WO2008079400A2 - Applications chirurgicales pour une protéine de liaison à bmp - Google Patents

Applications chirurgicales pour une protéine de liaison à bmp Download PDF

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Publication number
WO2008079400A2
WO2008079400A2 PCT/US2007/026315 US2007026315W WO2008079400A2 WO 2008079400 A2 WO2008079400 A2 WO 2008079400A2 US 2007026315 W US2007026315 W US 2007026315W WO 2008079400 A2 WO2008079400 A2 WO 2008079400A2
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WIPO (PCT)
Prior art keywords
bmp
bone
growth factor
bbp
seq
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PCT/US2007/026315
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English (en)
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WO2008079400A3 (fr
Inventor
Samuel S. Murray
Elsa J. Murray
Keyvan Behnam
Jeffrey C. Wang
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The Regents Of The University Of California
Department Of Veterans Affairs
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Priority claimed from US11/985,745 external-priority patent/US8193312B2/en
Application filed by The Regents Of The University Of California, Department Of Veterans Affairs filed Critical The Regents Of The University Of California
Priority to EP07868028A priority Critical patent/EP2115465A4/fr
Priority to JP2009542969A priority patent/JP5466017B2/ja
Priority to AU2007338627A priority patent/AU2007338627B2/en
Priority to US12/448,497 priority patent/US8415302B2/en
Priority to CA002673600A priority patent/CA2673600A1/fr
Publication of WO2008079400A2 publication Critical patent/WO2008079400A2/fr
Publication of WO2008079400A3 publication Critical patent/WO2008079400A3/fr
Priority to US13/857,711 priority patent/US9050300B2/en
Priority to US14/731,852 priority patent/US9610320B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor

Definitions

  • Growth factors are substances, including peptides, which affect the growth and differentiation of defined cell populations in vivo or in vitro. Normal bone formation occurs during development, bone remodeling occurs in adult life, and bone repair occurs in order to preserve the integrity of the skeleton. Bone formation, remodeling and repair involve bone resorption by osteoclasts and bone formation by osteoblasts. Cell differentiation and the activity of osteoblasts and osteoclasts are regulated by growth factors. Thus, any interference between the balance in cell differentiation and resorption can affect bone homeostasis, bone formation and repair.
  • Osteoblasts are derived from a pool of marrow stromal cells (also known as mesenchymal stem cells). MSC are present in a variety of tissues and are prevalent in bone marrow stroma. MSC are pluripotent and can differentiate into chondrogenic or osteogenic cells including osteoblasts, chondrocytes, fibroblasts, myocytes, and adipocytes.
  • BMP/NCP bone morphogenic protein/non-collagenous protein
  • BMP/NCP was never purified to homogeneity, but other investigators have used similar starting materials to clone a number of recombinant "BMPs.” However several of these molecules have little or no osteogenic activity. "BMPs” and other osteogenic factors have been studied for use in clinical applications. However, the cost of using minimally effective dosages of BMP-7 (also known as OP-1), for example has been a limiting factor in clinical use. Therefore, effective and affordable compositions and methods are desired for clinical applications relating to bone.
  • Adjuvant therapeutics to enhance bone healing are important in many aspects of orthopedics, but are especially important for spinal fusion where prompt and thorough osteogenesis is critical.
  • the most useful agents currently available include bone growth factors, such as bone morphogenetic proteins (BMPs). These proteins induce the recapitulation of endochondral bone formation among undifferentiated mesodermal cells.
  • BMPs bone morphogenetic proteins
  • these proteins induce the recapitulation of endochondral bone formation among undifferentiated mesodermal cells.
  • their usefulness is limited by expense and by local adverse effects such as unwanted ectopic bone formation and inflammatory responses associated with doses currently used for spinal fusion procedures.
  • BMP-2 bone generator for spinal fusion
  • Recent studies have shown that it may be used effectively for both anterior and posterior fusion procedures through the whole spinal column.
  • its high expense as well as the reported local side effects such as unwanted bone formation and dangerous swelling at the neck after the anterior cervical fusion procedure has prevented its extensive use for spinal procedures.
  • Focus has now shifted to increase the effectiveness of BMP-2 while decreasing its dose and controlling the side effects.
  • the main limitation is the need for better delivery systems that provide a sustained, biologically appropriate concentration of BMP-2 at the side of fusion bed. Delivery needs to be sustained, because BMPs have exceedingly short biological half-lives, usually the order of minutes or hours, rather than the days or weeks needed to stimulate a complete osteogenic response.
  • rBMP-2 has been described as having a half-life of only a few hours.
  • the inventions are related to a cyclic peptide designated BMP Binding Peptide (BBP) that avidly binds growth factors, such as rhBMP-2.
  • BBP BMP Binding Peptide
  • BBP increases the rate and degree to which rhBMP-2 induces bone formation.
  • BBP may accomplish this by increasing the residency rates of other bone growth factors.
  • BBP alone induces calcification of chondrogenic, osteogenic and osteoblastic cells.
  • Compositions and substrates including BBP, antibodies to BBP and methods of using BBP are useful in applications relating to bone.
  • the invention may include a method of treatment with agents for maintaining bone homeostasis, enhancing bone formation and/or enhancing bone repair.
  • the invention includes methods and devices for increasing the residency times of bone inducing substances, such as BMP. In one embodiment, the invention includes methods and devices for increasing the rate and overall osteogenic activity of bone inducing substances, such as BMP. Further, in one embodiment, the invention includes methods and devices for reducing time required for the effect of osteogenesis and calcification of bone inducing substances.
