MXPA03008571A - Stimulation of osteogenesis using rank ligand fusion proteins. - Google Patents

Abstract

A method of enhancing bone formation comprising administering an effective amount of 1) an oligomeric complex of one or more of RANKL, a RANKL fusion protein or an analog, derivative or mimic thereof, 2) an osteogenic compound capable of enhancing activity of one or more intracellular proteins in osteoblasts or osteoblast precursors, wherein said activity is indicative of bone formation, or 3) an osteogenic compound capable of inactivating one or more phosphatases in osteoblasts or osteoblast precursors, wherein said inactivation is indicative of bone formation. The method also may be used to treat a disease or condition manifested at least in part by the loss of bone mass by administering to a patient a pharmaceutical composition comprising an oligomeric complex or osteogenic compound disclosed herein.

Description

STIMULATION OF OSTEOGENESIS USING PROTEINS OF FUSION OF THE FACTOR RECEIVER ACTIVATOR LINK NUCLEAR KAPPA This invention was made in part with government support under the financial support of the National Institutes of Health AR32788, AR46123 and DE05413. The government has certain rights in the invention. This application claims the benefit of the provisional applications of the U.S.A. serial numbers 60/277, 855, 60/311, 163, 60/329, 231, 60/328, 876, and 60/329, 393, filed on March 22, 2000, August 9, 2001, 12 October 2001, October 12, 2001, and October 5, 2001, respectively, all of which are incorporated in the present invention as references.

BACKGROUND OF THE INVENTION The present invention relates to methods for improving bone formation processes by administering effective amounts of oligomeric complexes of one or more of RANKL, a RANKL fusion protein, an analog, derivative, or mimic of osteogenic compounds capable of of 1) enhancing the activity of intracellular proteins in osteoblasts or in osteoblast precursors, wherein said activity is indicative of bone formation, or 2) inactivating phosphatases in osteoblasts or in osteoblast precursors, wherein said inactivation is indicative of the formation of bone. The present invention further relates to the treatment, prevention or inhibition of bone loss or reduced bone formation caused by diseases such as osteoporosis. This further relates to the improvement of fracture repair and the promotion of bone ingrowth in orthopedic implants or bone fusion sites by facilitating the formation of bone caused by the administration of oligomeric complexes or osteogenic compounds described in the present invention. The invention further provides compositions for stimulating bone formation.

BACKGROUND OF THE INVENTION Various conditions and diseases which manifest themselves in bone loss or bone thinning are a critical and growing concern with regard to health. It has been estimated that as many as 30 million Americans and 100 million worldwide are at risk only from osteoporosis. Mundy et al. Science, 286: 1946-1949 (1999). Other known conditions involving bone loss include juvenile osteoporosis, osteogenesis imperfecta, hypercalcemia, hyperparatoidism, osteomalacia, osteohalisteresis, osteolytic bone disease, osteonecrosis, Paget's disease of bone, loss of bone due to rheumatoid arthritis, loss of periodontal bone, bone loss due to cancer, loss of bone mass related to age, and other forms of osteopenia. Additionally, new bone formation is needed in many situations, for example, to facilitate bone repair or replacement for bone fractures, bone defects, plastic surgery, dental implants or other types of implants and in other similar contexts. Bone is a dense connective tissue in a specialized way. The bone matrix is formed by osteoblast cells located on or near the surface of the existing bone matrix. The bone is reabsorbed (eroded) by another cell type known as the osteoclast (a type of macrophage). These cells secrete acids, which dissolve bone minerals, and hydrolases, which digest their organic compounds. Therefore, bone formation and remodeling is a dynamic process that involves a progressive interaction between the creation and erosion activities of osteoblasts and osteoclasts. Alberts, et al., Molecular Biology of the Cell, Garland Publishing, N. Y. (3rd ed., 1994), pp. 1 182- 1186. Current forms of therapies for bone loss are mainly anti-reabsorptive, since they inhibit bone resorption procedures, rather than encouraging bone formation. Among the prevention agents used or suggested for the treatment of osteoporosis due to their sustained ability to inhibit bone resorption are estrogen, selective modulators of the estrogen receptor (SERM), calcium, calciton, calcitonin (Sambrook) , P. et al., N. Engl. J. Med. 328: 1747-1753), alendronate (Saag, K. et al., N. Engl. J. Med. 339: 292-299) and other bisphosphonates. . Luckman et al., J. Bone Min. Res. 13,581 (1998). However, anti-resorptives can not correct the slow rate of bone formation frequently involved in net bone loss, and may have undesirable effects that are related to their impact on the inhibition of bone resorption / remodeling or other unwanted side effects. A key development in the field of bone cell biology is the recent discovery that the RANK ligand (RANKL, also known as a ligand for osteoprotegerin (OPGL), cytokine-induced TNF activation (TRANCE), a factor for Osteoclast differentiation (ODF)), expressed in stromal cells, osteoblasts, activated T lymphocytes and mammary epithelium, is the only molecule essential for differentiation of macrophages towards osteoclasts. Lacey, et al., Cell 93: 165-176 (1998) (Osteoprotegerin Ligand Is a Cytokine that Regulates Osteoclast Differentiation and Activation.) The cell surface receptor for RANKL is RANK, (Activator Receptor of Nuclear Factor (NF) kappa B - Activator of the Nuclear Factor Receptor (NF) -kappa B) RANKL is a type 2 transmembrane protein with an intracellular domain of less than approximately 50 amino acids, a transmembrane domain of approximately 21 amino acids, and an extracellular domain of approximately 240 to 250 amino acids RANK exists naturally in transmembrane and soluble form The deduced amino acid sequences for at least the murine, rat and human RANKL forms and variants thereof are known, see for example, Anderson, et al., Patent No. 6,017,729, Boyle, U.S. Patent No. 5,843,678, and Xu J. et al., J. Bone in. Res. (2000/15: 2178) which are incorporated herein by reference. OPGL) has sid or identified as a potent inducer of bone resorption and as a positive regulator of osteoclast development. Lacey, et al., Previously mentioned. In addition to its role as a factor in the differentiation and activation of osteoclasts, it has been reported that RANKL induces the formation of human dendritic cell (DC) clusters. Anderson et al., Previously mentioned and the development of mammary epithelium J. Fata et al., "The osteoclast differentiation factor osteoprotegerin ligand is essential for mammary gland development," Cell, 103: 41-50 (2000). However, it was previously unknown and it was not expected to find that RANKL may play a role in the anabolic processes of bone formation or it may be used in methods to stimulate osteoblast proliferation or bone node mineralization. Therefore, even when many elements have been discovered about osteoclasts and their manipulation for therapeutic purposes, not much is known about osteoblasts and bone formation. Therefore, there is a need, in general, for methods to improve bone formation and prevent or inhibit bone loss by anabolic stimulant procedures, to a degree greater than coordinated resorption.

BRIEF DESCRIPTION OF THE INVENTION Accordingly, among the objects of the present invention is the provision of methods and compositions that stimulate osteogenesis, including improved osteoblast activity, commitment of osteoblast precursors to the osteobysto phenotype and deposition of bone matrix in vivo. Therefore, methods are provided to improve bone formation as well as to treat diseases and conditions of bone loss by increasing bone formation, in any case, the processes of bone resorption are affected in another way. Briefly, therefore, the present invention is directed toward a method for improving bone formation. The method requires the administration of effective amounts of 1) oligomeric complexes of one or more of RANKL, a fusion protein of RANKL, an analog, derivative, or mimic, 2) osteogenic compounds capable of enhancing the activity of intracellular proteins in osteoblasts or osteoblast precursors, wherein said activity is indicative of bone formation, or 3) osteogenic compounds capable of activating phosphatases in osteoblasts or in osteoblast precursors, wherein said inactivation is indicative of bone formation. A method for treating a disease or condition manifested at least in part by the loss of bone mass is also provided. The method comprises the administration of a pharmaceutical composition comprising a fusion protein of RANKL or an analog, derivative or mimic thereof in an amount effective to promote bone formation. In another embodiment, a pharmaceutical composition comprising an osteogenic compound capable of enhancing the activity of intracellular proteins in osteoblasts or in osteoblast precursors may be used, wherein said activity is indicative of bone formation. In a further embodiment, a pharmaceutical composition comprising an osteogenic compound capable of activating the phosphatases in osteoblasts or in osteoblast precursors may be employed, wherein said inactivation is indicative of bone formation. Therefore the loss of bone mass is prevented, inhibited or counteracted. In another aspect, applicants have provided a composition for stimulating bone formation, the composition includes an effective amount of a RANKL fusion protein, an oligomeric complex, or an analog, derivative or mimic thereof in a carrier or excipient. pharmaceutically acceptable. Furthermore, compounds including effective amounts of osteogenic compounds in pharmaceutically acceptable carriers or excipients are further provided, wherein said osteogenic compounds are capable of 1) enhancing the activity of intracellular proteins in osteoblasts or in osteoblast precursors, wherein said activity is indicative of bone formation; or 2) inactivating phosphatases in osteoblasts or in osteoblast precursors, wherein said inactivation is indicative of bone formation. In one embodiment, the intracellular proteins are selected from IKB-a and γ-β. In a preferred embodiment, the intracellular proteins exhibit prolonged activity comprising intracellular kinases, and more preferably said kinases are ERK1 / 2, IKK, PI3 kinase, Akt, JNK, and p38. In a more preferred embodiment, the kinases are ERK1 / 2. In another preferred embodiment, the activity of one or more intracellular proteins constitutes the phosphorylation of said protein (s). Specifically, the phosphorylated proteins include ERK1 / 2, IKK, PI3 kinase, Akt, JNK, and p38. More preferably, the phosphorylated kinases are ERK1 / 2. In another aspect, the activity of one or more intracellular proteins can be detected by at least about 15-30 minutes after the incubation of the osteogenic compound with osteoblasts or osteoblast precursors. Preferably, the activity can be detected for 40 minutes, and more preferably it can be detected by at least 60 minutes after said incubation. In another embodiment, osteogenic compounds capable of inactivating one or more phosphatases in osteoblasts or in osteoblast precursors, wherein said inactivation is indicative of bone formation can be used in the methods and compositions of the present invention. Preferably, said phosphatase is selected from the group consisting of specific phosphatases of ERK1-, ERK2-, IKK-, kinase PI3-, Akt-, JNK-, and p38-, and more preferably the phosphatase is specific for ERK1 / 2. . In another preferred embodiment, the inactivation comprises the phosphorylation of a phosphatase. Preferred oligomeric compounds used in the methods and compositions described in the present invention include oligomeric complexes of GST-RANKL, AP-RANKL, RANKL-leucine zipper, and a derivative of RANKL comprising the "Fallow" domain of TALL-1. Other objects and features will be apparent in part and in part will be pointed out in the present invention below.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is the structure and sequence of the murine cDNA of RANKL and the protein used to produce the GST-RANKL fusion proteins discussed in Examples 1 and 25 below. Figure 2 describes a size exclusion chromatography of the GST-RANKL fusion protein under conditions that mimic the physiological medium. See example 1.

