CZ219093A3 - Insulin-like growth hormone binding protein derived from human bone - Google Patents

Abstract

Purified and isolated compositions of a binding protein for insulin-like growth factors (IGFs) I and II are provided. The binding protein, known as human bone derived IGF binding protein (hBD-IGFBP), potentiates the proliferative effects of IGF-II upon bone cells. Diagnostic assays are also provided for hBD-IGFBP, as well as pharmaceutical compositions and methods for treatment of bone disorders, wound healing, skin repair, and means for modulating IGF activity in bone.

Description

Technical field

The present invention relates to bone metabolism and in particular to bone metabolism processes that are mediated by a novel insulin-like growth factor binding protein (IGFBP) isolated from human bone. More specifically, the invention relates to IGFBP called human bone derived IGFBP (hBD-IGFBP), which potentiates the effect of insulin-like growth factor-II (IGF-II) on bone cell proliferation.

BACKGROUND OF THE INVENTION

IGF-I and IGF-II, the two most abundant growth factors present in human plasma, form a family of polypeptides that resemble proinsulin structurally and have both anabolic and acute insulin-like activities in many tissues (Daughaday et al., Endocrine Hev., 10: 68-91 (1989)). All previous studies in mice and rats emphasized IGF I as primary IGF, with IGF II as fetal hormone; Recent studies have considered the important role of IGF II in human bone metabolism. IGF II was found to be the most abundant growth factor in human bone and the most abundant growth factor produced by human bone cells. In addition, IGF II is one of several growth factors that is mitogenic to human bones. Also, recently purified IGFBP inhibitors, called IGBFPs, which inhibit basal bone cell proliferation by about 40 7b in the absence of serum, thus confirm that endogenous IGF production contributes substantially to cell proliferation in the absence of other growth factors.

-2A eventually, IGF II receptor blocking antibodies were found to inhibit basal bone cell proliferation, suggesting that IGF II is a key growth factor of bone cells. (Mohan et al., Growth, Genetics and Hormones, 6: 1-9 (1990) and Mohan et al., Clin.Orthopedics and Rel. Es., 263 * 30-48 (1990)).

Recently, it has also become clear that a family of structurally similar proteins that specifically bind IGF is required to modulate IGF action in various tissues. Four classes of human IGFBP (designated hIGFBP-1, hIGFBP-2, hIGFBP-3, hIGFBP-4) were isolated and complete amino acid sequences determined from the nucleotide sequences of isolated cDNA clones (see Mohan et al., Clin.Orthopedics and -Rel. Res. , 263: 30-48 (1990); Baxter et al., Prog. Growth Factor Res., 1: 49-68 (1989); Binkert et al., EM30 J. 8: 2497-2502 (1988); 152: 1289-1297 (1988), Brinkman et al., EMBO J. 7: 2417-2423 (1988); Lee et al., Mol.Endocrinol, 2: 404 -411 (1988); Wood et al., Mol.Endocrinol. 2: 11781185 (1988); Latour et al., M _o L. Endocrinol, 4: 1806-1814 (1990) and Shimasaki et al., Mol. Endocrinol 4: 1451-1458 (1990).

IGFBP-1 was isolated from various sources including amniotic fluid, placental membranes, decidua and HEP 62 hepatoma cells. The R-terminal amino acid sequences of IGF3P-1 proteins isolated from these different sources were found to be identical, and cloning and assembling of c ^ NA sequences encoding IGFBP-1 from HEP G2, human uterus and human placenta from cDNA libraries were described. IGFBP-2 was purified from conditioned media obtained from rat liver cells (3RL-3A) and Madin-Darby bovine kidney cells. The gene encoding IGFBP 2 was cloned from the 3RL-3A and human fetal liver libraries. IGF3P-3 was found

-3 in serum as a 150 kilodalton ternary complex between IGF-I or IGF-II, an acid-labile glycoprotein of about 85 kilodaltons, an IGFBP-III molecule which is an acid-stable glycoprotein of 53 kilodaltons. IGFBP-III has been purified for homogeneity from human serum and cloning and sequencing of cDNA encoding IGFBP-III has been described. IGFBP-IV was originally purified from conditioned human bone cell media as an inhibitor of IOFBP and rat serum. The cloning and sequencing of the IGFBP-IV cDNA clone isolated from a human bone cell library and from a liver cDNA library has been described recently. In addition to these four classes of IGFBP, Martin et al., J.Biol.Chem., 265 * 4124-4130 (1990), Roghabi et al., FE3S Lett., 255: 253-258 (1989) and Zapf et al. , J. Biol. Chem., 265: 14892-14898 (1990), describe the partial purification of IGFBP from human cerebrospinal fluid, from culture conditioned A8 2804 transformed fibroblast medium and from hypoglycemic serum, which shows a strong affinity for IGF-II more than IGF- AND.

Thus, a plurality of IGFBP variants are described which are produced from different sources and which exhibit diverse IGF binding properties as well as different biological functions. N _ap examples of IGFBP-I, IGFBP-III and IGFBP-bind both IGF-I and IGF-II with nearly equal affinity, whereas IGFBP-II and IGFBP purified from amniotic fluid, fibroblast cells and human serum bind IGF-II with higher affinity than IGF-I. In terms of functions, IGFBP-I has been shown to inhibit and potentiate the proliferative effect of IGFBP-1 on choriocarcinoma cells and human fibroblasts (Elgin et al., J. Biol. Chem., 84: 32543258 (1987) and Ritvos et al., Endocrinology, 122: 2150-2157 (1988)). IGFBP-3 has been shown to inhibit or stimulate the effect of IGF-1 depending on fibroblast culture conditions (De Mellow et al., Biochem. Biophys.Res.Comm., 156: 199-204 (1988)). Compared to *

-4IGFBP-1 and IGFBP-3 were the only IGFBP-4s to be shown to inhibit IGF-1 and IGF-II effects on bone cells (Mohan et al., Proc. Natl. Acad. Sci. USA, 86: 8338-8342 (1989)). Experience also suggests that production of different IGF3β is modulated in a differently specific manner in tissues. For example, IGFBP-1 production is modulated by insulin, while IGFBP-3 production is modulated by growth hormone (Baxter et al., Prog -rowth Factor Res., 1: 49-68 (1989)). These findings suggest that the multiplicity of different IGFBPs in conjunction with their unique regulation may cause modulation of IGF activities in a local, tissue-specific manner.

There remains a need in the art to determine the relationship of IGF-I and IGF-II functions in bone metabolism via IGFBP, and so there is a need to identify IGFBPs produced by bone cells and present in the human bone matrix.