  • the method may be applied to induce the local repair of bone or to treat bone related disorders, such as osteoporosis.
  • BBP may be used to induce the effects of other bone growth factors on bone fusion in membraneous bone (such as spinal fusion) or in endochondral bone.
  • the invention may include implants having agents or seeded with pluripotential or differentiated cells for inducing bone formation or repair.
  • the invention may also include the application of substances or differentiated cells at a site where bone formation or bone repair is desired.
  • This invention is advantageous at least in that BBP alone or in combination with other growth factors enhances calcification of chondrogenic or osteogenic precursor cells. Further, this invention is advantageous at least in that BBP enhances osteogenesis to occur faster to a greater extent, which may improve the clinical rate and effectiveness of treatment with BMP, and reduce doses and therefore the costs and side effects of BMP treatment alone.
  • FIG. 1A are BBP bovine (1) amino acid and (2) nucleic acid sequences, respectively;
  • FIG. 1 B is a partial amino acid sequence of the bovine BMP binding protein ("BBP") showing the cystatin homology region, the BMP-2 homology region, and the TGF- ⁇ receptor Il homology domain.
  • BBP bovine BMP binding protein
  • FIG. 2 is an amino acid sequence alignment of human BMP-2 and the BMP-2 homology region in bovine SPP-24; (i, identical; c, conservative substitution; sc, semi-conservative substitution).
  • FIG. 3 is an amino acid sequence alignment of bovine fetuin and human TGF- ⁇ receptor Il (above) and of human TGF- ⁇ receptor Il and the TGF- ⁇ receptor Il homology domain of bovine SPP-24 (corresponding to BBP) (bottom); (i, identical; c, conservative substitution; sc, semi-conservative substitution).
  • FIG. 4 is a radiogram of mouse hind quarters 21 days after implantation of 500 ⁇ g of BBP in atelocollagen (top) or atelocollagen alone
  • FIG. 5 is a histological section of mouse muscle 21 days after implantation of 500 ⁇ g of BBP in atelocollagen. (H & E stain. Original magnification 100 X.)
  • FIG. 6 are radiograms of mouse hind quarters 21 days after implantation of 5 ⁇ g of rhBMP-2 (left) or 5 ⁇ g of rhBMP-2 plus 500 mg of BBP
  • FIG. 7 are radiograms of mouse hind quarters 9 (top) and 12 (bottom) days after implantation of 5 ⁇ g of rhBMP-2 (left) or 5 ⁇ g of rhBMP-2 plus 500 mg of BBP (right).
  • FIG. 8 are histological sections of mouse hind quarters 9 days after implantation of 5 ⁇ g of rhBMP-2 alone (A) or 5 ⁇ g of rhBMP-2 plus 500 ⁇ g of BBP (B).
  • FIG. 9 is a surface plasmon resonance sensogram for the interaction of rhBMP-2 (affixed to the chip) and cyclized BBP at concentrations ranging from 1 x 10 "5 M 1 x 10 "4 M.
  • FIG. 10 is a bar graph depicting the percentage of rhBMP-2 retention over 1, 3 and 7 days in the presence or absence of BBP.
  • FIG. 11 includes amino acid sequences against which specific SSP- 24/BBP antibodies have been generated.
  • FIGS. 12 A & B depict flowcharts of exemplary methods of the invention.
  • FIGS. 13 A & B are schematic depictions of two embodiments of the invention.
  • FIG. 14 A is a chart showing the amino acid sequences for BPP in various species (SEQ ID NOS 11, 1 and 12-19, respectively, in order of appearance).
  • FIG. 14 B is a list of the nucleic acid sequences for BPP in various species (SEQ ID NOS 20-28, respectively, in order of appearance).
  • FIG. 15 is an anterior-posterior radiograph of a rat spine fused at L4- L5 with the application of BBP high dose (1000 ⁇ g) + rhBMP-2 low dose (1 ⁇ g) 8 weeks after treatment.
  • FIG. 16 is an anterior-posterior radiograph of a rat spine showing pseudoarthritis at right anno fusion at left L4-L5 with the application of rhBMP- 2 (1 ⁇ g) treatment.
  • FIG. 17 is a histological section of rat spinal region 8 weeks after treatment of a combination of BBP and rhBMP-2. (H & E stain. Original magnification 8.4 X.)
  • FIG. 18 is a histological section of rat spinal region 8 weeks after treatment with low dose rhBMP-2 (1 ⁇ g). (H & E stain. Original magnification 8.4 X.)
  • One embodiment of the invention comprises a peptide having the amino acid sequence of SEQ ID No: 1.
  • the bovine derived amino acid SEQ ID No: 1 has been designated BBP, and SEQ ID No: 2 corresponds to the bovine nucleic acid sequence encoding BBP.
  • One embodiment of the invention comprises a peptide having the amino acid sequence of SEQ ID No. 12, which is the sequence of human BBP.
  • SEQ ID No. 21 corresponds to the human nucleic acid sequence encoding human BBP.
  • BBP is a 19 amino acid, 2.1kD peptide, derived from a 18.5 kD fragment of a known 24 kD secreted phosphoprotein ("SPP-24"). SPP-24 is illustrated by SEQ ID No: 2. Notably, SPP-24 inhibits BMP-2 induced bone formation. BBP contains the cystatin-like domain of SPP-24. BBP is expressed at least in the liver and bone (including at least demineralized cortical bone and periosteum).
  • the BBP amino acid sequence is similar to the TGF- ⁇ /BMP-binding region of fetuin, a member of the cystatin family of protease inhibitors.
  • BBP avidly binds rhBMP-2 (recombinant human BMP-2) with a K 0 of x 10 '5 M.