Figure 3 is a histological presentation of the stimulation by GST-RANKL of bone formation ex vivo in a culture of total calvarial organ, as discussed in example 2. The arrows mark the thickness of the parietal bone. Figure 4 is a graphical representation of dose-dependent increase in calvarium thickness due to stimulation by GST-RANKL of in vitro bone formation, as discussed in example 2. White bars indicate an exposure dose 1, while the black bars indicate an exposure dose 2 to GST-RANKL. Figure 5 (a) is a histological presentation of the stimulation by GST-RANKL of bone formation in vivo in mice, shown at low amplification, as discussed in example 3. Figure 5 (b) is a presentation Histological examination of GST-RANKL stimulation of bone formation in vivo in mice, shown at high amplification, as discussed in example 3. Figure 5 (c) illustrates a dual-energy X-ray absorptiometry analysis (DEXA) of the tibial metaphysis compared to the bone mineral density of the animals that were administered GST-RANKL or an in vivo control vehicle, as discussed in Example 3. Scale of the bar = 1 mm. Figure 6 is a histological presentation of a mouse tibia at high amplification, demonstrating the in vivo activation of the osteoblasts in animals given GST-RANKL as discussed in example 4. The arrow in the left panel indicates activated osteoblasts, while the date in the right panel indicates cells that lie in the flat bone. Figure 7 is a graphic illustration of the impact of the controlled administration of GST-RANKL to the animals, illustrating the number of osteoclasts and activated osteoblasts, as discussed in example 5. The white bars indicate the number of osteoclasts, while the black bars indicate the numbers of activated osteoblasts. Figure 8 is a histological presentation of the stimulation by GST-RANKL of the formation of the mineralized bone nodule in the bone marrow cells cultured ex vivo, as discussed in example 6. The red histochemical reaction product represents the forming units of mineralizing colony of osteoblasts. Figure 9 is an illustration of the label incorporation of a double fluorochrome in vivo within the mineralizing bone, as discussed in example 4. MAR represents mineral deposition, BFR indicates bone formation, and (ex) and ( en) indicate the exocranial and endocranial surfaces of the calvary, respectively. Figure 10 is a Western blot image illustrating rapid activation of MAPK pathway members in murine osteoclast precursors after treatment of cells with GST-RANKL. The activity was measured at the time of the interaction of GST-RANKL / RANK (0 minutes) and 5, 15, and 30 minutes after the interaction. From the top, the second, fourth, and sixth panels show the total levels of JNK, p38, and ERK respectively. The first, third and fifth panels illustrate the phosphorylated (activated) forms of JNK, p38, and ERK respectively. Figure 11 is an image of a Western blot illustrating the activity of Akt in murine osteoclast precursors after treatment of the cells with GST-RANKL. Activation was monitored at the time of the GST-RANKL / RANK interaction, and 5 and 15 minutes after the interaction. The lower panel illustrates the total Akt levels at the specified time points, while the upper panel illustrates the phosphorylated forms of Akt. Figure 12 is an image of a Western blot illustrating the prolonged activity of kinases in the MAPK pathway in murine osteoblasts after treatment of cells with GST-RANKL compared to treatment with RANKL alone. The points in time for which the phosphorylation was measured included 0 minutes (time of stimulation of the cells with GST-RANKL or RANKL), and 5, 10, 20, 30, and 60 minutes after the GST binding had occurred. -RANKL / RANKL or RANKL / RANK. The kinases whose activities were measured included ERK, JNK, p38, and Akt. pERK designates the phosphorylated ERK, ERK designates the total amount of the same protein, pJNK designates the phosphorylated JNK, JNK designates the total amount of JNK, pp38 designates the phosphorylated p38, p38 denotes the total amount of p38, pAkt designates a the phosphorylated Akt, and Akt designates the total amount of the same protein. The first panel from the top is p-lkBa, which designates phosphorylated IkBa, while IkBa denotes the total amount of the same protein. Figure 13 is an image of a Western blot illustrating the prolonged activity of ERK1 / 2. The points in time at which the ERK1 / 2 activity was measured include 0, 5, 10, 20, 30, and 60 minutes after the GST-RANKL / RANK interaction. pERK designates phosphorylated ERK while ERK designates the total amount of the same protein. Figure 14 is a graphical presentation of an alkaline phosphatase (AP) activity after exposure to GST-RANKL. Figures 5 (a) and 15 (b) illustrate GST-RANKL as oligomeric complexes, whereas cleaved RANKL (with GST removed) does not exist in oligomeric form. 15a Sample that the cleaved RANKL migrates as a particular trimeric species (1n), whereas GST-RANKL exists as a polydispersed mixture of mono-trimeric (n) and oligomeric (2-100n) units non-covalently associated under the dynamic equilibrium. 15b Illustrates possible oligomeric structures. Figure 16 consists of confocal microscopy images showing that the RANKL / RANK complexes are internalized rapidly, while the GST-RANKL / RANK complexes remain on the cell surface for at least one hour. In the grouped images, the colocalization of RANK (green fluorescence) and of the cell surface (red fluorescence) appears yellow.Figure 17 is an image of an agarose gel illustrating the expression of type I collagen in response to treatment with GST-RANKL. "+" indicates the treatment of primary osteoblasts with GST-RANKL, while "-" indicates the absence of said treatment. The osteoblasts were exposed to GST-RANKL for 1, 2, 4, or 6 hours of exposure at the start of each successive treatment window of 48 hours. All crops harvested between 8-48 hours were exposed to GST-RANKL for 6 hours. The expression of β-actin was used as a control for the experiment. Figure 18 is an image of an agarose gel that illustrates the expression of Cbfal in the bone marrow of mice treated with GST-RANKL or GST alone (labeled "control"). The lower panel is the control experiment, which illustrates the expression of HPRT (hypoxanthine phosphoribosyl transferase). Figure 19 is a graphical representation of osteoblast proliferation as measured by the incorporation of BrdU (5-bromo-2'-deoxyuridine) in response to treatment with GST-RANKL. Figure 20 (a) is an image of a Western blot that shows that osteoblasts transduced with negative dominant ERK can not phosphorylate an ERK substrate, known as RSK. DN-ERK represents the dominant negative ERK. LacZ represents the β-galactosidase. Figure 20 (b) is an image of an agarose gel showing that osteoblasts transduced with negative dominant ERK can not overregulate the expression of type I collagen in response to GST-RANKL.

Abbreviations and definitions To facilitate the understanding of the invention, various terms are defined below. "MAP kinase" or "MAPK" are used interchangeably in the present invention, and are abbreviations for mitogen activated protein kinase (mitogen activated p / otein kinase). "ERK1 / 2" refers to ERK1 and ERK2, which are abbreviations for kinase 1 regulated by extracellular signal and kinase 2 regulated by extracellular signal (extracellular signai-regulated kinase and extracellular signai-regulated kinase 2), respectively. JNK is an abbreviation for N-terminal kinase of c-jun (c-jun N-terminal kinase). p38 is a 38 kDa kinase, which is a member of the MAPK family of kinases. Akt is a serine threonine kinase Akt. "IKB" is an abbreviation for IkappaB protein. Therefore, IKB-a is IkappaB e ??? - ß is IkappaB ß. "IKK" is an abbreviation for IkappaB (IKB) kinase. "RSK" is an abbreviation for the ribosomal protein S6 kinase of p90.

"RANKL" or "RANK ligand" are used interchangeably in the present invention to indicate a ligand for RANK (NFkB Receptor Activator - Activator of NFkB Receptor). "AP" is an abbreviation for alkaline phosphatase. "GST" is an abbreviation for glutathione-s-transferase. "HPRT" is an abbreviation for hypoxanthine phosphoribosyl transferase. "Cbfal" is an abbreviation for the nuclear binding factor 1. "LacZ" is an abbreviation for β-galactosidase. "Osteogenic potential" or "osteogenic activity" are used interchangeably in the present invention to refer to any compound that is capable of improving bone formation, as determined from bone formation assays. "BrdU" is an abbreviation for 5-bromo-2'-deoxyuridine. "TALL-1" is an abbreviation for a protein ("TNF- and APOL-related jeukocyte expressed ligand 1 - ligand 1 expressed by the leukocyte related to TNF and APOL. "The term" an effective amount "means an amount of the substance in question which produces a statistically significant effect. "effective amount" for therapeutic use is the amount of the composition comprising an active compound required in the present invention to provide a clinically significant increase in cure rates in the repair of a fracture, reversion or inhibition of bone loss in osteoporosis; prevention or delay of the onset of osteoporosis; stimulation and / or increased bone formation in the fracture of non-union regions and in those that disturb osteogenesis; increase and / or acceleration of bone growth within prosthetic devices; repair or prevention of dental defects; or treatment or inhibition of other conditions, diseases or bone loss defects, including but not limited to those discussed in the present invention above. Said effective amounts will be determined using routine optimization techniques and are dependent on the particular condition to be treated, the condition of the patient, the route of administration, the formulation, and the judgment of the practitioner and other factors evident to those skilled in the art. The dose required for the compounds of the invention (for example, in osteoporosis where an increase in bone formation is desired) is manifested as that which induces a statistically significant difference in bone mass between the treatment and control groups . This difference in bone mass can be observed, for example, as at least 1-2%, or any clinically significant increase in bone mass in the treatment group. Other measurements of clinically significant increases in healing may include, for example, an assay for the N-terminal polypeptide of collagen type I, tests for breaking strength and tension, breaking and torsion strength, doubling at 4 points, connectivity increased in bone biopsies and other biomechanical tests well known to those skilled in the art. The general guide for treatment regimens is obtained from the experiments carried out in animal models of the disease of interest. As used in the present invention, "treatment" includes both prophylaxis and therapy. Therefore, to treat a subject, the compounds of the invention can be administered to a subject already suffering from bone loss or to prevent or inhibit the occurrence of said condition.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, the Applicants have discovered that the oligomeric complexes of the RANKL fusion proteins, particularly the oligomers of GST-RANKL, or the variants, analogs, derivatives and mimics thereof, can be administered in an amount and such that they stimulate a net increase in the numbers of activated osteoblasts and promote the anabolic processes of bone formation. This discovery provides the basis for useful methods to facilitate bone replacement or repair, as well as for the treatment of diseases or conditions that involve the loss of bone mass by stimulating anabolic processes of bone formation.

The following detailed description is provided to assist those skilled in the art in the practice of the present invention. Even so, this detailed description should not be considered to unduly limit the present invention since the modifications and variations in the modalities discussed in the present invention can be realized by those skilled in the art without departing from the spirit and scope of the present invention discovery. . All publications, patents, patent applications, databases and other references cited in this application are found in the present invention incorporated by reference in their entirety as if each individual publication, patent, patent application, database or reference were specifically and individually Indicated to be incorporated as reference. The selection and / or synthesis of RANKL, its fragments, variants, analogs, mimics, fusion products and oligomeric complexes of said compounds, wherein said oligomeric complexes are capable of promoting bone formation as taught in the present invention, are within the ability of a person skilled in the art and contemplated as within the scope of this invention. For example, Boyle, mentioned above, provides a detailed discussion of the synthesis of various forms of RANKL in the present invention (referred to as "protein for osteoprotegerin binding"), and describes, for example, murine and human variants, recombinant forms of RANKL, fragments of RANKL, analogues, mimics and derivatives of RANKL, and fusion proteins thereof. Also included within the scope or of the invention are derivatives or analogs of RANKL which have been modified post-translationally (such as glycosylated proteins), as well as polypeptides, which are encoded by the nucleic acids shown to hybridize to a part. or to all coding regions of the RANKL polypeptide cDNA under conditions of high stringency. See, for example, Boyle and Anderson, et al., Previously mentioned. The murine RANKL nucleic acid and the amino acid sequences are provided in the present invention as SEQ ID NO.1 and SEQ ID NO. 2, respectively (see figure 1). However, the RANKL sequences from other species have been identified and are available at http://www.ncbi.nlm.nih.gov/. The human RANKL nucleic acid and the amino acid sequences have, for example, the following accession numbers: AF019047 and AAB86811. The rat RANKL nucleic acid and the amino acid sequences have, for example, these access numbers: NM_057149 and NP_476490. Accordingly, any of the RANKL molecules can be used in the methods of the present invention, and therefore are contemplated within the scope of the present invention. RANKL and related molecules can be synthesized by the use of nucleic acid molecules which encode the peptides of this invention into an appropriate expression vector which includes the coding nucleotide sequences using methods well known in the art. These DNA molecules can be prepared, and subsequently analyzed, for example, using automated DNA sequencing and the well-known codon-amino acid relationship of the genetic code. Said DNA molecule can also be obtained as genomic DNA or as cDNA using conventional oligonucleotide probes and hybridization methodologies. Said DNA molecules can be incorporated into expression vectors, including plasmids, which are adapted for the expression of the DNA and for the production of the polypeptide in a suitable host such as a bacterium, for example, Escherichia coli, yeast cell, insect cell or mammalian cell. See, for example, Examples 1 and 25. Methods for the production of said recombinant proteins, including fusion proteins, are well known in the art and can be found in standard molecular biology references such as Sambrook et al., Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, 1989 and Ausubel et al., Current Protocols in Molecular Biology, 3rd ed., Wiley and Sons, 1995, and updates, incorporated herein by reference. It is further known that certain modifications can be made without completely eliminating the activity of the polypeptide. The modifications include the removal, replacement and addition of amino acids. Polypeptides containing other modifications can be synthesized by one skilled in the art. Therefore, the effectiveness of the polypeptides can be modulated through various changes in the sequence or structure of amino acids. Furthermore, it should be understood that the aforementioned analogs or mimics can be modified using methods known in the art to improve characteristics such as solubility, safety, or efficiency. A necessary feature of these preferred compounds is the ability to stimulate bone formation when used in accordance with the methods of the applicants described in the present invention. Applicants have discovered that the administration of GST-RANKL oligomers results in improved anabolic processes of bone formation. As shown in Example 1 and Figure 2, size exclusion chromatography indicates that the RANKL fusion proteins are capable of existing as oligomeric complexes under physiological conditions. It is believed that GST-RANKL oligomers are formed as a result of the tendencies of RANKL and GST to be trimerized or dimerized, respectively. Accordingly, other fusion partners besides the GSTs can be used to form oligomeric complexes comprising the RANKL. Preferred fusion partners include alkaline phosphatase and leucine zippers, however any other proteins with a tendency to form oligomeric structures are contemplated within the scope of the present invention. In a preferred embodiment, the merger partners of the RANKL are added to the N-terminus of the RANKL. The formation of GST-RANKL used to form oligomeric complexes is described in Examples 1 and 25. Additionally, it is within the scope of one skilled in the art to generate other forms of RANKL oligomers by well-known techniques. For example, one can construct RANKL oligomers using alternative proteins or polypeptides that have an intrinsic tendency to auto-associate and / or form complexes of a higher order. One can also create said oligomers by chemical modification or by synthesis of a polymeric form of RANKL in which many copies are associated, for example, similarly to a string of beads. Said alternative embodiments are also within the scope of this invention. Alkaline phosphatase (AP), like GST, has a tendency to dimerize. PAs form a large family of enzymes that are common to all organisms. Humans have four AP isoforms, three of which are tissue-specific and one of which is non-specific and can be found in bone, liver, and kidney. The three tissue-specific APs include: AP placental (PLAP), germinal cell AP (GCAP), and intestinal AP. The construction of an AP-amino terminal-RANKL can be carried out in a manner similar to the construction of the GST-RANKL fusion protein. Examples of alkaline phosphatases that can be used include but are not limited to human placental AP-1, human placental AP-2, placental AP precursor of human, mouse secreted AP, mouse embryonic AP precursor, and human embryonic AP mouse with the corresponding access numbers: AAA51710, AAA51707, ACC97139, AAL17657, P24823, and AAA37531. In a preferred embodiment, human placental alkaline phosphatase is employed, however other APs may be used, which are isolated either from humans or from other mammalian species such as Mus musculus. It is believed that the use of many different alkaline phosphatases is convenient due to the ability of all APs to dimerize. Briefly, a cDNA encoding a desired isoform of AP can be isolated from a cDNA library and processed to the 5 'end (at the amino terminus) of a RANKL cDNA in a suitable expression vector, such as , for example, pcDNA 3.1, using appropriate restriction endonucleases, in such a way that the resulting DNA sequence is in frame, without stop codons intervening in it. The expression vector, comprising the nucleotide sequence encoding AP-RANKL can be introduced into the host cells of choice by any of a number of transfection or transduction techniques known in the art. See also example 17. Alternatively, a RANKL fusion protein may comprise a peptide with the ability to oligomerize, such as a leucine zipper domain. The leucine zipper domains were originally identified in various DNA binding proteins (Landschulz et al., Science 240: 1759, 1988). The leucine zipper domain is a term used to refer to a conserved peptide domain present in these (and other) proteins, which is responsible for the dimerization of proteins. The leucine zipper domain comprises a repeating heptad repeat, with four or five leucine residues interspersed with other amino acids. Examples of leucine zipper domains are those found in the yeast transcription factor GCN4 and in a heat-stable DNA binding protein found in rat liver (C / EBP; Landschulz et al., Science 243: 1681, 1989). It is known that leucine zipper domains are folded as short forks, wound in parallel (O'Shea et al., Science 254: 539, 1991). The general architecture of the fork wound in parallel has been well characterized, with a "knot-in-orifice" packing as proposed by Crick in 1953 (Acta Crystallogr 6: 689). The dimer formed by a leucine zipper domain is stabilized by the heptad repeat, designated (abcdefg) n in accordance with the notation of McLachlan and Stewart (J. Mol. Biol. 98: 293; 1975), in which residues a and d are generally hydrophobic residues, with d being a leucine, which is in alignment on the same face of a helix. The negatively charged residues are commonly found at positions g and e. Therefore, in a parallel wound fork formed from two helical leucine zipper domains, the "knots" formed by the hydrophobic side chains of the first helix are packaged within the "holes" formed between the side chains of the second helix.