Such IGFBPs are probably required in the regulation of bone metabolism and can be used in clinical trials to provide information in the diagnosis of defects in bone metabolism. IGFBPs that potentiate IgG-dependent bone growth could be particularly useful in therapeutic applications for the treatment of metabolic bone diseases; such as osteoporosis. Surprisingly, the present invention complements these and other needs.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for the clinical diagnosis and treatment of metabolic diseases related to IGF-mediated bone formation and cell proliferation. More specifically, the invention provides human bone derived IGF binding protein (hBD-IGFBP). hBD-IGFBP acts synergistically with IGF-II to potentiate IGF-II mediated cell proliferation under conditions where both IGF-II and

-5IGFBPs are administered simultaneously. Purified hBD-IGFBP also binds to high affinity hydroxyapatite and thus provides an agent for targeting molecules specifically to the bone, such as IGF-II and / or IGF-I, other growth factors and drugs that can affect bone resorption and _t formation. as useful in the treatment of, for example, bone fractures and bone diseases such as osteoporosis or osteosarcoma. hBD-IGFBP can also be used in the treatment of wound healing and skin repair. hBD-IGFBP can be used diagnostically as a marker of the degree of bone formation as well as during treatment of bone diseases with a therapeutic agent. hBD-IGFBP can also be used as a reagent for the clinical evaluation of IGF levels in samples in patients with bone metabolism and other diseases.

Description of the figures in the attached drawings

Figure 1 - Comparison of the N-terminal amino acid sequence of hBD-IGFBP with other known IGFBPs. Sequences are arranged to be as identical as possible.

Figure 2 - Competitive binding curves of hBD-IGFBP. The sample was assayed for protein binding activity using labeled IGF-II in the presence or absence of unlabeled IGF-I and IGF-II.

Figure 3 - Protein profile (A) and IGFBP activity profile (B) of human bone extract in the FPLC mono Q chromatography step. Three 50 ml aliquots of ^H A bound pooled fractions were applied to the IGF-II affinity column. The resulting three affinity bound fractions were pooled, and loaded onto a Mono-Q-anion exchange column. The protein profile is monitored by absorbance at 280 nm. Two ml, 2tnin fractions were collected. Fractions of fractions were diluted 10-fold and assayed for binding protein activity. IGFBP activity is expressed in the amount of specifically bound labeled IGF-II.

Fig. 4 - Ligand-blot analysis of hBD-IGFBP at different degree of purification. A 50 µL sample was subjected to SDS-PASE (3-2? Gradient), transferred to nitro cellulose membrane, overlaid with IGF-II labeled, and subjected to autoradiography. Threshold a, HA binding function of human bone extract lane b, IGF-II affinity binding fraction; lane c, Mono Q IGFBP peak Af lane d, Mono Q IGFBP peak B; lane e, Mono Q IGFBP peak S and lane f, Mono Q IGFBP peak D.

The invention provides purified and isolated human bone derived IGFBP that specifically binds IGF-II with a higher affinity than IGF-Ι. The protein may be purified in the desired homogeneity from a protein extract of, for example, human bone preparations, human bone cell conditioned medium, or human serum. Preferably, substantially pure hBD-IGFBP is at least about 50%, more preferably at least about 70-80%, and most preferably 9599 # or more homogeneous, especially for pharmaceutical use. Once purified, partially or even homogeneously as desired, hBD-IGFBP can then be used diagnostically as an immunogen, therapeutically, and so on.

The hBD-IGFBP produced in accordance with the present invention can be purified by hydroxyapatite by apatite chromatography followed by affinity chromatography on an IGF-II column and finally for greater purity by Mono Q anion exchange chromatography using the FPLC system. The expressed homogeneity of the purified protein is demonstrated, for example, by its single-band migration to DS-P-IGE and the production of a single amino acid sequence by N-terminal sequence analysis. Antibody column affinity chromatography using antibodies specifically directed against hBD-IGFBP can also be used in the purification scheme. Further purification can be accomplished by conventional chemical purification procedures such as liquid chromatography, gradient centrifugation, gel electrophoresis, exemplified herein. Protein purification methods are known in the art (see generally Scopes R., Protein Purification, Springer-Verlag, NY (1982), incorporated herein by reference) and can be applied to the hBD-IGFBP purification described herein.

Specific binding of hBD-IGFBP to IGF-II and IGF-I is demonstrated by a poly-ethylene glycol precipitation assay.

In this aspect, the invention provides a purified protein which is useful in studies of the structural function of IGF determinants that allow binding to specific receptors as well as hBD-IGFBP. A further use of the purified hBD-IGFBP of the invention is described in the description of other aspects of the invention.

The purified IGFBP of the invention is unique and different from all previously identified IGFBPs. Human IGF3P-1, IGFBP-2, IGFBP-3, and IGFBP-4 are characterized by amino acid sequences that have been published that are different from the hBD-IGFBP amino acid sequence. The β-terminal amino acid sequence described for IGFBD purified from cerebrospinal fluid, conditioned fibroblast medium, and human serum is also different from hBD-IGFBP.

The homogeneous human IGF3β of the invention (hBD-IGFBP) is characterized by an N-terminal amino acid sequence identical or significantly identical to that shown in Figure 1. For purposes of the invention, the N-terminal amino acid sequence substantially identical to that shown in FIG. 1 means a sequence identical to that in FIG. 1 except for the presence of conservative amino acid substituents or other amino acid substituents, insertions or deletions that do not substantially break the bond. identical protein to IGF-I or IGF-II, or otherwise does not significantly alter its function in the applications listed below.

In the production of hBD-IGFBP by the recombinant pathway, the gene encoding the hBD-IGFBD of the invention is cloned and expressed by insertion into a suitable expression vector, which is sequentially used to transform or transfect suitable host cells for expression of the recombinant hBD-IGFBP polypeptide. One or more synthetic oligonucleotide probes reflecting at least a portion of the terminal amino acid sequence of purified hBD-IGFBP, typically from about 14 to about 25 nucleotides, are used to elucidate the oNA library of human bone cells. The positive clone containing the longest insert is sequenced according to standard procedures. The deduced amino acid sequence is compared to the N-terminal amino acid sequence of the purified protein, and the predicted molecular weight and amino acid composition related to the detected amino acid sequence are compared to those observed for the purified hBDIGFBP. The clone is then used to produce recombinant hBD-IGF3β using standard procedures, as generally described, for example, by Bambrook et al., Molecular Gloning, Laboratory Laboratory, 1989 Gold Spring Harbor Press, NY, and this reference is incorporated herein by reference.

