was evaluated. The following bone parameters were measured from the entire endosteal surface of the reticular bone at 180x magnification. evaluated:   1)% single labeled surface   2)% double labeled surface   3) Mineral deposition rate (MAR, μm / day)   4) Bone formation rate (BFR / BS, μm^Three/ Μm^Two/Day)   The data obtained from this study (mean ± SD) is summarized as follows:   1. Leptin treatment of ob / ob mice is enhanced with increased% single and / or double-labeled Anterior and posterior, as indicated by the textured surface, mineral deposition rate, and bone formation rate Significantly enhanced tibial intracortical bone formation in both posterior sites, which was high with leptin treatment Confirmed the early discovery of osteoblast activity.   2. Leptin treatment for 14 days only partially enhances this enhanced intracortical osteogenic activity. Held.   3. A similar stimulus of bone formation by leptin is a mechanical indication of bone formation (secondary sponge Found on the tibial metaphysis as indicated by body) But it was hardly dramatic (for example, the number of endosteal osteoblasts Elevated 28 days after leptin treatment, but the increase was observed in primary corpus cavernosum It was significant when evaluated, but not statistically significant).   4. Number of endosteal osteoclasts in the secondary corpus cavernosum compared to earlier leptin experiments Was significantly increased by leptin. However, this rise is not Was not significant.   5. As previously observed,% reticular bone volume was significantly altered by leptin treatment Was not done.   6. At the epiphyses, leptin treatment is applied to a% single labeled surface, a% double label. This resulted in a significant increase in the reduced surface and bone formation rates.   7. Leptin stimulates bone formation with elevated serum following leptin treatment. Leptin approximating IGF-I levels and against osteoblasts in ob / ob mice Its potential role in the intervention of effects can be shown.   8. These results indicate bone defects associated with glucocorticoid excess (Dubuc et al.,Horm . Metab. Res .7: 102, 1975 and Bray etc.,Physiological Rev. 59: 719-809 And / or that diabetes can be normalized or ameliorated by leptin treatment. And suggest.   9. Combined treatment with anti-absorbents has potential for anabolic effects of leptin on bone Could be enhanced.   From the foregoing, certain aspects of the invention have been described herein for purposes of illustration. It will be appreciated that various modifications may be made within the scope of the present invention. U. Accordingly, the invention is not limited except as by the appended claims.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG), EA (AM, AZ, BY, KG, KZ , MD, RU, TJ, TM), AL, AM, AT, AU , AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, G B, GE, HU, IL, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, N Z, PL, PT, RO, RU, SD, SE, SG, SI , SK, TJ, TM, TR, TT, UA, UG, UZ, VN (72) Inventors Kuiper, Joseph El.             United States, Washington 98011, Bo             Cell Northeast One Hundred             Fifty Force Street 6640 (72) Inventors Weigl, David S.             United States, Washington 98110, Ba             Inbridge Island, Northeast             Baker Hill Road 6551 (72) Inventor Liu, Chun Shi.             United States, Washington 98115, United States             Attle, Northeast Eighties             Street 2820

Claims (1)

[Claims]   1. A method for stimulating a cell population comprising bone marrow mesenchymal cells,   Exposing the cell population to an effective amount of ob protein for expansion of osteogenic cells A method comprising:   2. 2. The method of claim 1, wherein the cell population is enriched for bone marrow mesenchymal cells. the method of.   3. 2. The method of claim 1, wherein hematopoietic cells have been removed from said cell population. .   4. 2. The method of claim 1, wherein said osteogenic cells are predominantly osteoblasts.   5. After the exposing step, the method further comprises the step of combining the cell population with an anti-absorbent. The method of claim 1 comprising:   6. After the exposing step, the method further comprises injecting the cell population into a mammal. The method of claim 1 wherein the method comprises:   7. A method for stimulating a cell population comprising bone marrow mesenchymal cells,   Transforming the cell population into a biological fluid obtained from an ob protein-treated mammal; A method comprising exposing.   8. 8. The method according to claim 7, wherein said biological fluid is serum.   9. The biological fluid enriches molecules effective for expansion of osteogenic cells 8. The method of claim 7, wherein the method is classified for use.   Ten. A method for stimulating a cell population comprising bone marrow mesenchymal cells,   The cell population is treated with endocrine, or CNS, exposed to the ob protein. Exposing to a culture medium conditioned by cell or tissue growth Method.   11． 11. The method according to claim 10, wherein said cells are pituitary or hypothalamic cells.   12． A method for promoting bone repair or bone healing in a mammal, comprising:   An amount effective to confer a clinically significant increase in bone repair or bone healing rate Administering to the mammal.   13. Stimulate bone growth into prosthetic devices or dental implants inserted into mammals A method for:   To confer a clinically significant increase in bone growth into the device or implant Administering to said mammal an effective amount of the ob protein.   14. A method for treating bone loss in a mammal, comprising:   Effective amount of ob protein to confer a clinically significant increase in bone mass Administering to the mammal.   15． A method for extending bone length in a mammal, comprising:   An effective amount of ob to confer a clinically significant increase in bone growth plate width A method comprising administering a protein to said mammal.   16． 16. The method of claim 15, wherein said mammal is an adolescent or pre-adolescent solid.   17． To stimulate active bone growth in mammals with fractures in bone growth plates The method of;   Provide the mammal with an effective amount of ob protein to restore bone length before fracture A method comprising administering.   18． Method for inducing osteogenesis in mammals with oncological resection of bone There;   Providing an effective amount of ob protein to induce bone formation at the site of the resection; A method comprising administering to a mammal.