Several studies have indicated that conservative amino acids can be replaced by individual residues of leucine with a minimal decrease in the ability to dimerize; multiple changes, however, usually result in the loss of this capacity (Landschulz et al., Science 243: 1681, 1989; Turner and Tjian, Science 243: 1689, 1989; Hu et al., Science 250: 1400, 1990 ). van Heekeren et al. reported that several different amino residues can be substituted for the leucine residues in the leucine zipper domain of GCN4, and further they found that some GCN4 proteins containing two leucine substitutions were weakly active (Nucí Acids Res. 20: 3721, 1992). It has been found that amino acid substitutions at residues a and d of a synthetic peptide representing the leucine zipper domain GCN4 change the oligomerization properties of the leucine zipper domain (Alber, Sixth Symposium of the Protein Society, San Diego, California). When all the residues in position a are changed to isoleucine, the leucine zipper domain still forms a parallel dimer. When, in addition to this change, all the leucine residues in position d are changed to isoleucine, the resulting peptide spontaneously forms a trimeric hairpin wound in parallel in solution. Substituting all the amino acids in the D position with isoleucine and one in the a position with leucine results in a peptide that tetramerizes. Peptides containing these substitutions are still referred to as leucine zipper domains. However, it should be noted that in a preferred embodiment, leucine zipper domains capable of dimerizing proteins are used as fusion partners of RANKL. The construction of a fusion of a fusion protein of the RANKL-leucine zipper can be carried out in a similar manner as for GST-RANKL and AP-RANKL. See example 18. In addition to bacteria, other suitable expression systems such as mammalian cells and insect cells can be used. One skilled in the art can easily make necessary adjustments in order to express a leucine zipper fusion protein-RANKL. In an alternative embodiment, a derivative of RANKL can be used to form oligomeric complexes. It has recently been discovered that a member of the TNF ligand family, TALL-1 (also known as BAFF, THANK, BLyS, and zTNF4) possesses the ability to oligomerize under physiological conditions (Liu et al., Cell, 108: 383- 394, 2002). Liu et al. have shown that the "skirt" region, so called due to the length of the loop that forms the skirt and that allows it to extend from the molecule, mediates the trimer-trimer interactions and the subsequent formation of the grouping. This skirt region is unique to the TALL-1 from the members of the TNF family and is created by a surface handle DE (the handle that connects the D and E chains of TALL-1) that is longer than any of the handles of the other proteins of the TNF family, which have been discovered until now. It is believed that oligomerization occurs through a non-covalent interaction of the long loop DE with the TALL-1 molecules that surround it, resulting in the formation of large clusters. Since RANKL and TALL-1 are both members of the TNF ligand family and possess similar β-chain core structures, according to the invention, RANKL is mutated to create a mutant RANKL molecule that spontaneously oligomerizes under physiological conditions. In one embodiment, the RANKL modification is designed so that its AS-handle (amino acids 245-249 containing the amino acid sequence SIKIP) is substituted with the AS-handle of TALL-1 (amino acid sequence KVHVFGDEL). See example 19. To further recapitulate the TALL-1 oligomerization domains, the following amino acid changes can be made throughout the RANKL molecule: 168T? I, 187Y? L, 194K? F, 212F? 252H? V, 279F? I, and 283R? E. See example 20. Mutations can be introduced into RANKL by site-directed mutagenesis performed by PCR, using, for example, the QiuckChange multi-site directed mutagenesis kit (available from Stratagene). To determine the oligomerization potential of said modified RANKL molecule, one can use the same assays used to evaluate GST-RANKL, such as size exclusion chromatography. One skilled in the art can make such mutations and test the structure and function of the mutated RANKL without conducting experimentation.

The in vitro or live assays can be used to determine the efficiency of the RANKL oligomeric complexes of the present invention to promote bone formation in human and animal patients as deemed convenient by the applicants. For in vitro binding assays, osteoblast-like cells can be used. Suitable osteoblast-like cells include, but are not limited to, primary bone marrow stromal cells, primary osteoblasts, ST-2 cells, C1 cells, ROS cells, and MC3T3-E1 cells. Many of the cell lines are available from the American Type Culture Collection, Rockville, Md., And can be maintained at the specified standard growth medium. For in vitro functional assays, oligomeric complexes can be evaluated by culturing cells with a range of compound concentrations and evaluating markers or with indications of bone formation such as activation of the osteoblast, deposition of bone matrix, the thickness of the calvary and the formation of the bone nodule. See example 2 below. In addition, the proliferation of osteoblasts, the expression of collagen type I and / or the expression of Cbfa can be used to evaluate bone formation. See example 14 below. In addition, a general protocol for the treatment of osteoblasts with a compound is well established in the art. See, for example, Wyatt et al., BMC Cell Biology, 2: 14, 2001. A cell line of choice in this article was MC3T3-E1, which has been used as an in vitro model of osteobiotic differentiation and maturation. The treatment of the cells, in this case with BMP-2, was carried out in the following manner. Cells were seeded at 50007 cm2 in 25 cm2 culture bottles in -MEM supplemented with 5% fetal bovine serum, 26 mM NaHCO3, 2 mM glutamine, 100 u / ml penicillin, and 100 g / ml streptomycin, and were grown in a humidified atmosphere with 5% C02 / 95% air at 37 ° C. The cells were reseeded every 3-4 days after their release with 0.002% pronase E in PBS. The cells in the treatment groups were grown for 24 hours, then incubated with BMP-2 (50 ng / ml) dissolved in PBS containing 4 mM HCl and 0.1% bovine serum albumin (BSA) at 37 ° C. for 24 and 48 hours. The control groups received equal volumes of vehicle only. Exemplary conditions for the treatment of osteoblast cells or osteoblast precursors with oligomers, such as GST-RANKL, are described below. The osteoblast precursor cells were incubated in the presence of the vehicle, GST (a negative control), or increasing concentrations of the purified oligomeric GST-RANKL (e.g., concentrations ranging from 1 ng / ml to 100 ng / ml). The bone morphogenetic protein (BMP) -2 was administered as a positive control. The test compositions were administered for a period of 12 hours only at the beginning of the culture or once at the beginning and once three days later, again for a duration of 12 hours. It should be shown that the conditions used will vary according to the cell lines and the compounds used, their respective amounts, and additional factors such as planting conditions and composition of the medium. Such adjustments are easily determined by one skilled in the art. Additionally, the oligomeric compositions of RANKL which promote the formation of bone in accordance with the methods of the applicants, can be evaluated in various animal models. See examples 3-6 and the descriptions below. One commonly used trial is a neonatal mouse calvary trial. Briefly, four days after birth, the frontal and parietal bones of Swiss ICR white mouse pupae are removed by microdissection and divided along the sagittal suture. The bones are then incubated in a specified medium, where the medium contains either the test or control compounds. After incubation, the bones are removed from the medium, fixed in 10% formalin regulated in their pH for 24-48 hours, decalcified in 14% EDTA for 1 week, processed through graded alcohols , and they are embedded in paraffin wax. Sections of three micra of the calvary are prepared and evaluated using histomorphometric analysis of bone formation or bone resorption. Changes in bone are measured in sections of 200 micras apart. Osteoblasts and osteoclasts are identified due to their distinctive morphology. In addition to this assay, the effect of the compounds on the growth of murine calvarial bone can also be evaluated live.

In one of said examples of this screening test, ICR Swiss white mice, 4-6 weeks old, were used using 4-5 mice per group. Briefly, the test compound or the appropriate control compound was injected into the subcutaneous tissue on the right calvary of the normal mice. Mice were sacrificed at day 14, and bone growth was measured by histomorphometric modes. The bone samples were cleaned from the adjacent tissues and fixed in 10% formalin regulated in their pH for 24-48 hours, disqualified in 14% EDTA for 1-3 weeks, processed through graded alcohols, and they were imbibed in paraffin wax. The three to five micron sections of the calvary were prepared, and the representative sections were selected for histomorphometric evaluation of the effects of bone formation and bone resorption. The sections were measured by using a transparent camera fixation to directly trace the microscopic image onto a digitized plate. The changes of bone were measured on sections cut 200 micras apart, in 4 adjacent fields of 1X1 mm both on the injected sides and on the non-injected sides of the calvary. New bone was identified by its characteristic staining characteristics, and osteoclasts and osteoblasts are identified by their distinctive morphology. The software for histomorphometry (OsteoMeasure, Osteometrix, Inc., Atlanta) can be used to process the digitized data to determine cell counts and to measure areas or perimeters.

Additional in vivo assays include dosing assays in intact animals, and dosing assays in acute ovariectomized animals (OVX) (prevention model), and in chronic OVX animal assays (treatment model). The prototype dosage in intact animals can be achieved by, for example, subcutaneous, intraperitoneal, transepithelial, or intravenous administration, and can be carried out by injection, or by other administration techniques. The period of time for the administration of the test compound may vary (for example, 28 days as well as 35 days may be appropriate). As an example, transepithelial or subcutaneous dosing assays in vivo can be carried out as described below. In a typical study, 70 three-month-old Sprague-Dawley rats were weighed to coincide in weight and divided into seven groups, with ten animals in each group. This included a baseline control group of animals slaughtered at the beginning of the study; and a positive group that was given a compound that was known to promote bone growth. Three dose levels of the test compound were administered to the remaining groups. The test compound, PBS, and the vehicle were administered subcutaneously once per day for 35 days. The animals are injected with calcein for nine days and two days before slaughter (to ensure adequate marking of newly formed bone). Body weights were determined weekly. At the end of 35 days, the animals were weighed and bled by orbital or cardiac puncture.

Calcium, phosphate, osteocalcin and CBC were determined in serum. Both bones of the leg (femur and tibia) and the lumbar vertebrae were removed, cleaned of the adjacent soft tissue, and stored in 70% ethanol or 10% formalin for evaluation, as was done by computerized tomography. peripheral quantitative (pQCT, Ferretti, J. Bone, 17: 353S-364S, 1995), dual-energy X-ray absorptiometry (DEXA, Laval-Jeantet A. et al., Calcif Tissue Intl, 56: 14-18, 1995 , and Casez J. et al., Bone and Mineral, 26: 61-68, 1994) and / or histomorphometry. The effect of the test compounds on bone remodeling can be evaluated as well. Test compounds can also be tested in acute ovariectomized animals. Such assays may also include a group treated with estrogen as a control. An example of the test in these animals is briefly described below. In a typical study, 80 three-month-old Sprague-Dawley rats are weighed to match their weights and are divided into eight groups, with ten animals in each group. This included a baseline control group of animals slaughtered at the beginning of the study; three control groups (no OVX and vehicle only, OVX and vehicle only, and OVX and PBS only); and a control OVX group that was given a compound that was known to promote bone mass. Three dose levels of the test compound were administered to the remaining groups of OVX animals.