In another aspect, the invention relates to hBD-IGFBP polypeptides and fragments. The hBD-IGFBP polypeptides and fragments can be isolated from the recombinant expression system or can be synthesized by the solid phase method of Merrifield, Ped. , 21: 412 (1962), Merrifield, J. An. Chem. Soc., 85: 2149 (1963) or Aran and Merrifield, in The Peptides, vol. 2, pp. 1-284 (1979) Academic Press, NY. These references are incorporated herein by reference, or using an automated peptide synthesizer. By "polypeptides" is meant sequences with at least about 3 amino acids, typically 6 or more, up to 100 to 200 amino acids, or more, including whole proteins. For example, the portion (i) of the hBD-IGFBP protein that binds hydroxyapatite and / or IGF-II can be identified by various methods such as the treatment of hBD-IGFBP with protease or chemical agents to achieve fragmentation and determine which fragment is capable of binding labeled IGF- Polypeptides can then be synthesized and used as antigens to inhibit IGF-II or to hydroxyapatite-hBD-IGFBP interaction, etc. It is to be understood that as used herein, references to hBD.IGFBP are meant proteins, polypeptides, and their fragments, unless the context implies otherwise.

In another aspect, the invention provides knowledge for the regulatory aspects of the hydroxyapatite / h3D-IGFBP / IGF-II interaction and thus the treatment, therapeutically and / or prophylactically, of diseases that can be directly or indirectly associated with hBDIGFBP or its ligands such as IGF-II . Due to the binding protein of the invention, agonists or antagonists can be identified that stimulate or inhibit the interaction of IGF-II, hydroxyapatite and other ligands with hBD-IGFBP. The metabolism and reactivity of the cells in response to hBP-IGFBP or IGF-II with ago-10 antagonists or antagonists are controlled, thus providing knowledge to alleviate or, in some cases, prevent disease.

Thus, the invention provides screening procedures for identifying agonists or antagonists of mediated ligand / hBD-IGFBP interactions. Such screening assays may utilize a wide variety of formats depending on the extent to which the ligand / binding protein interaction is examined. For example, such assays may be aimed at identifying compounds that bind to a binding protein and thus block or inhibit interaction with IGF-II or hydroxy apatite. Additional assays may be aimed at identifying compounds that may replace hBD-IGFBP. Still further assays may be used to identify compounds that inhibit or facilitate association of βGF to hBD-IGFBP and thereby mediate a cellular response to IGF.

In another aspect, the invention provides a protein that binds IGF-II with a higher selective affinity than IGF-I. Giant.

represents the competitive binding curve of hBD-IGFBP with Z 1 / I / IGF-II as ligand and IGF-I and IGF-II as compertitors. About 10 µg / ml IGF-I and 1 µg / ml IGF-II are required to displace 50% / ¹²⁵ I / IGF-II from IGFBP, which means that IGF-II was 10 times more potent than IG? -I in the replacement labeled indicator. When [ ¹² µl] IGF was used as the radiolabeled indicator, IGF-II was still more potent (4-fold) than IGF-I. These results suggest that IGFBP binds IGF-II with higher affinity than IGF-I.

Typically, the binding affinity of hBD-IGFBP for IGF-II will be extended fathom-9 -12 10 M to about 10 or more and more likely in the range of at least about 10 "° to ¹ 10 ~ ¹¹ M, whereas the binding affinity of hBD-IGFBP for IGF-I will be about 10 fold lower, i.

-11asi from 10 ^"8 M to about 10 ^-10 M. The selective affinity of hBD-IGFBP for IGF-II over IGF-I could explain why IGF-II is 10 to 15 times more abundant than IGF-I in human bone.

The invention provides hBD-IGFBP therapeutic and pharmaceutical compositions that take advantage of the hBD-IGFBP's high binding affinity for hydroxyapatite, which is at least about 1 to 10 µl or more. The hBD-IGFBP of the invention binds to hydroxyapatite even in the presence of strongly denaturing agents such as 4M guanidine HCl, while purified IGF-II does not bind to hydroxyapatite. Thus, the D.IGFSP provides a composition or vehicle for fixing IGF-II targeting to or to the bone. IGF-II can be chemically coupled to hBD-IGFBP by linkages that will be readily understood by those of skill in the art, but which should not substantially reduce the desired activity of the self protein.

Attachment of hBD-IGFBP to another molecule to be directed to bone tissue or cells, such as IGF or other molecules listed herein, can be accomplished by chemical coupling using well known laboratory procedures. By chemical linkage is meant that the protein molecules are bound, typically to each other, typically by covalent bonds. A preferred coupling method is the formation of at least one covalent bond between the hBD-IGFBP / IGF-II molecules. The binding may be direct, which includes bonds comprising synthetic linking groups, or indirect, by which is meant a bond having an inserted group, such as a protein or peptide, eg a plasma albumin, another spacer molecule thereof. For example, the linkage may be via heterobifunctional or homobifunctional crosslinkers such as carbodiimide, glutaraldehyde, N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) and derivatives, bis-maleimide, 4- (N-maleimidomethyl) cyclohexane-1-carbo -12-oxylate (SMCC) by cross-linking without exogenous crosslinkers using single-molecule reactive groups such as carbohydrate, disulfide, carboxyl or amino groups by oxidation or reduction of the native protein, or by treatment with an enzyme or the like. Methods for chemically crosslinking protein molecules are generally known in the art, and many hetero- and homobifunctional agents are described, for example, in US Pat. 4355023, 4657853, 4675980, 4925921 and 4970156 ⁷ and ImmunoTechnology Catalog and ⁿ andbook,

Pierce Chemical Co. (1989), which are incorporated herein by reference. Generally, such crosslinking should not substantially affect the desired functions of (i) IGF-II or h3D-IGFBP.

The hybrid, chimeric or fusion protein molecule IGF-II and hBD-IGFBP, or portions thereof, can also be prepared by recombinant DNA techniques as described, for example, in US Patent 489609 and in Prabro Sambroka et al., Supra, which are incorporated herein by reference.

The deposition of IGF-II / hBD-IGFBP complexes in bone allows, during bone resorption and dissolution of hydroxyapatite, release of complexes and initiation of new bone formation by stimulating osteoblast proliferation around the resorption site. Because of the high affinity of kBD-IGF3β for both IGR-II and hydroxyapatite, the invention provides therapeutic agents and compositions that are useful inter alia to direct IGF-II and / or IGF-I specifically to bone.

The novel hBD-IGFBP, hBD-IGFBP / IGF-II and other conjugates, anti-hBD-IGFBP antibodies and antagonists thereof, and pharmaceutical compositions prepared therefrom are particularly useful for administration in the treatment of a wide range of hSD-IGFBP-related diseases. and IGF-II.