  • BBP may also bind other molecules having similar binding domains to BMP-2, such as other TGF- ⁇ proteins (including but not limited to BMP-4, BMP-7 (OP- 1), BMP-6, BMP-8 (OP-2), BMP-9 and TGF- ⁇ ) and affect their retention rates and/or activity as well.
  • TGF- ⁇ proteins including but not limited to BMP-4, BMP-7 (OP- 1), BMP-6, BMP-8 (OP-2), BMP-9 and TGF- ⁇
  • BMP-2, BMP-4, BMP-6, BMP-7 and BMP-9 have been demonstrated to bind to the same receptors (with some variation in affinities). Therefore, given the sequence and binding similarities, binding with these additional BMPs is expected.
  • Additional growth factors useful in this invention may include: GDF5, and other BMPs such as, BMP-3, BMP-4, BMP-5, BMP- 6, BMP-7, BMP-8, BMP-9.
  • BBP alone induces calcification of vertebrate chondrogenic and osteogenic precursor cells.
  • BBP increases the increases the rate and degree to which rhBMP-2 induces bone formation.
  • BBP combined with BMP-2 in vivo causes osteogenesis to occur faster and to a greater extent and with smaller amounts of rhBMP-2, than compared with the effect of BMP- 2 alone. This result was unexpected given the role of SSP-2 which inhibits bone formation by BMP-2, as discussed above.
  • the BBP when implanted alone in mouse muscle, the BBP induces dystrophic calcification.
  • the process of bone formation in repair or ectopic bone formation the "mouse hindquarter" or “muscle pouch” model recapitulates endochondral bone formation.
  • the first step involves the production of cartilage, which is replaced by bone. This same process that occurs during endochondral bone formation in development, while some membraneous bone formation occurs directly without a cartilage intermediary.
  • a peptide comprising a fragment of BBP may be useful, if the fragment similarly increases degree or rate of osteogenesis by BMP-2 in mammalian cells, or increases degree or rate of calcification in vertebrate cells, or specifically mammalian chondrogenic or osteogenic progenitor cells.
  • BBP has modifications of the amino acid SEQ. ID No. 1 may also be useful in this invention.
  • the conserved amino acid sequences of BBP between species, deletional or insertional modifications, conservative or semi-conservative substitutional modifications are intended to be encompassed in the claimed BBP, to the extent that the modified amino acid sequences increase the residency time and or activity of BMP-2 or other TGF- ⁇ homologous molecules.
  • BBP is a ⁇ -pleated sheet-turn-sheet pleated sheet molecular motif ("B-T-B"). It is currently believed that growth factor binding amino acids reside in the T-section. Therefore, amino acid substitutions in the T-section may affect activity of BBP to a greater extent than substitutions in the B regions.
  • BPP is believed to increase the effect of growth factors, like BMP-2 by increasing the amount of time ("residency time") that a growth factor remains at the site of implantation, and/or the overall activity of BMP-2 or other TGF- ⁇ homologous molecules.
  • BMP alone for example, is rapidly removed from implantation sites, while implantation of BMP with BBP has been shown to increase residence time.
  • the increase in residency time may be attributed to, for example: 1) BPP's ability to increase the half-life of growth factors (such as by reducing the rate of proteolysis), and/or 2) decrease the diffusion rate of growth factors from an application site.
  • One embodiment of the invention comprises a peptide having the sequence of SEQ ID No. 11 : C-R-S-T-V-X-Y-S-X-X-X-V-X-X-V X-Y-Y-C, which is the mammalian consensus sequence for BBP.
  • Figure 14A shows the homology in amino acid sequence across bovine (SEQ ID No. 1 ; nucleic acid sequence set forth at SEQ ID No. 2), human (SEQ ID No. 12; nucleic acid sequence set forth at SEQ ID No. 21 (position 9 is either A or V), porcine (SEQ ID No. 13; nucleic acid sequence set forth at SEQ ID No. 22), ovine (SEQ ID No.
  • Figure 14A also shows highly conserved regions in chicken (SEQ ID No. 17; nucleic acid sequence set forth at SEQ ID No. 26), salmon (SEQ ID No. 18; nucleic acid sequence set forth at SEQ ID No. 27) and trout (SEQ ID No. 19; nucleic acid sequence set forth at SEQ ID No. 28).
  • X and Y are used to denote amino acid substitutions that are understood to be semi-conservative or conservative, respectively.
  • Conservative substitutions include amino acids selected from the same group, and semi-conservative substitutions include substitutions that are not believed to affect the BMP-2 binding domain or the function of the BBP.
  • the substitution at position 6 is conservative between human, rat and ovine, but semi-conservative with some other species because the amino acids reported at that position in different species are: Q and E (Q in porcine, rat, and mouse BBP, and E in chicken).
  • Q and E Q in porcine, rat, and mouse BBP, and E in chicken.
  • K and R are both classified as basic amino acids, Q is classified as an uncharged polar amino acid, therefore the substitution is not conservative.
  • substitution is semi-conservative, however, because the function of BBP is believed to be unaffected.
  • Semi- conservative substitutions are also found at positions 9, 10, 11 , and 16.
  • the amino acids A is found in bovine, human, porcine and ovine BBP, compared to K in rat and mouse BBP.
  • the amino acid E is reported for bovine, porcine, and ovine BBP
  • human BBP contains Q at that position
  • rat and mouse BBP contain the amino acid G.
  • K is reported for porcine BBP and R for ovine BBP.
  • W is found in bovine, porcine, ovine, rat and mouse BBP, whereas human BBP contains an H.