Since ovariectomy induces hyperphagia, all OVX animals are fed in pairs with animals without OVX treatment throughout the 35 days of study. The test compound, the positive control compound, the PBS or the vehicle were only administered transepithelially or subcutaneously once per day for 35 days. As an alternative, test compounds can be formulated in implantable concentrates that are implanted for 35 days, or can be administered transepithelially, such as by nasal administration. All animals are injected with calcein at empirically determined intervals, including but not limited to nine days and two days before slaughter. Body weights were determined weekly. At the end of the 35-day cycle, the blood and tissues of the animals were processed as described above. The test compounds can also be tested in chronic OVX animals. Briefly, 80 to 100 six-month-old Sprague-Dawley rats undergo surgery in which they are not treated (without OVX treatment), or ovariectomized (OVX) at the beginning of the experiment, and ten animals are sacrificed at the same time to serve as baseline controls. The body weights were monitored weekly. After approximately six weeks or more of bone depletion, 10 rats without OVX treatment and 10 OVX rats were randomly selected for slaughter as controls for the depletion period. Of the remaining animals, 10 rats without OVX treatment and 10 OVX rats are used as controls treated with placebo. The remaining animals are treated with 3 to 5 doses of the compound test for a period of 35 days. As a positive control, a group of OVX rats can be treated with an anabolic agent known in this model, such as PTH (Kimmei et al., Endocrinology, 132: 1577-1584, 1993). At the end of the experiment, the animals are sacrificed and the femurs, tibias, and lumbar vertebrae 1 to 4 were excised and collected. The left and right proximal tibias are used for measurements of pQCT, cancellous bone mineral density (BMD), and histology. The femurs are prepared for pQCT registration of the mid axis before the biomechanical evaluation. With respect to the lumbar vertebrae (LV), LV2 are processed for BMD (pQCT can also be performed), LV3 are prepared for undecalcified bone histology, and LV4 are processed for mechanical evaluation. In a further embodiment, applicants have discovered that the interaction between the oligomeric RANKL and its RANK receptor in osteoblasts or in osteoblast precursors results in a prolonged intracellular activity of the intracellular proteins. The mouse osteoblasts, when treated with GST-RANKL in vitro, show an activation, as characterized by the activation of the intracellular signaling pathways of NFkB and ERK. As evidenced by the applicants, the time course of the intracellular protein activity, especially of the ERK activity, is different from that observed in the osteoclast precursors, which also express RANK on the surface. In osteoclast precursors, ERK activity peaks 5-15 minutes after the interaction of RANK / GST-RANKL, and returns to baseline levels after 15-30 minutes. In contrast, ERK activity in osteoblasts has a peak at 10 minutes after the same interaction, and is still above baseline after 60 minutes. The prolongation of the time course is even more prominent in the osteoclast precursor cells, where the demonstrated activity of ERK has not reached its maximum even 60 minutes after the interaction of the oligomeric RANK / GST-RANKL. In addition to the different time course of ERK activity, osteoblasts and osteoblast precursor cells also exhibit prolonged activity of kinases such as IKK., PI3 kinase, Akt, p38 and JNK. This activity related to the osteoblasts contrasts with the interaction GST-RANKL with RANK on the osteoclasts, which results in a short-lived MAP kinases activity and bone resorption. Although we do not wish to stick to a particular theory, it therefore appears that the prolonged activity of the kinases observed in osteoblasts after stimulation with the oligomeric GST-RANKL plays a role in the anabolic processes of the bone. It is known that the intracellular signaling induced by the cytokine of the TNF family is attenuated by the internalization of the receptor-ligand complex (see, for example, Higuchi, M and Aggarwal, BB, J. Immunol., 152: 3550-3558, 1994). ). Therefore, applicants believe that the oligomeric complexes comprising the RANKL are not internalized as rapidly as the RANKL trimers, thus allowing a longer interaction with the receptor and prolonging intracellular signaling. See figure 16 and experiment 13. Accordingly, osteogenic compounds capable of promoting the activity of one or more intracellular proteins in osteoblasts or in osteoblast precursors, wherein said activity is indicative of bone formation, can be used in the methods of the present invention. Activated intracellular proteins include but are not limited to kinases. Preferably, the kinases comprise ERK1 / 2, PI3 kinase, IKK, Akt, and p38, and even more preferably, the kinases are ERK1 / 2. Other intracellular proteins include IKB-α and β-β. In another preferred embodiment, the activity comprises the phosphorylation of one or more intracellular proteins, and more preferably of the kinases. For the MAP kinase family, full activation requires dual phosphorylation on the tyrosine and threonine residues separated by a glutamate residue (known as the TEY motif, where T is threonine, E is glutamic acid, and Y is tyrosine ) by a particular kinase towards the 5 'end driven with the MAP kinase kinase (MKK) (by JyJAP kinase kinase). The requirement for dual phosphorylation ensures that MAP kinases are specifically activated by the action of MKK. Any of the assays available in the art can be used to determine if a kinase has been phosphorylated. Preferably, said assays include Western blot assays or kinase assays.

A Western blot can be carried out generally as follows. Once the cell lysates are generated, the intracellular proteins are separated based on their size by SDS-PAGE (sodium dodecyl sulfate-Dolyacrylamide gel electrophoresis - polyacrylamide gel electrophoresis with sodium dodecylsulfate). The separated proteins are transferred by electroblot to a suitable membrane (such as nitrocellulose or polyvinylidene fluoride) to which they adhere. The membrane is washed to reduce nonspecific signals, and then assayed with an antibody that recognizes only the specific amino acid that has been phosphorylated as a result of RANK signaling. After the additional wash, which removes the excess antibody, a second antibody is applied to the membrane, which recognizes the first antibody (bound to proteins specifically phosphorylated on the membrane) and which contains a reporter portion. The addition of an agent for development, which interacts with a reporter portion in the second antibody, results in the visualization of the bands. A kinase assay, for example for ERK1 / 2, can be carried out by using a known substrate for this kinase such as protein kinase S6 ribosomal p90 (RSK). Briefly, by way of example, the treated osteoblasts are washed in ice-cold PBS, for example, three times, and extracted with pH regulator for lysis in order to obtain the cell lysates. The supernatants obtained after the microcentrifugation of the cell lysates are incubated with the goat anti-RSK2 antibody (1: 200) together with the G-Sepharose protein at 4 ° C overnight. The beds are harvested by microcentrifugation, washed twice with pH regulator for lysis, followed by pH regulator for kinase. The activity of RSK2 phosphotransferase in the beds is measured by using the equipment for S6 kinase assay and [? -32?] ??? in accordance with the protocols provided by the manufacturer (Upstate Biotechnology, Inc.). An additional assay that can be applied to determine the activation of osteoblasts is a gel displacement test by electrophoretic mobility (EMSA). This assay monitors the nuclear translocation of a transcription factor complex (such as NFkB after activation of osteoblasts with GST-RANKL). Briefly, an EMSA can be carried out as follows. The treated osteoblast nuclei are isolated and their extracts are generated. Nuclear proteins are then incubated with a specific oligonucleotide probe that has been labeled with 32 P orthophosphate. After an appropriate time, the putative protein-DNA complexes are separated on a PAGE gel (without SDS present), in which it is dried and exposed to an X-ray film. If a specific complex has been formed (in this case a complex of NFkB proteins with a specific DNA sequence), a band will be visible in the developed film. Typically, appropriate controls are run in parallel with the experimental sample (s) in order to ensure that the band is specific for activated osteoblasts. For detailed procedures of Western blot, kinase assays, and EMSA, see for example Lai et al., Journal of Biological Chemistry, 276 (17): 14443-14450, April 27, 2001. Osteoclast activation can be detected by more at least 60 minutes after incubation of said cells with oligomers, such as GST-RANKL. In osteoblast precursor cells, activation reaches a peak after 5-10 minutes, and can be detected for more than at least 60 minutes. Accordingly, the activity of one or more intracellular proteins can be detected by at least about 30 minutes after the incubation of the osteogenic compound with the osteoblasts or osteoblast precursors. In a preferred embodiment, the activity is detected by at least about 40 minutes, and more preferably by at least about 60 minutes after said incubation. In another preferred embodiment, intracellular proteins whose activity is detected by at least about 30 minutes are kinases, and more preferably, the kinases are ERK1 / 2. To confirm that a compound that activates osteoblasts and / or stimulates the differentiation of osteoblast precursors can promote anabolic bone processes, said compound can be evaluated in a bone formation assay, where an increase in bone mass over the increase in background bone mass means a compound that has osteogenic activity. There are multiple assays for bone formation that can be used successfully to select potential osteogenic compounds of this invention. For example, cell-based assays for osteoblast differentiation and function, those based on measurable levels of collagen and those based on alkaline phosphatase activity, can be used. These tests are well known in the art and are easily carried out by one skilled in the art. In addition, multiple assays of bone formation in vivo and in vitro can be carried out with either osteoblasts or with osteoblast precursors since both cell types exhibit prolonged activity of the same kinases after stimulation with anabolic forms of RANKL. , such as GST-RANKL. In cases where intracellular activation assays and assays for bone formation are carried out with a library of compounds, it may be necessary to positively identify a compound that has been shown to be osteogenic. There are multiple ways to determine the identity of the compound. One process involves mass spectrometry, available from Neogenesis (http://www.neogenesis.com). Neogenesis's ALIS spectral search engine (automated ligand identification system) and software for data analysis allow a highly specific identification of a ligand structure based on the exact mass of the ligand. Mass spectrometry experiments can also be carried out by one skilled in the art to determine the identity of the compound. In another embodiment, osteogenic compounds capable of inactivating one or more phosphatases in osteoblasts or in osteoblast precursors, wherein said inactivation is indicative of bone formation can be used in the methods of the present invention. In a preferred embodiment, phosphatases inhibit kinases involved in osteogenesis, including p38, ERK, JNK, IKK, and Akt. More preferably, the phosphatases are specific for MAPK or specific for Akt, and even more preferably they are specific for ERK1 / 2. Although not wishing to be bound by a particular theory, this method is convenient for this purpose due to the fact that a kinase activity is strictly regulated by its corresponding phosphatase. In the case of ERK1 / 2, phosphatase is known as mitogen-activated protein kinase 3 phosphatase (MKP-3). This phosphatase belongs to a family of dual specific phosphatases, which are responsible for the removal of the phosphate groups from the threonine and tyrosine residues in their corresponding kinases (Camps et al., FASEB J., 14, p. 6-16, 1999). The rapid removal of the phosphate groups by the phosphatases ensures that the activation of the kinase is short-lived and that the level of phosphorylation is low in a resting cell. However, in order to activate the phosphatase and to remove the phosphate groups, it also needs to be phosphorylated. Therefore, inhibition of phosphatase activity results in activation or prolongation of ERK1 / 2 activity. A method for determining the ability of an osteogenic compound to inactivate phosphatases in osteoblasts / osteoblast precursors initially involves the activation of osteoblasts / osteoblast precursors with a substance known to activate these cells, such as GST-RANKL or BMP-2 (bone morphogenetic protein 2). This leads to the activation of the phosphatases, at which point the osteoblasts / osteoblast precursors are treated with a test compound and the cell lysates are obtained. The ability of the test compound to dephosphorylate (inactivate) the phosphatase (s) is determined by performing Western blots or kinase assays. See above. For further details on the evaluation of phosphatase activity, see Muda et al., J Biol Chem., 273: 9323-9329, 1998, and Camps et al., Science 280: 1262-1265, 1998. If it is determined that the compound possesses phosphatase inhibitory activity, this can also be further evaluated in one of the assays for bone formation to determine its osteogenic activity. These trials were also described above.

Pharmaceutical compositions and methods In a preferred embodiment of the invention, a method is provided for preventing or inhibiting bone loss or for promoting bone formation by administering 1) oligomeric complexes of one or more RANKL, a fusion protein of RANKL, an analog, derivative, or mimic, 2) osteogenic compounds capable of promoting the activity of intracellular proteins in osteoblasts or osteoblast precursors, wherein said activity is indicative of bone formation, or 3) osteogenic compounds capable of inactivating intracellular proteins in osteoblasts or osteoblast precursors, wherein said inactivation is indicative of bone formation. The bone formation compositions of the present invention can be utilized by providing an effective amount of said compositions to a patient in need thereof. In a preferred embodiment, said compositions are used to treat conditions selected from the group consisting of: osteoporosis, juvenile osteoporosis, osteogenesis imperfecta, hypercalcemia, hyperparatoidism, osteomalacia, osteohalisteresis, osteolytic bone disease, osteonecrosis, Paget's disease, rheumatoid arthritis , inflammatory arthritis, osteomyelitis, treatment with corticosteroids, periodontal disease, skeletal metastasis, cancer, bone loss related to age, osteopenia, and degenerative joint disease. For use for the treatment of animal subjects, the compounds of the invention can be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the type of treatment desired, for example, prevention, prophylaxis, therapy; the compounds are formulated in accordance with these parameters. A summary of such techniques is found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA. The administration of oligomers comprising the RANKL or osteogenic compounds of the present invention can be controlled pharmacokinetically and pharmacodynamically by the calibration of various administration parameters, including the frequency, dose, mode of duration and route of administration. Therefore, in one embodiment, the formation of bone mass is achieved by the administration of anabolic compositions such as an oligomeric complex of one or more of RANKL, a RANKL fusion protein, an analog, derivative or mimic in a non-continuous manner. , intermittent, such as by daily injection and / or ingestion. Generally, any osteogenic compound as described in the present invention can be administered intermittently to achieve the same effect. Variations in dosage, duration and mode of administration can also be manipulated to produce the required activity. For administration to animal or human subjects, the dose of the compounds of the invention is typically 0.01-100 mg / kg. However, dose levels are highly dependent on the nature of the disease or situation, the patient's condition, the practitioner's judgment, and the frequency and mode of administration. If the oral route is used, the absorption of the substance will be a factor that affects the bioavailability. A low absorption will have the effect that high concentrations, and therefore high doses, will be necessary in the gastrointestinal tract. It will be understood that the appropriate dosage of the substance to be properly evaluated by performing model tests on animals, where the effective dose level (eg, ED5o) and toxic dose (eg, TD50) as well as the dose level lethal (for example, LD50 or LD-io) are established in suitable and acceptable animal models.