Advantageously, the pharmaceutical compositions may be administered parenterally, i.e., subcutaneously, intramuscularly or intravenously, or topically, orally, via aerosol, by intranasal delivery; and the like, provided that the invention provides compositions for parenteral administration which include solutions of hBD-IGFBP, hBD-IGFBP / ICF-II and other conjugates, anti-hBD-IGFBP antibodies and antagonists thereof, or a mixture of hBD-IGFBP and IGF-II dissolved in an acceptable carrier, preferably an aqueous carrier. Many aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like. These compositions can be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable excipients which are required for approximate physiological conditions such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. concentration. of the desired hBD-IGFBP, hBD-IGFBP / IGF-II, or antibodies to hBD-IGFBP or other antagonists thereof in these compositions can vary widely, i.e., from less than about 0.00001%, usually at least about 0.001% up to about 0.05 to 0.1% by weight and will be chosen primarily according to the volume of the liquid, viscosities, etc., according to the particular mode of administration, administration, condition being treated, e.g. fracture repair, osteoporosis, treatment of surgical or traumatic wounds, tumors such as osteosarcoma or plion carcinoma etc. and the subject being treated, i. adult, child or newborn.

A typical pharmaceutical composition for intravenous infusion for treating adults suffering from mild osteodegenerative disease may be made to contain 250 ml of sterile Ringer's solution and about 50 mg to 5 grams of hBD-IGFBP or hBDrIGFBP / IGF-II. Current methods for preparing parenterally or orally administrable compounds will be known or apparent to those skilled in the art and are

See, e.g., Remington's Pharmaceutical Science, 16th Ed., Mack Publishing Company, Raston, PA (1982), which is incorporated herein by reference.

Compositions comprising hBD-IGFBP or hBD-IGFBP / IGF-II, or cocktails thereof, can be administered in a prophylactic and / or therapeutic treatment. In therapeutic applications, the compositions are administered to a patient already suffering from a hBD-IGFBP or IGF-II-derived disease in an amount sufficient to cure or at least partially alleviate the disease and its complications. An adequate amount to achieve this is defined as a therapeutically effective dose. The amount effective for this use will depend on the disease, i.e., bone degeneration such as osteroporosis, fracture, injury, tumor, etc., and the severity, age of the patient and general condition of the patient's health. Generally, the amount will range from 1.0 to 500 µg of hBD-IGFBP or hBD-IGFBP / IGF-II per kilogram of body weight and hour of infusion, at a dose of from 10 to 50 µg hBD-IGFBP or hBD-IGFBP / IGF- II per kilogram and hour of infusion, which is generally more used. Thus, the materials of the invention can be used in serious disease states, in terms of minimizing foreign substances and the absence of responses to foreign substances, and medical treatment by administering considerably higher doses of these pharmaceutical agents is possible and desirable.

In prophylactic applications, compositions comprising the present hBD-IGFBP or hBD-IGFBP / IGF-II or cocktails thereof are administered to patients not yet in a disease state to enhance the patient's disease resistance. Such an amount is defined as a prophylactically effective dose. In this use, the exact amount is again dependent on the patient's condition, etc., but generally ranges from 1 to 500 µg per kilogram and hour of infusion,

Preferably 10 to 50 µg per kilogram per hour. A preferred prophylactic use is for the treatment of patients at risk for severe osteodegenerative diseases.

Single or multiple administration can? be performed with the dose level and the dosage pattern selected by the attending physician. In any case, the pharmaceutical composition should provide an amount of hBD-IGFBP or hBD-IGFBP / IGF-II sufficient, for example, to treat a patient.

In yet another aspect, the invention provides an agent that effectively stimulates bone cell proliferation in response to IGF-II. Exogenous administration of IGF-II to bone cells under serum-free conditions increases their proliferation. This proliferative effect of IG1 * -II is potentiated by the addition of hBD-IGFBP in conjugation with IGF-II. This synergistic effect of the combination? hBD-IGFBP and IGF-II, which is greater than the additive effect achieved by combining the results obtained with each agent separately, have not been described for other IGF3β in any cell type. Thus, the invention provides therapeutic agents for treating bone diseases (eg, osteoporosis) where bone formation is impaired. The pharmaceutical compositions will comprise hBD-IGFSP and, if desired, IGF, with physiologically acceptable carriers and / or additives.

The present invention also provides agents that can generally be used in wound healing and skin repair to increase the strength and half-life of IGF, since hSD-IgGFBP can increase the half-life of IGF by protecting them from proteases, playing a role in targeting IGF specifically to bone and / or potentiate the proliferative action of IGF.

HBD-IGFBP + IGF complexes can be prepared in many ways, e.g. by incubating the purified concentrates

-17hBD-IGFBP and IGF at neutral pH overnight prior to administration. The concentration of hBD-IGFBP and IGF in the compositions can be very broad, but preferably these coneentrations are approximately equinolar. Other formulations are known to those skilled in the art in the context of the present disclosure.

When formulated separately, the hBDIGFBP composition may be administered separately or concurrently with the IGF-II composition. If administered separately, it will typically be administered first as hBD-IGFBP and then IGF-II. Such compositions may be administered in specific areas to stimulate local bone formation (e.g., fracture repair) or administered systemically, as in the treatment of bone diseases such as osteoporosis, to enhance general bone formation.

In another embodiment, the present invention provides the preparation of multiple potent IGF molecules. Structural-functional analysis of IGF and hBD-IGFBP can identify the region (s) of IGF that are utilized in binding to hBD-IGFBP. It is possible to produce IGF molecules modified with amino acid substitutions, insertions, or deletions such that the modified molecules bind to IGF receptors and hBDIGFBP with higher affinity, but not to IGFBP inhibitors such as IGFBP-4 and IGFBP-3. Such modified IGF molecules can serve as potent anabolic agents in triggering general wound healing and skin repair. In another embodiment, hBD-IGFBP fragments are produced. fragments will typically have the desired function, such as the ability to bind IGF-II or hydroxyapatite, while eliminating other portions of the molecules that are not essential for this function. The fragments may be used singly or together.

In addition to IGF, the hBD-IGFBPs of the invention are useful in targeting other desirable molecules to the bone. Molecules can affect bone formation or resorption directly or indirectly, as the lacquer will be apparent to those skilled in the art, a wide range of agents can be targeted to bone tissue in this manner. Representative Examples Include agents that stimulate bone formation, such as bone morphogenic protein (BMP), TGF7), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), agents that reduce bone formation, as may be desirable in certain tumors such as glucocorticoid or 1,25-dihydroxyvitamin Dj, compounds that increase bone resorption such as macrophage colony stimulating factor JM-CSF) and interleukins /, and compounds that reduce bone resorption such as bisphosphonate and calcitonin which are given as examples. The compounds may be coupled to hBD-IGFBP by a variety of methods including conjugation as described above, as well as fusion and chimeric proteins, as appropriate. A typical dose of the aforementioned targeting compounds, such as growth factors, will be released on the surface of the bone tissue at a concentration of about 10 pg / ml to about 50 ng / ml bone tissue.