  • a and V Two hydrophobic amino acids, A and V, are found at position 17. At position 18, two basic amino acids, R and H, are found.
  • One embodiment of the invention may be a composition including BBP which increases degree or rate of calcification in vertebrate cells, or more specifically mammalian chondrogenic or osteogenic precursor cells. Further, the invention may be including BBP which increases degree or rate of osteogenesis by BMP-2, and one of BMP-2 or demineralized bone matrix. Further, the composition may additionally or alternatively include other TGF- ⁇ family members, including but not limited to BMP-4 or BMP-7 separately or mixtures thereof.
  • the invention may include a medicament for use in inducing the rate or degree of osteogenesis in a vertebrate including a therapeutically effective dosage of BBP alone or in combination with a growth factor, such as BMP or DBM.
  • the invention may further include, a medicament for use in inducing the rate or degree of calcification in a vertebrate including a peptide comprising BBP.
  • BBP or the combination of BPP and one or more other growth factors may be placed on or in a substrate or carrier, such as a collagen sponge or orthopedic implant or prosthesis, for implantation at a site where bone formation and/or implant integration is desired.
  • a substrate or carrier such as a collagen sponge or orthopedic implant or prosthesis
  • BBP pharmacological (biological activity) properties.
  • BBP alone or in combination with other TGF-family members such as BMP-2, BMP-4 and BMP-7, or demineralized bone matrix may be used in clinical or research methods for inducing bone formation, maintaining bone homeostasis and/or enhancing bone repair.
  • BBP may be used alone or in combination to treat developmental or homeostatic bone disorders (such as osteoporosis), bone injury (such as fracture healing flat (e.g., membranous) and long (e.g., endochondral) bones, non-union fractures and reconstructive surgery.
  • the invention may also be used in treating periodontitis, periodontal regeneration, alveolar ridge augmentation for tooth implant reconstruction, treatment of non-union fractures, sites of knee/hip/joint repair or replacement surgery.
  • BMP has been demonstrated to be effective in providing spinal fusion, but has limitations to widespread use due to adverse side effects, such as causing inflammation and ectopic bone formation, particularly in the cervical spine.
  • BBP may be used in conjunction with lower doses of BMP to preserve its efficacy, an increase its retention at the application site.
  • BBP has been demonstrated to bind BMP, including recombinant human BMP-2 enabling a two fold retention of rhBMP-2 for up to seven days in vitro and in vivo.
  • Clinical indices of a method or compounds ability to maintain bone homeostasis is evidenced by improvements in bone density at different sites through out the body as assessed, at least by DEXA scanning.
  • Enhanced bone formation in a healing fracture is routinely assessed by regular X-ray of the fracture site at selected time intervals. More advanced techniques for determining the above indices, such as quantitative CT scanning or quantitative histological methods (e.g., tissue is processed, stained, and microscopically examined and bone defined an measured with image analysis) may be used. Further, measures of bone density, bone area, bone mineral content, formation of ectopic bone, and increases in the opacity of tissue upon X-ray examination, expression of alkaline phosphatase activity, calcium incorporation, mineralization or expression of osteocalcin mRNA may be used to observe the effects of BBP calcification and/or osteogenesis
  • the invention may also include the use of agents which inhibit osteoclastic bone resorption.
  • Agents which may be useful in this invention to effect osteoclastic bone resorption include, but are not limited to, bisphosphonates, the selective estrogen receptor modulators, calcitonin, and • vitamin D/calcium supplementation.
  • the invention may also include the use of agents which induce osteoblastic bone formation.
  • Agents which may be useful in this invention include, but are not limited to PTH, sodium fluoride and growth factors, such as insulin-like growth factors I and II.
  • in vivo models used to show the calcification effects of BBP alone or osteogenic effects in combination with BMP have been used previously in demonstrating similar behaviors of other compounds.
  • in vivo models have also previously been able to successfully predict the in vivo osteogenic effects of compounds such as BMP and insulin like growth factors (IGF).
  • IGF insulin like growth factors
  • a therapeutically effective dose of BBP or a TGF- ⁇ family member useful in this invention is one which has a positive clinical effect on a patient or desired effect in cells as measured by the ability of the agent to enhance calcification or osteogenesis, as described above.
  • the therapeutically effective dose of each agent can be modulated to achieve the desired clinical effect, while minimizing negative side effects.
  • the dosage of the agent may be selected for an individual patient depending upon the route of administration, severity of the disease, age and weight of the patient, other medications the patient is taking and other factors normally considered by an attending physician, when determining an individual regimen and dose level appropriate for a particular patient.
  • This invention is advantageous and unexpected us in at least the dosage of BMP-2 required to induce a given rate or degree of osteogenesis may be reduced when BMP-2 is combined with BBP. This is advantageous at least in reducing the cost of treatment, as BMP can be costly for some applications. Further, reducing treatment levels of BMP (or other bone growth factors) by treating in combination with BMP may also reduce side effects which are related to the amount of BMP used, including for example, inflammation within the soft tissue of the neck or ectopic bone formation. Thus, the use of BBP as a bone growth factor binding agent to increase exogenous growth factor retention, can aid in overcoming negative aspects of growth factor use by reducing the amount of growth factor needed to achieve healing.
  • the therapeutically effective dose of an agent included in the dosage form may be selected by considering the type of agent selected and the route of administration.
  • the dosage form may include a agent in combination with other inert ingredients, including adjutants and pharmaceutically acceptable carriers for the facilitation of dosage to the patient, as is known to those skilled in the pharmaceutical arts.