In addition, if a substance has been proven to be efficient in such animal tests, controlled clinical trials should be conducted. In general, for use in treatment, the compositions of the invention can be used alone or in combination with other compositions for the treatment of bone loss. Such compositions include anti-reabsorbents such as a bisphosphonate, a calcitonin, a calcitrol, an estrogen, SERM and a calcium source, or a supplemental agent for bone formation such as parathyroid hormone or its derivatives, a bone morphogenetic protein, osteogenin , NaF, or a statin. See Patent of E.U.A. No. 6, 080, 779 incorporated in the present invention as reference. Depending on the mode of administration, the compounds will be formulated within the appropriate compositions. The formulations can be prepared in a manner suitable for systemic administration or for topical or local administration. Systemic formulations include, but are not limited to those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection) or can be prepared for transdermal, transmucosal, or oral administration. The formulation will generally include a diluent as well as, in some cases, adjuvants, pH regulators, preservatives and the like. For transepithelial administration, the appropriate penetrants for the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For oral administration, the compounds can also be administered in liposomal compositions or as microemulsions. Suitable forms include syrups, capsules, tablets, as understood in the art. For injection, the formulations can be prepared in conventional forms as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquid before injection or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol and the like. Said compositions may also contain amounts of non-toxic auxiliary substances such as wetting agents or emulsifying agents, pH regulating agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, and so on. The oligomers comprising the RANKL and the osteogenic compounds described in the present invention can also be administered locally to the sites in the patients, both in humans and in other vertebrates, such as domestic animals, rodents and cattle, where the formation and growth of the bone is desired using a variety of techniques known to those skilled in the art. For example, these may include sprays, lotions, gels or other vehicles such as alcohols, polyglycols, esters, oils and silicones. Such local applications include, for example, at a site of a bone fracture or defect to repair or replace the damaged bone. Additionally, the oligomeric complexes and osteogenic compounds of the present invention can be administered, for example, in a suitable vehicle, at the junction of an autograft, allograft or prosthesis and native bone to aid in the attachment of the graft or prosthesis to the bone. native. The pharmaceutically acceptable excipients include, but are not limited to, physiological saline, Ringer's solution, tocopherol, phosphate solution or pH regulator, saline with regulated pH, and other vehicles known in the art. The pharmaceutical compositions may also include stabilizers, antioxidants, colorants, and diluents. The pharmaceutically acceptable carriers and additives are chosen such that the side effects from the pharmaceutical compound are minimized and the performance of the compound is not suppressed or inhibited until such a degree of treatment is not effective. The following examples are illustrative of the invention, but should not be considered as limiting the various aspects of the invention thus illustrated.

EXAMPLES EXAMPLE 1 Expression of RANKL as a GST-RANKL fusion protein. The cDNA encoding residues 158-316 of murine RANKL was cloned into pGEX-4T-1 (Amersham, Genbank accession number U13853 - see listing of the national library of medicine at http: //ncbi.nlm.nih. gov under nucleic acids.) to the 3 'end of glutathione S-transferase using the restriction endonucleases Sali and Notl. After induction of expression of the medium protein by IPTG (0.05 mM) in Escherichia coli BL21 (DE3) (Invitrogen), the cells were ground in a pH regulator for lysis comprising 150 mM NaCl, 20 mM Tris-HCl pH 8.0, and 1 mM EDTA. The lysates were incubated with glutathione sepharose (Amersham) for affinity purification of the GST-RANKL fusion protein, followed by excessive washing with a pH regulator containing 150 mM NaCl and 20 mM Tris-HCI pH 8.0. After competitive elution (reduced glutathione 10 mM) from the affinity column, the isolated protein was then subjected to ion exchange chromatography, eluted with a salt gradient having a range from 0-500 mM, and dialyzed against a saline solution and physiological pH. The purified GST-RANKL was then assayed for endotoxin contamination by a lime amoebocyte lysate assay, and quantified for bioactivity by an osteoclastogenesis reading in vitro. Under conditions that mimic the physiological medium, GST-RANKL formed large oligomeric complexes, as demonstrated by size exclusion chromatography. See Figure 2. Most of the protein, as determined by the area under the curve in Figure 2, exists as oligomeric complexes of GST-RANKL.

EXAMPLE 2 Ex vivo stimulation of bone formation in calvary organ culture! full. An assay for bone formation was carried out as described in the U.S. Patent. No. 6, 080, 779 column 10, II. 29-55 incorporated in the present invention as a reference. The calvaries of neonatal mice were placed in organ culture in the presence of the vehicle, GST (a negative control), or of increasing concentrations of purified GST-RANKL obtained as described in example 1. Bone morphogenetic protein (BMP) ) -2 was administered as a positive control. The test compositions were administered for a period of 12 hours only at the beginning of the culture (1X) or once at the beginning and once three days later, again for a duration of 12 hours (2X). After seven days, the thicknesses of the calvaries were determined histomorphometrically and compared between the various control and experimental groups to evaluate bone formation. Previously, the bones of the calvary were removed from the incubation medium, fixed in 10% neutral formalin with pH regulated for 12 hours, disqualified in 14% EDTA for 3 days, dehydrated through gradual alcohols, and they were embedded in paraffin to perform histological sections. The calvaries were selected coronally through the central portion of the parietal bone, perpendicular to the sagittal suture. The coronal sections representative of the comparable anatomical position were subjected to histomorphometric evaluation (OsteoMeasure, Osteometrics Inc., Atlanta, GA) of the calvaria thicknesses. See Figure 3. GST-RANKL induced a dose-dependent increase in the thickness of the calvary when 1X or 2X was administered. See figure 4. At the highest doses evaluated (100 ng / ml) the thickness of the calvaries had doubled.

EXAMPLE 3 In vivo stimulation of bone formation in mice. Mice, C3H / HeN (Harian, Indianapolis, IN) were administered 100 micrograms of GST (control) or 100 micrograms of GST-RANKL as obtained in example 1, subcutaneously, once a day for nine days. Histological examination of the tibia revealed a marked increase in bone mass and a net increase in the number of activated osteoblasts in mice treated with GST-RANKL compared to control mice. See figures 5 (a) and 5 (b), taken at a low resolution amplification and a high resolution amplification, respectively. The figures revealed a marked increase in the cortical thickness and in the increase of the transverse architecture of the primary spongiosa, in relation to the control animals that received GST. The dual-energy X-ray absorptiometry (DEXA) analysis of the mice that were administered GST or GST-RANKL was also performed using standard procedures. The results (see Figure 5 (c)) show a significant increase in bone mineral density of the group treated with GST-RANKL compared to the control group.

EXAMPLE 4 In vivo activation of osteoblasts. The C3H / HeN mice (Harían, Indianapolis, IN) were administered GST (control) or GST-RANKL, following the procedure established in example 3. The histological examination of the tibia at high amplification revealed a marked activation of the Osteoblasts in mice treated with GST-RANKL compared to control mice. Quiescent osteoblasts are evident in control animals as thin cells that line the bone, while activated osteoblasts are evident in animals treated with GST-RANKL as buff, cuboidal cells along the surface of the bone. See Figure 6. The measurement of the bone formation ratio during in vivo administration of GST-RANKL, against control with GST, was achieved by intraperitoneal administration of 20 mg / kg calcein in 2% NaHCO 3. seven and two days before euthanasia to allow the incorporation of two fluorescent brands within the mineralizing bone matrix. After dissection, the calvaries were fixed in 70% EtOH and embedded in polymethyl methacrylate to make histological sections. 9 fluorescent micrographs of the coronal sections of the parietal bone taken in the middle between the coronal and lambdoidal sutures are shown in figure 9, with the external surface of the calvary facing upwards in the figure and the internal surface facing downwards. The amount of bone synthesized during the five-day period is that which is encompassed within the two series of parallel fluorescent bands. While the magnitude of bone formation in the control animals that only received GST is insufficient to produce distinctive double markings, there is a clear deposition of bone during the five days between the first and second markings in the animals treated with GST- RANKL.

EXAMPLE 5 The administration of GST-RANKL stimulates the proliferation of osteoblasts without substantially affecting osteoclastogenesis. The purified GST-RANKL fusion product was administered subcutaneously to the C3H / HeN mice (Harian, Indianapolis, IN), in incremental doses of 5, 50, 500, 1500, 5000 μg / kg, once a day, per 7 days. The GST in moles equivalent to the highest dose of RANKL serves as a negative control. The mice were sacrificed and the long bones were fixed, decalcified and stained for tartrate-resistant acid phosphatase (TRAP) activity. The activity of TRAP is a specific phenotypic marker of the osteoclast in the context of bone. The number of osteoblasts and activated osteoclasts per mm of the osseous surface was quantified histomorphometrically.

As seen in Figure 7, GST-RANKL administered intermittently (ie, by daily injection), resulted in a dose-dependent increase in activated osteoblasts, but not in the number of osteoclasts. GST had no obvious impact on osteoblasts or osteoclasts.

EXAMPLE 6 Promotion of the differentiation of osteoblast precursors as evidenced by formulation of the ex vivo bone nodule. Equal numbers of bone marrow cells were placed from mice treated with GST-RANKL (100 and from mice treated with GST, as discussed in Example 3, under osteoblastogenic conditions for 28 days to determine if the number was increased of osteoblasts and their committed precursors capable of forming bone After 28 days, the cells were stained with Alizarin red to identify the mineralized bone nodules and with hematoxylin to identify the colony forming units. from mice treated with GST-RANKL they generated substantially more mineralized bone nodules than their counterparts to which GST was administered (see Figure 8).

EXAMPLE 7 GST-RANKL rapidly activates MAP kinases in precursors of murine osteoclasts. Wild type C57BL / 6 mice were obtained from Harlan Industries (Indianapolis, IN). For the isolation of osteoclast precursors, bone marrow macrophages (BMM) were isolated from total bone marrow of mice four to six weeks of age and incubated in boxes for tissue culture at 37 ° C in CO2 at 5%. After 24 hours in culture, the adherent cells were harvested and deposited on a Ficoll Hypaque gradient and the cells were harvested at the gradient interface. Cells were reseeded at 65,000 / cm2 in minimal essential medium, supplemented with 10% heat-inactivated fetal bovine serum at 37 ° C in 5% CO2 in the presence of recombinant mouse M-CSF (10 ng / ml). The cells were treated with GST-RANKL on day 4 or 5. In the experiments directed at the evaluation of Akt activity, the cells were cultured in serum-free medium and in M-CSF-free medium for 24 hours before stimulation with GST-RANKL. The immunoblot (Western blot) of the osteoclast precursors was carried out in accordance with the following instructions. The BMM monolayers treated with cytokine and the control monolayers were washed twice with ice-cold PBS. The cells were lysed in the pH buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate. , 1 mM Na3P04, 1 mM NaF, and 1X protease inhibitor cocktail. Fifty μ9 of the cell lysates were boiled in the presence of pH regulator for sample with SDS (0.5 M Tris-HCl, pH 6.8, c / s 10% SDS, 10% glycerol, c / s bromophenol blue 0.05%) for 5 minutes and separated on SDS-PAGE, using 8% gels. Proteins were transferred to nitrocellulose membranes using a semi-dry blot apparatus (Bio-Rad, Richmond, CA) and incubated in blocking solution (dehydrated milk 5% fat in tris-saline with regulated pH containing 0.1% Tween 20) for 1 hour to reduce non-specific binding. The membranes were then exposed to the primary antibodies overnight at 4 ° C, washed three times, and incubated with goat anti-mouse IgG or rabbit secondary antibody conjugated with horseradish peroxidase for 1 hour. The membranes were washed extensively, and an improved chemiluminescence detection assay was carried out according to the manufacturer's instructions (Amersham). The results of the immunoblot assay are illustrated in Figure 10. As can be seen from this figure, the total cellular amounts of JNK, p38, and ERK did not change significantly at any point in the assay. Phosphorylation (activation) of ERK and p38 was detected 5 minutes after the stimulation with GST-RANKL, observing a peak at ten minutes after the interaction RANK / GST-RANKL, and was not detectable 30 minutes after the interaction . JNK was phosphorylated 15 minutes after stimulation with GST-RANKL, however the protein was rapidly dephosphorylated so that at 30 minutes after stimulation with GST-RANKL, the phosphorylated forms of JNK were not detectable. The data indicated transient and short-lived activity of ERK, JNK, and p38 in murine osteoclast precursors after stimulation with GST-RANKL.

EXAMPLE 8 GST-RANKL rapidly activates Akt in murine osteoclast precursors. The osteoclast precursors were isolated, maintained, and manipulated as described in Example 7. The immunoblot protocol was also the same as in Example 7, except that a primary antibody was specific for phospho-Akt, obtained from the cellular signaling Figure 11 shows that there was a detectable phosphorylation of Akt at the time of stimulation by GST-RANKL, indicating a rapid activation of this protein. Akt is a substrate for PI3 kinase, and in its active state is involved in anti-apoptotic signaling. Akt activity increased over time, i.e., the number of phosphorylated Akt molecules in the osteoclast precursors increases with time. Therefore, the activity of Akt was greater at 5 minutes than at 0 minutes, and had a peak activity at 15 minutes after stimulation with GST-RANKL.

EXAMPLE 9 The activity of MAP-kinases induced by GST-RANKL is prolonged in murine osteoblasts. The primary osteoblasts were isolated from neonatal murine calvaries by sequential enzymatic digestion. Briefly, the calvaries were fragmented and incubated at room temperature for 20 minutes with gentle agitation in an enzymatic solution containing 0.1% collagenase, 0.05% trypsin, and 4 mM NA2EDTA in phosphate buffered saline with regulated pH (PBS). calcium and magnesium. This procedure was repeated to produce a total of six digests. The cells isolated from the last four to six digested were cultured in MEM containing 15% FBS, 50 μ ?, ascorbic acid and 10 mM β-glycerophosphate. The cells were maintained at 37 ° C in a humidified atmosphere containing 6% CO2, with daily replenishment of the medium and the cytokines. After treatment with cytokine at the indicated times and doses, the cells were lysed in regulator for pH RIPA containing 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.2% sodium deoxycholate, and 1 mM EDTA, with 1 mM Na3P0, 1 mM NaF, and a cocktail of 1X protease inhibitor added immediately before use. The protein concentration was quantified and standardized by a Micro BCA protein assay (Pierce). The lysates were denatured by heat in the Laemmli regulator, resolved by SDS-PAGE, and transferred onto nitrocellulose. The levels of total and phosphorylated ERK, JNK, p38, Akt, and IkBa were determined using the primary and secondary antibodies according to the protocols established by the manufacturer, with conventional chemiluminescent detection. The membranes were cleaned between hybridizations with PBS containing 10 μ-mercaptoethanol. and 2% SDS. The results of the immunoblot assay in which the activity of MAP kinases is measured after stimulation with GST-RANKL or after equimolar stimulation with RANKL are shown in FIG. 12. The stimulation with GST-RANKL was carried out as it was described in example 7. Kinases whose dephosphorylation was measured include ERK, JNK, p38, and Akt. Again, as observed in the osteoclast precursors, the amount of total protein did not change significantly in the cells at any time point. However, all the evaluated kinases exhibited prolonged activity in osteoblasts. Both ERKs were activated for 5 minutes after the stimulation with GST-RANKL, and their activity could be detected 60 minutes after the stimulation. The activity of JNK, p38, and Akt was detected at the time of stimulation with GST-RANKL, and could be detected at least 60 minutes after the stimulation. In addition, phosphorylation of IkB was detected 10 minutes after the stimulation and increased until the end of the trial (60 minutes), indicating translocation increases of NFkB towards the nucleus. The data suggest that the activity pattern of MAP kinase is different from the activity of the same kinases in osteoclasts. The prolonged activity observed in osteoblasts seems to play a role in accelerated anabolic bone processes. In addition, treatment with RANKL was not able to induce prolonged activity of the kinases as observed with GST-RANKL.