In another aspect, the invention provides a diagnostic marker for assessing bone formation in clinical samples taken from patients having metabolic bone diseases or bone neoplasia. Considering that hBD-IGFBP is an important modulator of IGF-II activity and that IGF-II is an important human growth factor, hBD-IGFBP levels can be used to monitor bone metabolic diseases where aberrant hBD-IGFBP levels represent the presence of the disease. Thus, the invention also provides reagents / containing a marker for the clinical diagnosis of bone formation for monitoring

-19 Bone formation during treatment of bone diseases with therapeutic agents. The hBD-IGFBP or antibody composition can be used to detect and quantify hβD-IGFBP in biological fluids such as human plasma, serum or urine.

As will be appreciated by those skilled in the art, a variety of types of immunoassays are suitable for use in the present invention. For example, direct and indirect binding assays, competitive assays, sandwich assays, and the like, which are generally described in, eg, US Patent Nos. 4642285, 4376110, 4016043, 3879262, 3852157, 3850752, 3839153, 3791932, and Harlow and Lane, Aitfcbodies , A Laboratory Manual, Cold Spring Harbor Publications, NY (1988), all of which are incorporated herein by reference. In one assay, the hBD-IGFBP format is quantified directly by measuring the binding of antibodies to hBD-IGFBP, and these antibodies are then detected with, e.g., labeled anti-IgG, IgM and / or IgA human antibodies. In another embodiment, a patient's hBD-IGFBP can be measured by competition for binding to labeled or unlabeled h3D-IGFBP. A large number of radiolabeled substances can be used, examples of which are radionuclides, particles (eg gold, ferritin, magnetic particles, red blood cells), flourated substances, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (especially haptens), chemiluniscence substances etc., but radionuclides are preferably used.

Also, hBD-IGFBP or antibodies for use in such assays may be coupled to an insoluble or solid support such as an ELISA microtiter well, microspheres, filter membrane, insoluble or precipitated polymer, etc. to function as affinity resins. Aitiser or monoclonal antibodies to hBD-IGFBP are generally of inhuman origin such as antisera from rabbit, goat, mouse, etc. Also, kits for use for detecting hBD-IGFBP can be provided, wherein hBD-IGFBP and / or antibodies can be provided, usually in in lyophilized form, in a container, cell alone or in conjunction with other agents, labeled and / or anti-antibodies, and the like. The hBD-IGFBP polypeptide and antibodies, which may be conjugated to or unconjugated to a labeled substance, are contained in buffer kits such as TRIS, phosphate, carbonate buffer, etc. '. stabilizers, biocides, inert proteins, e.g., dusk, albumin, or the like. It is often desirable to include an inert excipient or extender for diluting the active ingredients, wherein the additive may be present in an amount of from about 1 to about 99% of the total composition.

Antibodies for diagnostic or therapeutic use can be produced in a variety of ways. The preparation of non-human monoclonal antibodies, eg, mice, is well known and can be performed, for example, by immunizing an animal with a recombinant or synthetic hBD-IGFBP molecule or a selected portion thereof (eg, a peptide). For example, a selected screening can identify a region of the hBD-IGFBP molecule that is predominantly responsible for IGF recognition, if desired. Antibodies producing cells obtained from immunized animals are immortalized and screened, or first screened for the production of antibodies that bind hBD-IGFBP and then immortalized.

The following examples serve to illustrate the invention and are not intended to limit it in any way.

21 Examples of embodiments of the invention

Example 1

Preparation of human bone extract for purification of hBD-IGFBP

The human femoral heads obtained during total hip replacement surgery were stored frozen at -20 ^° C until use. The bones were cut with a band saw and ground in a Wiley mill to extract hBD-IGFBP. Bone proteins were extracted by demineralizing the femoral head powder with 10% ethylenediamine tetraacetate (EDTA) in the presence of 4M guanidine HCL and protease inhibitors (Guanedin EDTA extract) after initial extraction with water and 4M guanidine HCL as described by Mohan et al. (Biochem. Biophys.Acta, 884: 234242 (1986)). Guanidine EDTA extract was then concentrated in Anicon using ®5 (5 kilodalton molecular weight throughput) and used for purification of hBD-IGFBP.

Example 2

Purification and characterization of hBD-IGFBP from human bone extract

Human bone extract was subjected to hydroxyapatite (HA) chromatography in 4M guanidine HCl as described by Mohan et al., Ibid. The IGF-II affinity column is prepared by binding 250 µg of IGF-II purified from human bones to cyanogen-activated S _e pharose 4B beads. The pooled HA binding fraction was concentrated in an Anicon cell using a YM5 membrane to about 300 ml and dialyzed against a 20-fold volume of potassium phosphate buffer (0 mM potassium phosphate, pH 6.0) containing inhibitors

22proeases (100 mM epsilon caproic acid, 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride). The IGF-II affinity column is equilibrated with potassium phosphate buffer and then a 50 ml aliquot (approximately 3-4 mg total protein / ml) of the dialyzed HA bound fraction of the combined bone extract is loaded onto the column. The column is then washed extensively with potassium phosphate buffer for complete removal of unbound proteins. Bound proteins are eluted with 20 to 25 ml of 30 mM tris-acetate and pH 7.2 / 4 M guanidine-HCl. The affinity bound fraction is concentrated in an Anioon cell using a YM 5 membrane and then dialyzed against 30 mM Tris-HCl (pH 8.0) buffer. The dialyzed affinity bound fraction was applied to a Pharmacia FPLC Mono Q anion exchange column pre-equilibrated with tris-HCl buffer. Bound proteins are eluted with a linear gradient of 0-1 M NaCl in 20 mM Tris-HCl buffer over 100 min.

hBD-IGFBP activity was determined by the polyethylene glycol precipitation method. Briefly, 50 _{of the} ul sample that is? tested are incubated with 2500 to 50000 cpm Ί- labeled IGF-I or IGF-II for 60 minutes at room temperature in 250 / ul 0 _t M HEPES / 0.1% bovine serum albumin / 0.1% Triton X100 / 44 mm Na ₂ CO ₃ / 0.02% NaN 4, pH 6. To this mixture was added 100 µL of 2% immune serum globulin and 500 microliters of 25% polyethylene glycol and then centrifuged.

Under these conditions, polyethylene glycol precipitates a large complex between IGF-I or IGF-II and hBD-IGFBP, but does not precipitate unbound IGF-I or IGF-II. The amount of ¹²⁵ I-IGF-II in the PEG precipitate is then calculated. The non-specific binding is then determined by performing the assay in the presence of an excess of unlabeled IGF-I or IGF-II and an amount of ¹² µL-IGF-I or

The IGF-II that precipitated is subtracted from the above value.