  • Therapeutic formulations of BBP may be prepared for storage by mixing the BBP having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers, in the form of lyophilized cake or aqueous solutions.
  • Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; anti-oxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins.
  • Other components can include glycine, blutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol) (PEG).
  • glycine, blutamine, asparagine, arginine, or lysine monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins
  • chelating agents such as EDTA
  • sugar alcohols such as mannitol or sorbitol
  • salt-forming counterions such as sodium
  • nonionic surfactants such as Tween, Pluronics or polyethylene glycol) (PEG).
  • the dosage form may be provided in preparations for subcutaneous (such as in a slow-release capsule), intravenous, intraparitoneal, intramuscular, peri- or intraskeletal for example. Any one or a combination of agents may be included in a dosage form. Alternatively, a combination of agents may be administered to a patient in separate dosage forms. A combination of agents may be administered concurrent in time such that the patient is exposed to at least two agents for treatment. [0059] Additional Agents. The invention may include treatment with an additional agent which acts independently or synergistically with BBP to enhance calcification osteogenesis.
  • BBP may be combined with BMP, bisphosphonates, hormone therapy treatments, such as estrogen receptor modulators, calcitonin, and vitamin D/calcium supplementation, PTH (such as Forteo or teriparatide, EIi Lilly), sodium fluoride and growth factors that have a positive effect on bone, such as insulin-like growth factors I and Il and TGF- ⁇ .
  • hormone therapy treatments such as estrogen receptor modulators, calcitonin, and vitamin D/calcium supplementation, PTH (such as Forteo or teriparatide, EIi Lilly)
  • PTH such as Forteo or teriparatide, EIi Lilly
  • sodium fluoride that have a positive effect on bone, such as insulin-like growth factors I and Il and TGF- ⁇ .
  • BBP is currently thought to act upon BMP-2 at least by increasing its residency time with a substrate.
  • One embodiment of the invention is a method of detecting the ability of BBP to enhance the residency time of a TGF- ⁇ homologous molecule including applying an amount of the TGF- ⁇ homologous molecule at a first and second selected location. Further, applying a selected amount of BBP at the first selected location, and finally detecting the amount of the TGF- ⁇ homologous molecule at the first and second location after a selected time period; and calculating the difference between the amount of the TGF- ⁇ homologous molecule at the first and second location.
  • the invention may include a method of enhancing the rate or degree of osteogenesis in vertebrate tissue including application of BBP which increases degree or rate of osteogenesis by BMP-2 in mammalian cells and one of a TGF- ⁇ family member, such as BMP-2 or demineralized bone matrix.
  • the invention may include a method of inducing calcification of vertebrate tissue, or more specifically vertebrate chondrogenic or osteogenic precursor cells, including application of BBP.
  • the invention may include a method of enhancing the rate or degree of osteogenesis in vertebrate tissue including administering chondrogenic or osteogenic precursor cells to the patient at a location proximate to the desired location of osteogenesis; further, administering BBP, and administering one of a TGF- ⁇ family member, such as BMP-2 or demineralized bone matrix.
  • a method of enhancing the rate or degree of osteogenesis in vertebrate tissue including administering chondrogenic or osteogenic precursor cells to the patient at a location proximate to the desired location of osteogenesis; further, administering BBP, and administering one of a TGF- ⁇ family member, such as BMP-2 or demineralized bone matrix.
  • the invention may include a method of enhancing the rate or degree of calcification in vertebrate tissue including administering osteogenic cells to the patient at a location proximate to the desired location of calcification and further, administering BBP.
  • the invention may include method of enhancing the rate or degree of osteogenesis in a vertebrate including treating vertebrate undifferentiated mesynchymal stem cells with one of a TGF- ⁇ family member, such as BMP-2 or demineralized bone matrix to induce osteogenesis of the cells. Further, treating the vertebrate mesynchymal stem cells with BBP; and administering the vertebrate mesynchymal stem cells to the patient at a location proximate to the desired location of osteogenesis.
  • a TGF- ⁇ family member such as BMP-2 or demineralized bone matrix
  • mammalian cells such as mesenchymal stem cells can be harvested, from the patient or a cell donor.
  • the cells may be injected in a location where bone formation or repair is desired (such as a fracture site or implant site where bone growth is needed), or first treated with BBP and/or BMP.
  • the cells may then be re-administered to the patient, either systemically or at a selected site at which osteogenesis of calcification is desired.
  • the patient may by treated locally or systemically with at least one additional agent which effects osteogenesis or calcification.
  • FIG. 12A and B depict flowcharts of exemplary methods of the invention, the steps of which may be performed in any order.
  • One embodiment of the invention may include an article of manufacture comprising BBP immobilized on a solid support.
  • the solid support may further include a TGF- ⁇ family member, such as BMP-2 or demineralized bone matrix.
  • One embodiment of the invention may include an implant for use in vivo including, a substrate where at least the surface of the implant includes BBP.
  • the implant may further include MSC, chondrocytic or osteoblastic progenitor cells.
  • the implant may be formed into the shape of a pin, screw, plate, or prosthetic joint, for example.
  • FIGS. 13A & B depict two embodiments of the present invention.
  • the invention may include implants or grafts (200) for use in the body comprising, a substrate having a surface (201), wherein at least the surface of the implant includes BBP (203) in an amount sufficient to induce, calcification or osteogenesis in the surrounding tissue.
  • the implant may include mesynchymal stem cell, chondrogenic or osteogenic cells expressing BBP, and/or BMP-2, demineralized bone matrix, or collagen cultures.