EXAMPLE 10 The activity of ERK1 / 2 induced by GST-RANKL is prolonged in murine osteoclast precursors. The osteoclast precursors were isolated and maintained in accordance with the procedures set forth in Example 9. The immunoblot was carried out in the same manner as the immunoblot in Example 9. As seen in Figure 13, the ERK activity in Osteoclast precursors were prolonged and increased over time. While in the osteoblasts the activity was prolonged but did not change significantly during the time, the activity of ERK in the osteoblast precursors was detected initially at 10 minutes after the stimulation with GST-RANKL, and was increased up to 60 minutes after of the activation, which was the length of time by which the test was carried out.

EXAMPLE 11 AP activity after exposure to GST-RANKL in osteoblasts. The primary calvarium osteoblasts were cultured in MEM containing 15% FBS, 50 μ ?, ascorbic acid and 10 mM β-glycerophosphate. The cells were maintained at 37 ° C, with daily replenishment of the medium and cytokines. The activity of alkaline phosphatase (AP) of the osteoblast, a direct measure of the differentiation and function of the osteoblast, was quantified by the addition of a colorimetric substrate, p-nitrophenyl phosphate 5.5 mM. The cells were then exposed to GST-RANKL, administered at different regimes. Pulsatile exposure to GST-RANKL 50 ng / ml was provided at 1, 3, 6, 8, or 24 hours of total exposure through a 48-hour treatment window. After 4 of said treatments of 48 hours, the activity of AP was quantified (± S.D.) and normalized to the total protein levels. As can be seen from Figure 14, the maximum anabolic effect was observed when exposure to GST-RANKL was provided by an 8-hour treatment window, once every 48 hours. Therefore, GST-RANKL induced an increase in AP activity when administered intermittently.

EXAMPLE 12 Oligomerization of GST-RANKL. GST-RANKL was subjected to proteolysis to isolate the cleaved RANKL fragment from its GST fusion partner. Briefly, GST-RANKL was incubated with the 3C human rhinovirus type 3 protease (Amersham Pharmacia Biotech) for 4 hours at 4 ° C in 50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 10 mM EDTA, and DTT 1 mM. The uncleaved fusion protein and the protease labeled with GST were removed by passing them through an affinity matrix with glutathione. All purified recombinant proteins were tested for endotoxin contamination by a lime amebocyte lysate assay (Bio Whittaker), and analyzed by mass spectrometry to confirm their identity. Both GST-RANKL and cleaved RANKL were dialyzed against a saline solution and physiological pH, and fractionated by gel filtration on Superose-6 26/60 using an AKTA exploratory chromatography system (Amersham Pharmacia). The elution volumes were calibrated to a molecular weight using the following standards: ribonuclease A (13,700), chymotrypsinogen A (25,000), ovalbumin (43,000), bovine serum albumin (67,000), aldolase (158,000), catalase (232,000) , ferritin (440,000), thyroglobulin (669,000), and dextran blue 2000 (2,000,000). Fractions containing the protein from different elution volumes were subjected to Western analysis using a primary monoclonal anti-GST antibody. As shown in Figure 15 (a), the cleaved RANKL migrated as a particular trimeric species (1 n), whereas GST-RANKL migrated as a poiidisperse mixture of mono-trimeric (1n) and oligomeric (2-1 OOn) non-covalently associated under dynamic equilibrium. Crystallographic evidence has established that GST has an innate tendency to dimerize, whereas RANKL spontaneously trimerizes. A particular GST-RANKL trimer, consisting of 3 RANKL molecules and 3 GST molecules, therefore contains a free GST that is not bound to a neighboring GST, resulting in a 3: 2 stoichiometry that engenders a propensity to oiigomerization. Higher order branched oligomers are formed when the GST of a GST-RANKL trimer is given a dimer with GST from a neighboring GST-RANKL trimer (see Figure 15 (b)).

EXAMPLE 13 Internalization of GST-RANKL. Primary murine osteoblasts were maintained in -MEM containing 10% fetal bovine serum, and cultured in MEM containing 15% FBS, 50μ, ascorbic acid and 10mM β-glycerophosphate for differentiation. The cells were maintained at 37 ° C in a humidified atmosphere containing 6% C02, with daily replenishment of the medium and cytokines. Primary murine osteoblasts were cultured on coverslips in a-MEM containing 10% fetal bovine serum and treated with GST-RANKL or with RANKL excised for the indicated times. For the phospholipid membrane staining, cells were incubated for 20 minutes with fluorescent staining for lipophilic carbocyanin membrane Vybrant Dil (Molecular Probes). Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X, blocked with 1% BSA / 0.2% non-fat dehydrated milk in PBS, and stained for RANK with a polyclonal anti-RANK antibody. . The serial optical sections were obtained using a Radiance2100 laser scanning confocal microscope (BioRad). Established fixations of the microscope were calibrated to black level values using cells stained with an isotypic control Ig. The GST-RANKL was cleaved as described in example 12. The primary osteoblasts in culture were exposed to cleaved RANKL or to 5 nM GST-RANKL. At the indicated times, the cell surface was stained with a lipophilic fluorescent dye, and the RANK was stained with an anti-RANK antibody. Confocal microscopy was used to locate RANK (green fluorescence) and the cell surface (red fluorescence). In the clustered images, the colocalization of RANK and the cell surface appears yellow (superposition of green fluorescence and red fluorescence). The GST-RANKL: RANK complexes remained on the cell surface for at least one hour, corresponding to the sustained intracellular signaling of RANK. In contrast, the cleaved RANKL-RANK complexes were completely internalized in one hour, correlating with the absence of RANK signaling induced by RANKL cleaved at this time. The results are shown in figure 16.

EXAMPLE 14 Expression of collagen type I and Cbfa 1 in response to GST-RANKL. For the in vivo experiments, the mice were administered with 5 μg kg of GST-RANKL or GST alone as a control by subcutaneous injection and were euthanized one hour later. For in vitro experimentation, the primary osteoblasts were exposed to 100 ng / ml GST-RANKL or to GST alone as a control. The RNA was isolated with the RNeasy total RNA system (Qiagen) and digested with deoxyribonuclease to remove the genomic DNA. The messenger DNA was subsequently isolated from total RNA with the Oligotex mRNA purification system (Qiagen) and analyzed with the Platinum system for quantitative RT-PCR by one-step thermoscript (Life Technologies). Briefly, 1 μg of RNA was retro-transcribed into cDNA using murine gene-specific oligonucleotide primers designed to extend the exon-intron boundaries: Cbfa 1 sense 5 '-CCGCACGACAACCGCACCAT-3' (SEQ ID NO. 3), Cbfa 1 antisense 5'-CGCTCCGGCCCACAAATCTC-3 '(SEQ ID No. 4), and cc1 chain of type I collagen type 5'-TCTCCACTCTTCTAGTTCCT-3' (SEQ ID NO: 5) and chain 1 of collagen type I antisense 5 ' -TTGGGTCATTTCCACATGC-3 '(SEQ ID NO.6). Reverse transcription was carried out at 60 ° C for 30 minutes, followed by denaturation at 95 ° C for 5 minutes. The amplification by touchdown PCR came immediately. As a control, the expression levels of hypoxanthine phosphoribosyl transferase (HPRT) were evaluated concomitantly. The reaction products were fractionated electrophoretically in 2% agarose, and the results were presented from the linear range of the assay. Type I collagen, synthesized by osteoblasts, is the main organic component of bone. As shown in Figure 17, primary osteoblasts gradually regulate the expression of collagen as they differentiate in culture. Intermittent exposure to GST-RANKL accelerates this process, inducing vigorous expression of collagen within 12 hours of initial exposure to it. Cbfal is the master transcription factor for osteoblastogenesis, and its absence results in complete loss of osteoblasts and bone formation in mice (see, for example, Otto et al., Cell 89, pp. 765-771 , 1997, and Komori et al., Cell 89, pp. 755-764, 1997). As shown in Figure 18, Cbfal expression is promoted in the bone marrow within the first hour of systemic administration of GST-RANKL relative to the expression of control animals that received only GST.

EXAMPLE 15 GST-RANKL stimulates the proliferation of osteoblasts. The proliferation rate of osteoblasts in vitro was evaluated by the incorporation of 5-bromo-2'-deoxyuridine (brdU) into the DNA. Briefly, the cells were cultured in the presence of 10 μ BrdU for 48 hours, in the presence or absence of 100 ng / ml of GST-RANKL, or one molar equivalent of GST alone as a control. The incorporation of BrdU was quantified by ELISA (Amersham Pharmacia Biotech) using an anti-BrdU antibody labeled with peroxidase. The spectrophotometric measurements were carried out at 450 nm following the addition of the colorimetric substrate 3,3 -5,5'-tetramethylbenzidine. As shown in Figure 19, treatment with GST-RANKL promoted the proliferation rate of osteoblasts by up to 4 times during a 48-hour test period.

EXAMPLE 16 The activation of ERK is involved in the anabolic effects of GST-RANKL. A kinase-defective ERK1 cDNA (see Robbins et al., J. Biol. Chem., 268, pp. 5097-5106, 1993) used in this experiment was a result of mutation of alanine nucleotides at positions 211 and 212 with cytokine and guanine, respectively, resulting in a replacement of lysine 71 with arginine (Erk1 K71 R). ERK1 K71 R works in a dominant negative fashion to block both ERK1 and ERK2 activities (see Li et al., Immunol., 96, pp. 524-528, 1999). The ERK1 K71 R DNA was cloned into the restriction endonuclease sites Ncol and BamHI of the retroviral vector SFG as previously described (see Ory et al., Proc. Nati, Acad. Sel. USA, 93, pp. 11400- 11406, 1996). For the generation of the retroviral particles pseudotyped with the vesicular stomatitis virus (VSV) -g luco protein G, the retroviral vector SFG-ERK1 K71R was transfected into the packing cell line 293GPG expressing the Mui V gag-pol glycoprotein and VSV-G under tetracycline regulation. The conditioned medium was harvested after the withdrawal of tetracycline from days 3 to 7, and it was found to contain a viral titer of >; 5X106 colony forming units / ml. Before transduction, the medium was filtered through a membrane of 0.45 μ? T ?, and hexadimethrine bromide (polybrene) was added at a concentration of 8 μg / ml. As a negative control, a retrovirus carrying a LacZ cDNA was generated in the same manner. It was shown that transduction with pseudotypic VSV-retroviruses had no impact on the differentiation or function of the osteoblast (see Kalajzic et al., Virology, 284, pp. 37-45, 2001 and Liu et al., Bone 29, pp. 331-335, 2001). For retroviral transduction, the primary murine osteoblasts were cultured at a density of 60 cells per mm 2 in 150 mm culture boxes, and exposure to 25 ml of conditioned medium containing > 5X10 colony forming units / ml per 24 hours. The efficiency of the transduction exceeded 90%, as evidenced by the staining of the osteoblasts transduced with the LacZ retrovirus with X-gal. As seen in Figure 20 (a), osteoblasts transduced with negative dominant ERK failed to phosphorylate RSK, a known substrate for ERK cascading downstream of the process in response to treatment with GST-RANKL. In addition, Figure 20 (b) shows that osteoblasts transduced with negative dominant ERK could not overregulate the expression of collagen type I in response to GST-RANKL.

EXAMPLE 17 Expression of RANKL as an AP-RANKL fusion protein. The cDNA encoding residues 158-316 of the murine RANKL was cloned into the appropriate vector using the appropriate restriction endonucleases. A cDNA encoding human alkaline phosphatase 1 was isolated from a cDNA library and processed to the 5 'end (at the amino terminus) of a RANKL cDNA in a mammalian expression vector, such as, for example, pcDNA3.1, using appropriate restriction endonucleases, in such a way that the resulting DNA sequence is in frame, without stop codons intervening. The resulting vector is transduced into a mammalian cell line, such as, for example, CHO cells by standard methods. The purified AP-RANKL is then tested for endotoxin contamination by lime amoebocyte lysate assay, and quantified for bioactivity by in vitro osteoclastogenesis reading. Human AP 1 is a secreted protein, and as a result, the AP fusion protein is secreted into the medium. After a sufficient period of time for the AP-RANKL to be expressed and secreted by the mammalian cells in vitro, the medium is purified by affinity to isolate the AP-RANKL. The empirical mass of the AP-RANKL fusion protein is determined by mass spectrometry. The ability of AP-RANKL to form oligomeric complexes is evaluated by size exclusion chromatography.