-23To determine the apparent molecular weight of hBD-IGFBP,

25 by ligand blot analysis using ⁷ I-IGF-II as a tracer. In this procedure, 50 µl of the sample was electrophoresed under non-reducing conditions in 3-27% SDS polyacrylamide gel plates. After transfer of the samples to nitrocellulose by electroblotting, the nitrocellulose membranes are incubated with radiolabeled IGF-II. After washing unbound radiolabeled IGF-II, the membrane is subjected to autoradiography as described by Hossenloop et al., (Anal.Bioohem., 154: 138-143 (1986)).

The amino acid composition of the sample was determined by an Applied Biosysterna model 420 analyzer and the N-terminal sequences were determined using an Applied Biosysterna model 470A vapor phase protein sequencer (Mohan et al., Biochim.Biophys.Acta, 966: 44-55 (1988)).

Giant. 3 represents the protein profile and IGFBP activity profile of the affinity bound phase in the Mono Q chromatography step. Neither protein absorbance peak nor IGFBP activity peaks were present in the region where authentic IGF3P-4 eluted (fraction 9-15, 0.1M NaCl), this confirms that IGFBP-derived bone is not IGF3P-4. However, four protein absorbance peaks were found, eluting at different NaCl concentrations. Of the four protein peaks, the first two peaks (A and B) contain significant IGFBP activity, while the last two peaks (C and D) have little IGFBP activity.

Ligandblotting and affinity radiolabelling were used to test the apparent molecular weight of IGFBP present in human bone extract.

Figure 4 shows ligand-blots in which HA binding, IGF-II binding, and Mono Q protein peaks were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

-24 transferred to nitrocellulose and examined with [ ¹²⁵ I] IGF-II marker. The major IGFSP present in the HA bound and IGF-II bound fractions of my apparent molecular weight of 29 kDa. Furthermore, these fractions also show a broad, low intensity band between 68 and 43 kDa molecular weight markers. The major 29 kDa IGFBP is separated from the higher molecular weight IGFBP by Mono Q chromatography. Mono Q peak A shows a major band at 29 kDa and a minor band at 24 kDa. Mono Q peak B shows a broad diffuser band; between 68 and 43 kDa markers and a small band at 29 kDa. Mono Q peak C (represents the peak protein absorbance peak) and D shows only a weak band at 29 kDa. These data confirm that the major IGFBP in human bone extract is 29 kDa IGFBP.

Both the amino acid composition and the 29 kDa N-terminal IGFBP amino acid sequence in Mono Q peak A appear to be unique, having limited similarity to other known. IGFBP (Table 1, 2).

-25Table 1

The amino acid composition of hBD-IGFBP and known IGFBP

peak A IGFBP-1 IGFBP-2 IGFBP-3 IGFBP-4 asx β, ι 6.8 6.6 6.8 8.0 glx 5.9 13.2 13.9 10.2 12.2 ser 9.3 9.0 3.5 10.2 5.9 gly 9.0 7.3 11.8 θ, 7 9.7 his < 5.1 2.6 3.8 2.7 4.6 arg 6.3 4.3 6.9 7.2 8.0 thr 6.3 3.8 3.8 3.4 2.5 ala 7.9 11.1 7.3 6.8 6.8 for 5.8 7.7 9.3 8.7 8.9 tyr 3, * 2.6 1.7 3.4 1.3 wall 6.6 3.8 5.5 5.3 3.8 met 3.5 1.3 3.1 0.8 1.7 cys 5.5 7.7 6.6 6.8 8.4 ile 3.3 3.8 1.4 2.3 2.5 leu 6.8 7.3 9.0 7.2 8.0 phe 2.9 1.7 1.0 1.9 2.1 lyw 4.0 3, no 4,5 7.2 5.1 trp 2.1 0.3 0.4 0.4

100 mm diameter sections of the immobilone membrane were wetted with methanol, placed in a filter unit and held in place with rubber O-rings. After washing the immoblon membrane with water, BP in peak A is fixed to the membrane by filtration through 1 ml of peak A. Half of the membrane is used for amino acid composition studies.

-26Tahulka 2

The amino terminal sequence of RBD-IGFBP in Mono Q peak A

Rest amino acid (prnol)

1) L = 59.4 2) G = 50.1 3) F - 34.8 4) F = 48.0 5) V = 49.1 6) X 7) V = 27.1 8) E = 20.6 9) P = 22.2 10) D = 15.2 11) D = 18.7 12) K = 13.4 13) A = 22.8 14) A = 32.8 15) L = 28.5

50 prnol of hBD-IGFBP was used to analyze the N-terminal amino acid sequence. The average of repeated results was 86.3% · X = not known.

Thus, the 29 kDa IGFBP was designated as human bone derived IGBBP (hBD-IGFBP). In addition to the main sequence (Leu-Gly-Phe-Phe-Val-X-Val-Glu-Pro-Asp-Asp-Hys-Ala-AlaLea) there was an evident presence of another sequence in the Mono Q peak that lacks one or two amino acids on the N-terminus. Purified hBD-IGFBP appears to be susceptible to proteolytic cleavage since storage of purified 29 kDa IGFBP at 5 ° C results in the disappearance of the 29 kDa band overnight and the appearance of a major band at 24 kDa and low molecular weight bands, several. In the N-terminal sequence analysis of the 24 kDa band, the sequence ^ (Leu-Gly-Phe-X-Val-X-X-Glu-Pro-X-X-Lys) was obtained.

-27 probable sequence of 29 kDa IGFBP. Prolonged storage of 24 kDa IGFBP results in its disappearance and the appearance of several low molecular weight bands appearing to contain very little IGFBP activity as determined by ligand-blot analysis, consistent with these results are previously reported IGFBP-related protease data .

Mono Q peak B appears to have different amino acid composition and does not potentiate? action of IGF-II in bone cells. Attempts to sequence peak B were unsuccessful. Sequence determination of the first few cycles of the major protein peak (fraction 45, peak C) yielded many amino acids with an unreadable sequence. Therefore, Mono Q peaks C and D which contain very little IGFBP activity may represent degradation products of 29 kDa IGFBP.