  • the implant may be in the form of, but are not limited to pins, screws, plates or prosthetic joints which may be placed in the proximity of or in contact with a bone (202) that are used to immobilize a fracture, enhance bone formation, or stabilize a prosthetic implant by stimulating formation or repair of a site of bone removal, fracture or other bone injury (204).
  • the invention may also include the in vitro (such as on cultures of collagen or chondrocytes) or in vivo application of at a least BBP containing composition or BBP expressing cells (206) in the proximity of or in contact with a bone (202), an implant (200) at a site of bone removal, fracture or other bone injury (204) where osteogenesis and/or calcification is desired.
  • the BBP composition may be applied in combination with other agents such as BMP-2, demineralized bone matrix, or collagen cultures.
  • stem cells for treating bone related disorders in humans has also been examined. Infusion of osteoblastic progenitor stem cells from a healthy individual into a diseased individual has been shown to improve bone density in these patients. Cells may be pretreated with BMP and BPP, or applied concurrently therewith.
  • the invention may include a monoclonal or polyclonal antibody having selective binding to any portion of BBP, or the BBP portion of the BBP precursor, SSP-24.
  • BBP or fragments thereof may be fused (for example by recombinant expression or in vitro covalent methods) to an immunogenic polypeptide and this, in turn, may be used to immunize an animal in order to raise antibodies against BBP.
  • Antibodies are recoverable from the serum of immunized animals.
  • monoclonal antibodies may be prepared from cells from the immunized animal in conventional fashion. Immobilized antibodies may be useful particularly in the detection or purification of BBP.
  • Two examples of specific peptide sequences against which rabbit polyclonal antibodies have been generated include: (1) An antibody against the peptide sequence
  • IQETTCRRESEADPATCDFQRGYHVPVAVCRSTVRMSAEQV (FIG. 11- SEQ. ID No. 3) that reacts with both bovine and human SSP-24, the BBP precursor.
  • This antibody was generated in rabbits immunized with the synthetic peptide indicated above.
  • An antibody directed against the sequence "CGEPLYEPSREMRRN” (FIG. 11 -SEQ. ID No. 4) that was also produced in rabbits immunized with a synthetic peptide corresponding to the indicated sequence. This second antibody reacts with bovine SSP-24.
  • the N-terminal cysteine is not a part of the native SSP-24 sequence; but is preferably included to allow the peptide to be conjugated to chromatographic resins for affinity chromatography. Additional peptide sequences may be identified for specific binding to BBP, and sequences may be selected so as to create an antibody having selective binding with BBP, but so as to not interfere with BBP binding, such as the region of BBP which binds with BMP-2 (including SEQ ID No. 3 and 4) or other TGF- ⁇ family members. These specific peptide sequences can be used to generate monoclonal antibodies, which preparation methods are known in the art.
  • Antibodies against the sequences above, corresponding sequences in the mouse, human, and rat genome, or any derivatives of the immunogenic sequences are also useful in this invention. These antibodies are useful in at least to the extent that they recognize the BBP amino acid sequence with high specificity. Such antibodies may also be useful in inhibiting protein specific interactions of BBP with other molecules where the antibody binds to a location on the peptide which interacts with other molecules. The inhibition of BBP activity in situations where the rate or degree of chondogenesis or osteogenesis may be modified.
  • antibodies specific for BBP may be useful in decreasing the degree or rate of osteogenesis by BMP-2 in vertebrate cells or decreasing degree or rate of calcification in vertebrate cells, or more specifically in mammalian chondrogenic or osteoblastic precursor cells.
  • One embodiment of the invention may also include a method of using BBP selective antibodies to detect the presence of SSP-24/BBP in sample (including but not limited to a cell culture, tissue sample, peptide fraction, Western blot) including exposing the sample to the BBP selective antibody and visualizing the complex of SSP-24/BBP and BBP antibody.
  • sample including but not limited to a cell culture, tissue sample, peptide fraction, Western blot
  • BBP antibodies may be used for the affinity purification of the BBP from recombinant cell culture or natural sources. BBP antibodies that do not detectably cross-react with other growth factors can be used to purify BBP from these other family members.
  • the invention may include a nucleic acid construct comprising a DNA or RNA nucleic acid sequence encoding BBP, or modified sequences corresponding to the modified amino sequences described above.
  • the invention may also include, an expression vector operatively linked to a nucleic acid sequence encoding BBP, or precursor SSP-24 Further, a transformant may be obtained by introducing the nucleic acid construct encoding for BBP, or its precursor SSP-24 into a host cell.
  • Practice of this invention may include the use of an oligonucleotide construct comprising a sequence coding for BBP and for a promoter sequence operatively linked in a mammalian or a viral expression vector.
  • Expression and cloning vectors contain a nucleotide sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomes, and includes origins of replication or autonomously replicating sequences. Such cloning vectors are well known to those of skill in the art.
  • Expression vectors may contain an inducible or constitutive promoter which is recognized by the host organism and is operably linked to the BBP nucleic acid.
  • the nucleic acid may be operably linked when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein which participates in the secretion of the polypeptide.
  • One embodiment of the invention may also include a method of using DNA or RNA nucleic acid sequences complimentary and having specific binding for the DNA or RNA sequences encoding BBP to detect the presence of BBP DNA or RNA in a sample, respectively (including but not limited to a cell culture, tissue sample, nucleic acid fraction, or Southern or Northern blot) including exposing the sample to the complimentary BBP DNA or RNA sequences and visualizing the complex of hybrids.
  • Example 1 EXTRACTION AND SEPARATION OF NON- COLLAGENOUS BONE PROTEINS (NPCS).