EXAMPLE 18 Expression of RANKL as a fusion protein GCN4-RANKL. The cDNA encoding residues 158-316 of the murine RANKL was cloned into the appropriate vector using the appropriate restriction endonucleases. A DNA sequence encoding the GCN4 peptide was processed to the 5 'end (at the amino terminus) of a RANKL cDNA in a suitable expression vector, such as, for example, pGEX-6P-1 (Accession number U78872 ), using appropriate restriction endonucleases, in such a way that the resulting DNA sequence is in frame, without stop codons intervening. Following the induction of protein expression mediated by IPTG (0.05 mM) in Escherichia coli BL21 (DE3) (Invitrogen), the cells are ground in a pH regulator for lysis comprising 150 mM NaCl, 20 mM Tris-HCl pH 8.0, and 1 mM EDTA. The lysates were purified by affinity to isolate the GCN4-RANKL fusion protein. The isolated protein was then subjected to ion exchange chromatography, eluted with a salt gradient having a NaCl range of 0-500 mM, and dialyzed against physiological saline and pH. The GCN4-RANKL was then assayed for endotoxin contamination by a lime amoebocyte lysate assay, and quantified by bioactivity by in vitro osteoblastogenesis reading. The empirical mass of the GCN4-RANKL fusion protein was determined by mass spectrometry. The ability of GCN4-RANKL to form oligomeric complexes was evaluated by size exclusion chromatography.

EXAMPLE 19 Expression of a RANKL derivative comprising the skirt region of TALL-1. Residues 58-316 containing the murine RANKL were mutated so that their AS-handle (amino acids 245-249 containing the amino acid sequence SIKIP) was replaced with the AS-handle of TALL-1 (amino acid sequence KVHVFGDEL). Mutations can be introduced into the RANKL site-directed mutagenesis performed by PCR, using the site-directed multiple mutagenesis computer QuickChange (available from Stratagene). The mutated RANKL was cloned into the appropriate vector, such as, for example, pGEX-6P-1 (accession number U78872) using the appropriate restriction endonucleases such that the resulting DNA sequence is in frame, without stop codons intervening. Following the induction of protein expression mediated by IPTG (0.05 mM) in Escherichia coli BL21 (DE3) (Invitrogen), the cells are ground in a pH regulator for lysis comprising 150 mM NaCl, 20 mM Tris-HCl pH 8.0, and 1 mM EDTA. The lysates were incubated with glutathione sepharose (Amersham) for affinity purification of the mutated RANKL protein, followed by excessive washing with a pH regulator comprising 150 mM NaCl and 20 mM Tris-HCl pH 8.0. After competitive elution (reduced glutathione 10 mM) from the affinity column. The isolated protein was then subjected to ion exchange chromatography, eluted with a salt gradient having a NaCl range of 0-500 mM, and dialyzed against saline and physiological pH. The purified RANKL derivative was then tested for endotoxin contamination by lime amoebocyte lysate assay, and quantified for bioactivity by in vitro osteoblastogenesis reading. The empirical mass of the mutant RANKL was determined by mass spectrometry. The ability of the mutated RANKL to form oligomeric complexes was evaluated by size exclusion chromatography.

EXAMPLE 20 Expression of a RANKL derivative comprising the TALL-1 skirt region and additional amino acid changes. Residues 158-316 containing the murine RANKL were mutated so that their AS-handle (amino acids 245-249 containing the amino acid sequence SIKIP) was replaced with the AS-handle of TALL-1 (amino acid sequence KVHVFGDEL). The following amino acid changes are made along the RANKL molecule to increase the similarity with the structure TALL-1: 168T ^ I, 187Y -? L, 194K? F, 212P? Y, 252H * V, 279F, and 283R? E. Mutations can be introduced into RANKL by site-directed mutagenesis performed by PCR, using the site-directed mutagenesis multiplexer QuickChange (available from Stratagene). The mutated RANKL was cloned into the appropriate vector, such as, for example, pGEX-6P-1 using the appropriate restriction endonucleases such that the resulting DNA sequence is in frame, without stop codons intervening. Following the induction of protein expression mediated by IPTG (0.05 mM) in Escherichia coli BL21 (DE3) (Invitrogen), the cells are ground in a pH regulator for lysis comprising 150 mM NaCl, 20 mM Tris-HCl pH 8.0, and 1 mM EDTA. The Used ones were incubated with glutathione sepharose (Amersham) for affinity purification of the mutated RANKL protein, followed by an excessive washing with pH regulator comprising 150 mM NaCl and 20 mM Tris-HCl pH 8.0.

After competitive elution (reduced glutathione 10 mM) from the affinity column. The isolated protein was then subjected to ion exchange chromatography, eluted with a salt gradient having a NaCl range of 0-500 mM, and dialyzed against saline and physiological pH. The purified RANKL derivative was then tested for endotoxin contamination by lime amoebocyte lysate assay, and quantified for bioactivity by in vitro osteoblastogenesis reading. The empirical mass of the mutant RANKL was determined by mass spectrometry. The ability of the mutated RANKL to form oligomeric complexes was evaluated by size exclusion chromatography.

EXAMPLE 21 Ex vivo stimulation of bone formation in total calvarial organ culture. An assay for bone formation was carried out as described in the U.S. Patent. No. 6, 080, 779 column 10, II. 29-55 incorporated in the present invention as a reference. The neonatal mouse calvaries were placed in organ culture in the presence of the vehicle, AP (a negative control), or increasing concentrations of purified AP-RANKL. The bone morphogenetic protein (BMP) -2 was administered as a negative control. The test compositions are administered for a period of 12 hours only at the start of the culture (1X) or once at the start and once three days after, again for a duration of 12 hours (2X). After seven days, the calvarial thickness was determined histomorphometrically and compared between the various control and experimental groups to evaluate bone formation.

EXAMPLE 22 In vivo stimulation of bone formation in mice. The C3H / HeN mice (Harían, Indianapolis, IN) were given 100 micrograms of AP (control) or 100 micrograms of AP-RANKL subcutaneously, once a day for nine days. Histological examination of the tibia was carried out to evaluate the increase in bone mass and a net increase in the number of activated osteoblasts in the mice treated with AP-RANKL compared to the control mice. Dual-energy X-ray absorptiometry (DEXA) analysis of mice given AP or AP-RANKL was also performed using standard procedures to evaluate the change in bone mineral density in mice treated with AP-RANKL compared with mice treated with AP.

EXAMPLE 23 Ex vivo stimulation of bone formation in total calvarial organ culture. An assay for bone formation was carried out as described in the U.S. Patent. No 6, 080, 779 column 10, II. 29-55 incorporated in the present invention as a reference. The neonatal mouse calvaries were placed in organ culture in the presence of the vehicle, GCN4 (a negative control), or increasing concentrations of purified GCN4-RANKL. The bone morphogenetic protein (BMP) -2 was administered as a negative control. The test compositions are administered for a period of 12 hours only at the start of the culture (1X) or once at the beginning and once three days later, again for a duration of 12 hours (2X). After seven days, the calvarial thickness was determined histomorphometrically and compared between the various control and experimental groups to evaluate bone formation.

EXAMPLE 24 In vivo stimulation of bone formation in mice. Mice, C3H / HeN (Harían, Indianapolis, IN) were given 100 micrograms of GCN4 (control) or 100 micrograms of GCN4-RANKL subcutaneously, once a day for nine days. The histological examination of the tibia was then carried out to evaluate the increase in bone mass and a net increase in the number of activated osteoblasts in the mice treated with GCN4-RANKL compared with the control mice. Dual-energy X-ray absorptiometry (DEXA) analysis of the mice given GCN4 or GCN4-RANKL was also performed using standard procedures to evaluate the change in bone mineral density of mice treated with GCN4-RANKL compared to mice treated with GCN4.

EXAMPLE 25 Expression of RANKL as a GST-RANKL fusion protein. The cDNA encoding residues 158-316 of murine RANKL was cloned into pGEX-6p-1 (Amersham, Genbank accession number U78872 - see listing of the national library of medicine at http: //ncbi.nlm.nih. gov under nucleic acids.) to the 3 'end of glutathione S-transferase using the restriction endonucleases Sali and Notl. After induction of expression of the medium protein by IPTG (0.05 mM) in Escherichia coli BL21 (DE3) (Invitrogen), the cells were ground in a pH regulator for lysis comprising 150 mM NaCl, 20 mM Tris-HCl pH 8.0, and 1 mM EDTA. The lysates were incubated with glutathione sepharose (Amersham) for affinity purification of the GST-RANKL fusion protein, followed by excessive washing with a pH regulator containing 150 mM NaCl and 20 mM Tris-HCI pH 8.0. After competitive elution (reduced glutathione 10 mM) from the affinity column, the isolated protein was then subjected to ion exchange chromatography, eluted with a salt gradient having an Interval from 0-500 mM, and dialyzed against a saline solution and physiological pH. The purified GST-RANKL was then assayed for endotoxin contamination by a lime amoebocyte lysate assay, and quantified for bioactivity by in vitro osteoclastogenesis reading. Under conditions that mimic the physiological medium, GST-RANKL formed large oligomeric complexes, as demonstrated by size exclusion chromatography (data not shown). Most of the protein was presented as oligomeric complexes of GST-RANKL (data not shown).

EXAMPLE 26 Twenty six week old C57BL / 6 mice were randomized into two experimental groups. Group 1 of mice (10) received 100 ug of GST-RANKL by injection into the intramedullary cavity of the right femur. Group 2 of mice (10) received an equimolar volume of the GST vehicle by injection into the intramedullary cavity of the right femur. Mice were anesthetized with a cocktail of ketamine / xylazine (100 mg / kg ketamine and 10 mg / kg IP xylazine) and placed in left lateral recumbency. The main trochanter and the lateral femoral condyle of the right femur were identified and the intramedullary injection site was equidistant between these marks. Injections were made with 29-gauge needles in tuberculin syringes. On day 9, the mice were re-anesthetized with a cocktail of ketamine / xylazine (100 mg / kg of ketamine and 10 mg / kg of xylazine IP) and the dual-energy x-ray absorptiometry analysis (DEXA, Piximus) was performed. performed on each animal. The plain radiographs were taken immediately after the DEXA analysis (Faxitron, KV 0.15, time = 20 seconds). The animals were sacrificed by asphyxia with CC½ and both femurs were harvested for histological analysis. The femurs were fixed in 10% formalin with pH regulated for 48 hours and decalcified for 1 week. The DEXA analysis showed a significant difference in total bone mineral density (TBDM) between the group treated with GST-RANKL and the control group (see Table 1). No significant difference was observed either in the group treated with GST-RANKL or in the control group when the bone mineral density of the right and left femurs was compared (see table 2). There were no significant differences in skeletal density when simple radiographs of both groups were compared.

TABLE 1 BMD per group The means and standard deviations are reported. Proof of p-values for significant changes between groups. They are based on unpaired t tests.

TABLE 2 femoral BMD per side The means and standard deviations are reported for the right and left femurs for each group. P-value test for significant differences between the right and left sides. They are based on unpaired t tests.

Other features, objectives and advantages of the present invention will be apparent to those skilled in the art. It is intended that the explanations and illustrations presented in the present invention bring to light other experts in the art with the invention, its principles, and its practical application. Those skilled in the art can adapt and apply the invention in its numerous forms, as it can be better suited to the requirements of a particular use. Accordingly, it is not intended that the specific embodiments of the present invention, as set forth, be exhaustive or limiting of the present invention.

Claims (2)