Because the N-terminal sequence of the Mono Q purified hBD-IGFBP peak provides additional sequences in addition to the main sequence that lack one or two amino acids (probably due to the co-purification of the IGFBP protease that degrades this IGFBP) and because the cysteine residue has not been derivatized at position 7 of the present hBD.IGFBP sequence and aspartic acid at position 10 by cysteines (cysteine residues were protected in many different members of the IGFBP family). Thinning of Mono Q purified from human bone derived IGFBP at 5 ° C leads to the disappearance of 29 kDa IGFBP and the appearance of a lower molecular weight IGFBP by SDS-PAGE. When low molecular weight IGFBPs were sequenced, valine and aspartic acid were not found at positions 7 and 10 with no signals at these positions. These findings confirm that these hBD-IGFBPs have the sequence in one embodiment of L-G-F-F-V-X-C-E-P-G-D-K-A-A-L. An alternative sequence for hBD-IGFBP, which also falls within the above findings, is: L-G-S-F-V-H-C-E-P-C-D-E-K-A-L, and this sequence is similar to the BP-5 sequence of Kiefer et al.

-28Biophys.Res.Comm. 176: 219-225 (1991); Shimasaki et al., J. Biol. 266: 10646-10653 (1991) and as described by Drop, Sndocrinol. 130: 1736-1377 (1992). The sequence may be subjected to various variations based on native or introduced substitution (s), additions or deletions, which may be allelic variations or may be produced by the partial sequence techniques included herein. It should be noted that using the methods described herein, the protein can be isolated and purified, and the sequence determined by various known methods. Further, the N-terminal sequence allows the construction of degenerate oligonucleotide probes to clone a gene that encodes the h3D-IGFBP of the invention.

Example 3

Use of hBD-IGFBP in a Bone Cell Proliferation Study

The serum-free culture medium IGF-mediated bone cell proliferation study described by Mohan et al. , (Biochem. Biophys, Aeta, 884: 234-242 (1986)), cited by reference, measure the incorporation of (1 H) thymidine into trichloroacetic acid precipitated material. This assay was performed using the MC3T3-E1 osteoblast cell line. Approximately 10,000 cells were plated per well in serum-free Dulbecco-modified Eagle's medium in 48-well culture dishes and used for the 1 H-thymidine study as generally described by Mohan et al., Ibid.

In this assay, hBD-IGFBP alone has little mitogenic activity as determined by incorporation of 1H-thymidine into trifluoroacetic acid insoluble macromolecules

-29 (Table 3, below). However, when hBD-IGFBP is added, along with submaximal concentrations of IGF-II, to serum-free mouse bone cell cultures, hBD-IGFBP potentiates the proliferative action of IGF-II. IGFBP-3 showed an increase in IGF-I action only when added to the cultures several hours prior to the addition of IGF-I (Mohan et al., Proc.

Nati. Ac a -. Sci. US A, 86: 8338-8342 (1989) and De Mellow et al., Biochem.Biophys.Res. Comm., 156: 199-204 (1988)) and no other IGFBPs are expected to show potentiation of IGF activity under conditions where IGF and IGFBP are added simultaneously. These data confirm that: hBD-IGFBP is not a more passive carrier for IGF but also positively regulates IGF action. Although the mechanism (s) by which hBD-IGFBP potentiates IGF-II stimulated (1 H) thymidine incorporation is not known, any mechanisms of possible explanation are not limiting. N © example hBDIGFBP directs? IGF-II into the cell membrane for easier access of IGF-II to its receptors (especially via the RGD sequence as in IGFBP-1). Or, hBD-IGFBP can increase the affinity of IGF-II to its receptors by binding to IGF-II and / or increasing the viability of IGF-II by protecting it from proteases.

-30Table 3

Potentiating Effect of h3D-IGFBP on IGF-II Induced Cell Proliferation / Thymidine Incorporation (% Control) Experiment 1 Experiment; 2 trial 3 treatments

B3D control h3D-IGF3P

IGF-II + IGFBP-H3D IGF-II ^X

100+ 15

128+ 18

138 + 18

195 + 32

100 +22

213 + 29

265 + 43

672 + 74

100 + 12

116 + 9

214 +24

320 + 40

Single serum cultures of the mouse osteoblast cell line MC3T3-E1 were incubated for 18 hours with effectors before addition. (1 H) -thymidine. Final concentrations of IGF-II and h3D-IGFBP were 3 and 10 ng / ml, respectively. Values are average + std. 6 wells. The (1 H) thymidine-incorporation in bovine serum albumin (BSA) of the treated control cultures was 1404+ 217, 237 + 53 θ 730 + 91 in three experiments.

^The interaction between hBD-IGFBP and IGF-II was highly significant (P 0.00001) by three methods of analysis between hBD-IGFBP and IGF-II experiments using the CSS computer program.

-31Example 4

Purification of hBD-IGFBP from Conditioned Bone Cell Medium

Since bone cells produce h3D-IGF3β, serum-free conditioned medium separated from the bone cell cultures can also be used to purify hBDIGFBP. The conditioned bone cell medium is concentrated in Anicon using YM5 (5 kilodalton molecular weight separator), acidified with acetic acid to a final concentration of 1M and subjected to gel filtration on Sephadex G-100 to separate IGF from ICFBP. Proteins were eluted with 1M acetic acid. Fractions containing IGFBP were pooled, lyophilized, reconstituted with phosphate buffered saline; and processed on an IGF-II affinity column. Affinity-bound proteins are then subjected to FPLC Mono Q anion exchange chromatography to separate hBD-IGFBP from other IGFBPs.

Example 5

Quantitative diagnostic study for hBD-IGFBP hBD-IGFBP from human bone or expressed by recombinant methods is used to produce polyclonal and / or monoclonal antibodies, and these antibodies are then used in the hBD-IGFBP quantitative assay. hBD-IGFBP is mixed with complete Freud's adjuvant. and injects rabbits, guinea pigs, rats or mice according to standard protocols for making antibodies. The animals are then injected with hBD-IGFBP mixed with incomplete Freud's adjuvant every 3-4 weeks. For polyclonal antiserum, animals are bled after 3-4 injections and titer for

The antibodies to hBD-IGFBP are determined by radioimmunoassay or similar methods. Monoclonal antibodies are produced by immortalizing antibodies by producing cells obtained from immunized animals using well known techniques. Purified hBD-IGFBP is radiolabeled and used as a signal producing marker. The higher titer monoclonal antibody or antiserum is then used to develop the hBD-IGFBP radioassay to measure hBD-IGFBP levels in serum, urine or other biological fluids.

In general, hBD-IGFBP production is increased by treating bone cells with agents that increase bone cell proliferation. Thus, hBD-IGFBP can be used as a diagnostic marker for disease styles associated with bone drum proliferation, such as osteoporosis. Accordingly, low serum hBD-IGFBP indicates osteoporosis associated with low bone formation. Since hBD-IGFBP potentiates IGF-II action, high serum hBD-IGFBP may also be associated with some cancers.