  • NCPs were extracted from defatted, demineralized human cortical bone powder with 4 M GuHCI, 0.5 M CaCI 2 , 2 mM N-ethylmalemide, 0.1 mM benzamidine HCI, and 2 m M NaN 3 for 18 hr at 6 0 C. Residual collagen and citrate-soluble NCPs were extracted by dialysis against 250 mM citrate, pH 3.1 for 24 hours at 6 0 C.
  • the residue was pelleted by centrifugation (10,000 x g at 6 0 C for 30 min), defatted with 1 :1 (v/v) chloroform: methanol for 24 hr at 23 0 C, collected by filtration and dried at 22 0 C.
  • the material was resuspended in 4 M GuHCI, dialyzed against 4 M GuHCI, 0.2% (v/v) Triton X-100, 100 mM Tris-HCI, pH 7.2 for 24 hr at 6 0 C, then dialyzed against water, and centrifuged at 10,000 x g for 30 min at 6 0 C.
  • the pellet was lyophilized and subsequently separated by hydroxyapatite chromatorgraphy.
  • Results Sequence Identification and Analysis: The fraction of bBMP/NCP which eluted from hydroxyapatite at 180 mM phosphate was separated by SDS-PAGE electrophoresis and the material with a M r of 18.5 kD was submitted for MALDI/TOF MS analysis. The major protein component of this material was determined to be a fragment of SPP-24 on the basis of six peptides with sequences identical to regions of that protein. (Hu, et al., Isolation and molecular cloning of a novel bone phosphoprotein related in sequence to the cystatin family of thiol protease inhibitors. J. Biol. Chem. 270:431-436, 1995.) The sequences of these peptides are shown in Table 1.
  • Table 1 Identification of the 18.5 kD protein by MALDI/TOF mass spectroscopy and peptide fingerprinting.
  • FIG. 1 B is a partial amino acid sequence of the bovine SSP-24, the BMP-2 homology region, and the TGF- ⁇ receptor Il homology domain. Underlined amino acids have been confirmed to be present by mass spectroscopy. (GenBank Accession Number U08018; Hu, et al.)
  • FIGS. 2 and 3 Comparisons of the two regions of interest to human BMP-2 and human TGF- ⁇ receptor Il are shown in FIGS. 2 and 3.
  • FIG. 2 is an amino acid sequence alignment of human BMP-2 and the BMP-2 homology region in bovine SPP-24.
  • FIG. 3 is an amino acid sequence alignment of bovine fetuin and human TGF- ⁇ receptor Il (top) and of human TGF- ⁇ receptor Il and the TGF- ⁇ receptor Il homology domain of bovine SPP-24 (corresponding to BBP)(bottom). Alignment of the SPP-24, fetuin, human BMP-2, and human TGF- ⁇ receptor Il sequences was accomplished using the T-Coffee program.
  • Example 2 IN VIVO ACTIVITY OF BBP
  • Methods The osteogenic activity of material was tested using male Swiss-Weber mice aged 8 to 10 weeks were used (Taconic Farms, Germantown, NY). Prior to the assay, the BBP was solublized and lyophilized into 2 mg of atelocollagen. The dried material was placed in a #5 gelatin capsule and sterilized by exposure to chloroform vapor.
  • mice were anesthetized using 1% isoflurane delivered in oxygen at 2 l/min through a small animal anesthesia machine (VetEquip, Pleasanton, CA). Animals were affixed to a surgery board and the fur over the hindquarters shaved. The skin was cleaned with 70% ethanol and a midline incision made over the spine adjacent to the hindquarters. Blunt dissection with scissors was used to expose the quadriceps muscle on one side. A small pouch was made in the muscle using the point of scissors and the #5 capsule containing the test material was inserted into the pouch. The skin was then closed with three 11 mm Michel surgical clips and the animal returned to its cage for monitoring.
  • rhBMP-2 Various amounts of rhBMP-2 and BBP were combined and prepared for implantation. All possible combinations of the following amounts were used in pilot studies, rhBMP-2: 0 ⁇ g, 0.05 ⁇ g, 0.5 ⁇ g, 5 ⁇ g, and 50 ⁇ g; BBP: 0 ⁇ g, 50 ⁇ g, and ⁇ g 500 mg. Samples of 5 ⁇ g of rhBMP-2 were used in more extensive subsequent studies because that amount consistently produced an amount of ectopic bone that was neither too large nor too small for reliable analysis.
  • FIG. 4 is a radiogram of mouse hind quarters 21 days after implantation of 500 ⁇ g of BBP in atelocollagen (top) or atelocollagen alone (bottom). When implanted alone with carrier, BBP induced calcification.
  • FIG. 5 is a histological section of mouse muscle 21 days after implantation of 500 ⁇ g of BBP in atelocollagen. Note the dystrophic calcification primarily associated with intramuscular adipose tissue. (H & E stain. Original magnification 100 X.)
  • FIG. 6 are radiograms of mouse hind quarters 21 days after implantation of 5 ⁇ g of rhBMP-2 (left) or 5 ⁇ g of rhBMP-2 plus 500 mg of BBP (right). Note the increased opacity associated with the samples containing both rhBMP-2 and BBP.
  • implants that contained both the peptide and rhBMP-2 produced detectable cartilage and bone earlier than implants of BMP-2 alone.
  • FIG. 7 are radiograms of mouse hind quarters 9 (above) and 12 (below) days after implantation of 5 ⁇ g of rhBMP-2 (left) or 5 ⁇ g of rhBMP-2 plus 500 mg of BBP (right). Note the appearance of calcification in the sample from the day 9 sample containing both rhBMP-2 and BBP but not the sample containing BMP-2 alone.