NOVELTY OF THE INVENTION CLAIMS

1- The use of an effective amount of an oligomeric complex of one or more of RANKL, a fusion protein of RANKL, an analogue, derivative or mimic, to prepare a medicament for improving the processes of bone formation when training is desired bone. 2. The use as claimed in claim 1, wherein the improvement is selected from the group consisting of increasing the number of activated osteoblasts and increasing the proliferation of osteoblasts. 3. The use as claimed in claims 1 and 2, wherein the processes are selected from the improvement of the differentiation of the osteoblast precursor and the improvement of the proliferation of the osteoblast precursor. 4. The use as claimed in any of claims 1-3, wherein the desired bone formation comprises one or more of bone formation at a bone fracture site, the formation of bone at the junction of a bone and an allograft, autograft, bone prosthesis, or a vertebral body fusion. 5. - The use as claimed in any of claims 1-4, wherein the analog, derivative or mimic comprises a recombinant RANKL protein or a fragment thereof. 6. - The use as claimed in any of claims 1-5, wherein the fusion protein is selected from GST-RANKL, AP-RANKL, and leucine zipper-RANKL. 7. The use as claimed in any of the preceding claims, wherein the RANKL derivative comprises the RANKL protein comprising the TALL-1 skirt region. 8. The use of an oligomeric complex of one or more of RANKL, a RANKL fusion protein, an analog, derivative or mimic for the preparation of a pharmaceutical composition for treatment in a patient, including mammals and humans, of a condition manifested at least in part, by the loss of bone mass. 9. The use as claimed in claim 8, wherein the pharmaceutical composition is intermittently administrable. 10. The use as claimed in claims 8 and 9, wherein the fusion protein is selected from at least one of GST-RANKL, AP-RANKL, and leucine zipper-RANKL. 11. The use as claimed in any of claims 8-10, wherein the RANKL derivative comprises the RANKL protein comprising the TALL-1 skirt region. 12. - The use as claimed in any of claims 8-1 1, further comprising providing concomitantly an inhibiting agent of bone resorption. 13. The use as claimed in claim 12, wherein the bone resorption inhibiting agent is selected from the group consisting of a bisphosphonate, a calcitonin, a calcitro, an estrogen, a SERM and a calcium . 14. The use as claimed in any of claims 8-13, further comprising providing concomitantly one or more additional agents for bone formation. 15. The use as claimed in claim 14, wherein one or more additional agents for bone formation are selected from the group consisting of a parathyroid hormone or its derivatives, a bone morphogenetic protein, osteogenin, and a statin 16. The use as claimed in any of claims 8-15, wherein the disease or condition is selected from the group consisting of osteoporosis, juvenile osteoporosis, osteogenesis imperfecta, hypercalcemia, hyperparatoidism, osteomalacia, osteohalisteresis, osteolytic disease. of bone, osteonecrosis, Paget's disease, rheumatoid arthritis, inflammatory arthritis, osteomyelitis, treatment with corticosteroids, periodontal disease, skeletal metastasis, cancer, bone loss related to age, osteopenia, and degenerative joint disease. 17. - The use of an osteogenic compound capable of improving the activity of one or more intracellular proteins in osteoblasts or osteoblast precursors when bone formation is desired, to prepare a medication to improve the processes of bone formation. 18. The use as claimed in claim 17, wherein the improvement is selected from the group consisting of increasing the number of activated osteoblasts and increasing the proliferation of osteoblasts. 19. The use as claimed in claims 17 and 18, wherein the processes are selected from the improvement of osteoblasts precursor differentiation and from the improvement of osteoblast precursor proliferation. 20. The use as claimed in any of claims 17-19, wherein the desired bone formation comprises the formation of bone at a site of bone fracture, bone formation at the junction of a bone and an allograft, autograft , bone prosthesis, or in a vertebral body fusion. 21. Use as claimed in any of claims 17-20, wherein one or more intracellular proteins comprise a kinase. 22. The use as claimed in any of claims 17-21, wherein the enhanced activity comprises the phosphorylation of a kinase. 23. - The use as claimed in claims 21 and 22, wherein the kinase is selected from the group consisting of ERK1, ERK2, PI3 kinase, IKK, Akt, JNK, and p38. 24. - The use as claimed in claims 21 and 22, wherein the kinase is selected from ERK1 and ERK2. 25. - The use as claimed in any of claims 17-24, wherein the intracellular protein is selected from IKB-ct e ??? - ß. 26. - The use as claimed in any of claims 17-25, wherein the activity of one or more intracellular proteins is detected for at least about 30 minutes, preferably for at least about 40 minutes, and particularly for at least about 60 minutes after the incubation of said osteogenic compound with said osteoblasts or osteoblast precursors. 27. - The use of an osteogenic compound capable of enhancing the activity of one or more intracellular proteins in osteoblasts or osteoblast precursors for the preparation of a pharmaceutical composition for treating a disease or condition in patients, including mammals and humans, manifested at least in part, by the loss of bone mass. 28. - The use as claimed in claim 27, wherein the pharmaceutical composition is intermittently administrable. 29. - The use as claimed in claims 27 and 38, which further comprises providing concomitantly an inhibiting agent of bone resorption. 30. The use as claimed in claim 29, wherein the bone resorption inhibiting agent is selected from the group consisting of a bisphosphonate, a calcitonin, a calcitro, an estrogen, a SERM and a calcium. . 31. The use as claimed in any of claims 27-30, further comprising providing concomitantly one or more additional agents for bone formation. 32. - The use as claimed in claim 31, wherein one or more additional agents for bone formation are selected from the group consisting of a parathyroid hormone or its derivatives, a bone morphogenetic protein, osteogenin, and a statin 33. The use as claimed in any of claims 27-32, wherein the disease or condition is selected from the group consisting of osteoporosis, juvenile osteoporosis, osteogenesis imperfecta, hypercalcemia, hyperparatoidism, osteomalacia, osteohalisteresis, osteolytic disease. of bone, osteonecrosis, Paget's disease, rheumatoid arthritis, inflammatory arthritis, osteomyelitis, treatment with corticosteroids, periodontal disease, skeletal metastasis, cancer, bone loss related to age, osteopenia, and degenerative joint disease. 34. - The use as claimed in any of claims 27-33, wherein one or more intracellular proteins comprise a kinase. 35 - The use as claimed in any of claims 27-33, wherein the enhanced activity comprises the phosphorylation of a kinase. 36 - The use as claimed in claims 34 and 35, wherein the kinase is selected from the group consisting of ERK1, ERK2, PI3 kinase, IKK, Akt, JNK, and p38. 37. The use as claimed in claims 34 and 35, wherein the kinase is selected from ERK1 and ERK2. 38. - The use as claimed in any of claims 27-37, wherein the activity of one or more intracellular proteins is detected for at least about 30 minutes, preferably for at least about 40 minutes, and particularly for at least about 60 minutes after the incubation of said osteogenic compound with said osteoblasts or osteoblast precursors. 39. The use as claimed in any of claims 27-33, wherein one or more intracellular proteins are selected from IKB-a and γ-β. 40. The use of an osteogenic compound capable of inactivating one or more phosphatases in osteoblasts or osteoblast precursors when bone formation is desired, wherein said inactivation indicates bone formation, to prepare a medicament for improving the processes of bone formation. 41. The use as claimed in claim 40, wherein the improvement is selected from the group consisting of increasing the number of activated osteoblasts and increasing the proliferation of osteoblasts. 42. The use as claimed in claims 40 and 41, wherein the processes are selected from the improvement of the differentiation of the osteoblast precursor and from the improvement of the osteoblast precursor proliferation. 43. The use as claimed in any of claims 40-42, wherein the desired bone formation comprises one or more of bone formation at a bone fracture site, bone formation at the junction of a bone and bone. an allograft, autograft, bone prosthesis, or a vertebral body fusion. 44. The use as claimed in any of claims 40-43, wherein the inactivation comprises the dephosphorylation of a phosphatase. 45. The use as claimed in any of claims 40-44, wherein the phosphatase is selected from the group consisting of specific phosphatases of ERK1, ERK2, PI3 kinase, IKK, Akt, JNK, and p38. 46. - The use as claimed in any of claims 40-44, wherein the phosphatase is selected from specific ERK1 phosphatases and specific ERK2 phosphatases. 47. The use of an osteogenic compound capable of inactivating one or more phosphatases in osteoblasts or osteoblast for the preparation of a pharmaceutical composition, to be administered to a patient, including mammals and humans, to treat a disease or condition manifested at least in part , for the loss of bone mass. 48. - The use as claimed in claim 47, wherein the pharmaceutical composition is intermittently administrable. 49. - The use as claimed in claims 47 and 48, which further comprises providing. concomitantly an inhibiting agent of bone resorption. 50. The use as claimed in claim 49, wherein the bone resorption inhibiting agent is selected from the group consisting of a bisphosphonate, a calcitonin, a calcitrol, an estrogen, a SERM and a calcium. 51. The use as claimed in any of claims 47-50, further comprising providing concomitantly one or more additional agents for bone formation. 52. The use as claimed in claim 51, wherein one or more additional agents for bone formation are selected from the group consisting of a parathyroid hormone or its derivatives, a bone morphogenetic protein, osteogenin, and a statin 53J- The use as claimed in any of claims 47-52, wherein the disease or condition is selected from the group consisting of osteoporosis, juvenile osteoporosis, osteogenesis imperfecta, hypercalcemia, hyperparatoidism, osteomalacia, osteohalisteresis, osteolytic disease of bone, osteonecrosis, Paget's disease, rheumatoid arthritis, inflammatory arthritis, osteomyelitis, treatment with corticosteroids, periodontal disease, skeletal metastasis, cancer, bone loss related to age, osteopenia, and degenerative joint disease. 54. The use as claimed in any of claims 47-53, wherein the inactivation comprises the dephosphorylation of a phosphatase. 55.- The use as claimed in any of claims 47-54, wherein the phosphatase is selected from the group consisting of specific phosphatases of ERK1, ERK2, PI3 kinase, IKK, Akt, JNK, and p38. 56.- The use as claimed in any of claims 47-54, wherein the phosphatase is selected from ERK1-specific phosphatase and ERK

2-specific phosphatase. 57. A composition for stimulating bone formation comprising an effective amount of an oligomeric complex of one or more RANKL, RANKL fusion protein, analogue, derivative or mimic. 58. The composition according to claim 57, further characterized in that it additionally comprises a pharmaceutically acceptable excipient or carrier. 59. The composition according to claims 57 and 58, further characterized in that the stimulation of bone formation is selected from the group consisting of increasing the number of activated osteoblasts and increasing the proliferation of osteoblasts. 60. - The composition according to any of claims 57-59, further characterized in that the stimulation of bone formation is selected from the improvement of the differentiation of the osteoblast precursor and from the improvement of the proliferation of the precursor of osteoblasts. 61. - The composition according to any of claims 57-60, further characterized in that the stimulation of bone formation comprises the stimulation of bone formation in one or more of a bone fracture site, in the union of a bone. bone and an allograft, autograft, bone prosthesis, or a vertebral body fusion. 62. - The composition according to any of claims 57-61, further characterized in that the analog, derivative or mimic comprises a recombinant RANKL protein or a fragment thereof. 63. The composition according to any of claims 57-62, further characterized in that the fusion protein is selected from one or more of GST-RANKL, AP-RANKL, and leucine zipper-RANKL. 64. - The composition according to any of claims 57-63, further characterized in that the RANKL derivative comprises the RANKL protein comprising the skirt region of TALL-1. 65. The composition according to any of claims 57-64, further characterized in that it additionally comprises one or more resorption inhibiting agents. 66. - The composition according to claim 65, further characterized in that the bone resorption inhibiting agent is selected from the group consisting of a bisphosphonate, a calcitonin, a calcitro !, an estrogen, a SERM and a calcium . 67. The composition according to any of claims 57-66, further characterized in that it additionally comprises one or more additional agents for bone formation. 68. - The composition according to claim 67, further characterized in that one or more additional agents for bone formation is selected from the group consisting of parayroid hormone or its derivatives, a bone morphogenetic protein, osteogenin, or a statin . 69. A composition for stimulating bone formation comprising an effective amount of an osteogenic compound capable of enhancing the activity of one or more intracellular proteins in osteoblasts or osteoblast precursors, wherein said activity indicates bone formation. 70. The composition according to claim 69, further characterized in that it additionally comprises a pharmaceutically acceptable excipient or carrier. 71. - The composition according to claims 69 and 70, further characterized in that the stimulation of bone formation is selected from the group consisting of increasing the number of activated osteoblasts and increasing the proliferation of osteoblasts. 72. The composition according to any of claims 69-71, further characterized in that the stimulation of bone formation is selected from the improvement of the differentiation of the osteoblast precursor and from the improvement of the proliferation of the precursor of osteoblasts. 73.- The composition according to any of claims 69-72, further characterized in that the stimulation of bone formation comprises the stimulation of bone formation in one or more of a bone fracture site, in the union of a bone. bone and an allograft, autograft, bone prosthesis, or a vertebral body fusion. 74. The composition according to any of claims 69-73, further characterized in that it additionally comprises one or more resorption inhibiting agents. 75. The composition according to claim 74, further characterized in that the bone resorption inhibiting agent is selected from the group consisting of a bisphosphonate, a calcitonin, a calcitrol, an estrogen, a SERM and a calcium. 76.- The composition according to any of claims 69-75, further characterized in that it additionally comprises one or more additional agents for bone formation. 77. The composition according to claim 76, further characterized in that one or more additional agents for bone formation is selected from the group consisting of parathyroid hormone or its derivatives, a bone morphogenetic protein, osteogenin, or a statin. . 78. The composition according to any of claims 69-77, further characterized in that the intracellular protein comprises a kinase. 79. - The composition according to any of claims 69-78, further characterized in that the enhanced activity comprises the phosphorylation of a kinase. 80. The composition according to claims 78 and 79, further characterized in that the kinase is selected from the group consisting of ERK1, ERK2, PI3 kinase, IKK, Akt, JNK, and p38. 81. The composition according to claims 78 and 79, further characterized in that the kinase is selected from the group consisting of ERK1 and ERK2. 82. The composition according to any of claims 69-77, further characterized in that the intracellular protein is selected from IKB-cc e ??? -. 83. The composition according to any of claims 69-82, further characterized in that the activity of one or more intracellular proteins is detected for at least about 30 minutes, preferably for at least about 40 minutes, and particularly for at least about 60 minutes after the incubation of said osteogenic compound with said osteoblasts or osteoblast precursors. 84. A composition for stimulating bone formation comprising an effective amount of an osteogenic compound capable of inactivating one or more phosphatases in osteoblasts or osteoblast precursors, wherein said inactivity indicates bone formation. 85 - The composition according to claim 84, further characterized in that it additionally comprises a pharmaceutically acceptable excipient or carrier. 86. The composition according to claims 84 and 85, further characterized in that the stimulation of bone formation is selected from the group consisting of increasing the number of activated osteoblasts and increasing the proliferation of osteoblasts. 87. The composition according to any of claims 84-86, further characterized in that the stimulation of bone formation is selected from the improvement of the differentiation of the osteoblast precursor and from the improvement of the proliferation of the precursor of osteoblasts. 88. The composition according to any of claims 84-87, further characterized in that the stimulation of bone formation comprises the stimulation of bone formation in one or more of a bone fracture site, in the union of a bone. bone and an allograft, autograft, bone prosthesis, or a vertebral body fusion. 89. The composition according to any of claims 84-88, further characterized in that it additionally comprises one or more resorption inhibiting agents. 90. The composition according to claim 89, further characterized in that the bone resorption inhibiting agent is selected from the group consisting of a bisphosphonate, a calcitonin, a calcitrol, an estrogen, a SERM and a calcium. 91. The composition according to any of claims 84-90, further characterized in that it additionally comprises one or more additional agents for bone formation. 92. The composition according to claim 91, further characterized in that one or more additional agents for bone formation is selected from the group consisting of parathyroid hormone or its derivatives, a bone morphogenetic protein, osteogenin, or a statin. . 93. The composition according to claims 84-92, further characterized in that the inactivation comprises the dephosphorylation of a phosphatase. 94. The composition according to claims 84-93, further characterized in that the phosphatase is selected from the group consisting of specific phosphatases of ERK1, ERK2, IKK, PI3 kinase, Akt, JNK, and p38. 95. The composition according to claims 84-93, further characterized in that the phosphatase is selected from the group consisting of specific ERK1 phosphatases and specific ERK2 phosphatases.