Example 6

Quantitative IGF diagnostic assay using · hBD-IGFBP

As described in this example, recombinant or purified hBD-IGFBP can also be used to quantify IGF levels in a biological sample such as serum. 2-5 ng of purified hBD-IGFBP is incubated with 40,000 cpm of ¹² µl-IGF in the presence or absence of unlabeled competitor. IGF standards or samples containing unknown amounts of IGF 'are used as competitors.

-33After 60 minutes incubation? at room temperature, the hBD-IGFBP · IGF complex is precipitated by the addition of polyethylene glycol in the presence of bovine gamaglobulin. After centrifugation for 30 minutes at 1185 x g, the supernatant fraction is counted in a gamma counter. The standard curve is performed at different concentrations of unlabeled IGF and the amount of IGF in the unknown sample is calculated using the standard curve. Thus, the amount of biologically active free IGF was determined by this assay using purified hBD-IGFBP, which has a high affinity for IGF.

Example 7 hBD-IGFBP Bone Fixation of IGF-II

Human bone contains a relatively large amount of IGF-II. However, as shown in Table 4, labeled IGF-II alone does not provide specific binding to hydroxyapatite (the table shows only non-specific binding that is less than 10% of the total added) or to collagen, and unlike IGF-II, labeled hBD- IGFBP shows specific binding to hydroxyapatite. The binding of hSD-IGFBP to hydroxyapatite was specific because the major serum binding protein, ie, IGF3P-3, had no similar activity and because hBD-IGFBP did not bind to collagen, another major component of bone. Furthermore, the binding of hBD-IGFBP to the hydroxyapatite was strong, so that the h3D-IGFP3-hydroxyapatite complex could not be dissociated with 4M guanidine HCl (4M guanidine HCl showed a dissociation interaction between the antibody-antigen complex). Preincubation of labeled IGF-II and hBD-IGFBP prior to addition to the hydroxyapatite column significantly increases IGF-II binding to the hydroxyapatite column. ^{x a} hBD-IGFBP activity facilitating IGF-II binding to hydroxyapatite was specific in that IGFBP-3 did not

Any such activity is consistent with the conclusion that, under normal conditions, IGF-II is fixed in bone by hBD-IGFBP.

Table 4 hBD-IGFBP facilitates IGF-II binding to hydroxyapatite

Lignin% of the marker bound to the collagen type I hydroxyapatite

¹²⁵ iig-ii <10 <10 ¹²⁵ r / hSD-IGF3 [beta] 60 <Ίθ ¹²⁵ I / IGF-H + hBD-IGFBP 45 <10 ^t25 I / IGFBP-3 <10 <10 ^{, 125} I / IGF-II + IGFBP-3 <10 <10

Example 8

Regulation of IGFBP-5 production in human bone cells in vitro

To determine whether human bone cells produce hBD-IGFBP in vitro, Northern blots of all RNA extracted from serum-free cultures of MG63, TE85, TE89, aaOs2 and U2 human osteosarcoma cells were hybridized using an oligonucleotide probe to the N-terminal sequence of hBD- IGFBP and using a human IGF3P-5 cDNA probe (Dr. Shimasaki, La Jolla, CA). These studies confirm that all cell lines that were tested express hBD-IGFBP mRNA. According to Western-ligand-blot analysis, IGF-I and IGF-II increase hBD-IGFBP production several times in U2 cells. The biological characterization of h3D-IgFBP confirms that this protein potentiates the proliferative action of IGF-II in bone cells.

Proliferation of human osteoblasts and progesterone-stimulated IGF-II production in vitro was monitored.

In this experiment, the effect of progesterone on other components in the IGF regulator system (IGF-I, IGFBP and IGF receptors) in human osteoblasts similar to the osteosarcoma cell line MG63 was studied. In all experiments, MG63 cells were plated at a density of 7500 cells / cm in DMEM containing 1% calf serum. After overnight incubation, the medium was changed to serum free before addition of progesterone or vehicle (ethanol). At the time of the study, cells were incubated with 100 mM progesterone for 0.5 hours, hours, 4 hours and 6 hours. Northern-blot analyzes demonstrate an increase in mRNA levels for IGF-II, IGF-I, hBD-IGFBP and t. γp-1 and type-2 IGF receptor after 30 minutes to 6 hours compared to any controls. IGF3P-4 inhibitor mRNA levels, however, were decreased within 30 minutes after the addition of progesterone. This was not attributed to changes in IGFBP-3 mRNA levels. Progesterone, a steroid hormone that has been found to stimulate human bone cell proliferation thus increases? IGFBP-5 production in human bone cells. The stimulatory effect of progesterone on bone cell proliferation should be due not only to increased IGF production but also to increased IGF receptor expression, increased hBD-IGFBP (potentiates IGF action), and decreased IGF3P-4 inhibitory production.

Claims (19)

PATENT N O'R OKY _? and rz Purified and isolated insulin-like growth factor binding protein, hBD-IGFBP. 2. Purified hBD-IGFBP of molecular weight ca. 29 kDal, which is obtained from human bone or conditioned bone cell medium, and which binds IGF-II and hydroxyapatite. The purified hBD-IGFBP of claim 2, which potentiates the ability of IGF-II to stimulate bone cell proliferation. The hBD-IGFBP of claim 2, which has a higher specific binding affinity for IGF-II than IGF-I. The hBD-IGFBP of claim 2, which binds: hydroxyv -9 apatite with a binding affinity of at least 10 M. The purified hBD-IGFBP of claim 2, which is conjugated to a compound that affects bone formation. A pharmaceutical composition comprising a substantially pure hBD-IGFBP protein of about 29 kDal or a fragment thereof and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 7, further comprising IGF-I or IGF-II. The pharmaceutical composition of claim 8, wherein the IGF-I or IGF-II is conjugated to hBD-IGFBP. The pharmaceutical composition of claim 7, which is formulated for topical administration to a patient. 11. The pharmaceutical composition according to claim 7, which is ^_ formulated for parenteral administration to a patient. Use of substantially pure h3D-IGFBP for the manufacture of a pharmaceutical composition for modulating the effects of IGF in a patient. Use of hBD-IGFBP conjugated to IGF-II for the manufacture of a medicament for modulating the effects of f according to claim 12. 14th ^r sing IGF-II for the manufacture of a pharmaceutical preparation according to claim 13. Use according to claim 12, for the manufacture of? a pharmaceutical composition for treating degenerative bone disease. The use of claim 15, wherein the bone disease is osteoporosis. 17 · Use essentially pure; hBD-IGFBP for the preparation of a pharmaceutical preparation? to facilitate wound healing and fracture repair. -37a 18. Use of substantially pure h3D-IGFP or a fragment thereof conjugated to a compound for the preparation of a composition to facilitate delivery of the compound to bone tissue * The use of claim 18, wherein the compound is a compound affecting bone formation or resorption.