  • FIG. 8 are histological sections of mouse hind quarters 9 days after implantation of 5 ⁇ g of rhBMP-2 alone (A) or 5 ⁇ g of rhBMP-2 plus 500 ⁇ g of BBP (B). Note the abundant cartilage in the BMP + BBP specimen whereas the BMP alone specimen shows the earlier stages of inflammation and mesodermal cell proliferation.
  • Example 3 SURFACE PLASMON RESONANCE TO DETERMINE THE INTERACTION OF BMP-2 AND THE SYNTHETIC PEPTIDE
  • the peptide was dissolved in running buffer at concentrations ranging from 1 x 10 '5 to 1 x 10 "4 M. Flow rates from 5 to 50 ⁇ l/min and injection volumes of 20 to 100 ⁇ l were employed.
  • the regeneration solution was 10 ⁇ M glycine-HCI, pH 2.0.
  • FIG. 9 is a surface plasmon resonance sensogram for the interaction of rhBMP-2 (affixed to the chip) and cyclized BBP at concentrations ranging from 1 x 10 "5 M 1 x 10 ⁇ 4 M.
  • the estimated dissociation constant (K 0 ) for the interaction was 3 x 10 '5 M.
  • Example 4 RESIDENCE TIME STUDY: BBP and rhBMP-2 [00111] Methods: Labeled rhBMP-2 was mixed with BBP or vehicle and applied to collagen sponges. The sponges were implanted into muscle pouches in rodents. At specified times (1 , 3 and 7 days), the implants were removed and the amount of BMP remaining determined. Four animals were used in each group. [00112] Results: BBP increased retention of rhBMP-2 by a factor of about two. FIG. 10 is a bar graph depicting the percentage of rhBMP-2 retention over 1 , 3 and 7 days in the presence or absence of BBP. [00113] Discussion: Increasing the retention of BMP at an implant site may improve the effectiveness of the BMP, and also reduce the amount required for the same therapeutic result.
  • Example 5 IN VIVO ACTIVITY OF human BBP
  • Example 5 The methods of Example 5 were utilized to test the activity of hBBP in eight mice in the hindquarter ectopic bone formation assay method using 5 ⁇ g rhBMP-2 alone (control) or 5 ⁇ g rhBMP-2 plus 0.05 mg human BBP (hBBP). After 4 weeks, the animals were killed and the hindquarter removed. X-ray and DEXA analysis were conducted.
  • hBBP with BMP resulted in a greater amount of calcification induction than BMP alone.
  • Example 6 The effect of BBP on spinal fusion as an alternative of adjunct to BMP in a relevant animal model.
  • Results At week 8, the fusion rates were: 14% in Group 1 (BBP only); 80% in Group 2 (BBP plus low-dose BMP); and 40% in Group 3 (collagen plus low-dose BMP). This compares to 0% for historical negative controls at 100% for historical positive controls.
  • Example 7 The adjunctive effect of a binding peptide on BMP in an animal spinal fusion model.
  • Example 8 The adjunctive effect of a binding peptide on BMP in an animal spinal fusion model.
  • a prospective 8 week interventional trial employing a rat model of spinal fusion to test the effect on bone morphogenetic protein binding peptide (BBP) on rhBMP-2 induced bone healing.
  • BBP bone morphogenetic protein binding peptide
  • rhBMP-2 reduces the amount of rhBMP-2 required to achieve a satisfactory clinical outcome.
  • BBP was effective as an adjunct to increase the effect of BMP-2 and enabled decreasing the dose of the protein necessary to provide fusion.
  • Each sponge was placed in a sterile microfuge tube and either a suspension of BBP (500 ⁇ g or 1000 ⁇ g) in water or sterile water alone was applied.
  • the sponges were allowed to air dry in a tissue culture hood overnight and then placed at -70° C for an hour and lyophilized overnight.
  • the tubes (with small holes in the top to allow for lyophilization) were placed in autoclave pouches and sealed. The materials were then sterilized by exposure to chloroform vapor for at least 4 hours.
  • the pouches were opened and the sponges were removed from the tubes and placed in a second tube containing the designed amount of rhBMP- 2 or water. The sponges were able to completely absorb the solution.
  • the posterolateral intertransverse process spinal fusion at L4-L5 in the rat is a well established procedure in our Iaboratory13-17. Briefly, the animal was anesthetized with isofluane and the surgical site was shaved and prepped with Betadyne and alcohol. A 3 cm longitudinal midline incision was made through the skin and subcutaneous tissue over L4-L5 down to the lumbodorsal fascia. Then, two separate 2 cm longitudinal paramedial incisions were made in the erector spinae muscles on both sides of L4-L5. The transverse processes of L4-L5 were exposed, cleaned of soft tissue, and decorticated with a high-speed burr.
  • the site was irrigated with saline and the therapeutic test material was placed.
  • the lumbodorsal fascia was closed with 4-0 Prolene (Ethicon, Somerville, NJ).
  • the skin was closed with 4-0 Prolene and meticulous post-operative care was provided.
  • the level fused was manually palpated with Adson forceps and compared with the adjacent nonfused levels. Each specimen was graded as fused or non-fused by three independent observers. The spine was designated as "not fused” if any of the three observers graded the spine as not fused.
  • HCI standard 10% decalcifying solution
  • Sagittal sections were cut carefully at the level of the transverse process. The specimens were imbedded in paraffin and sections of each specimen obtained. These sections were stained with hematoxylin and eosin. They were evaluated by an independent observer as fused and not fused.
  • SPSS software A p score of less than or equal to 0.05 was set for statistical significance.
  • BBP + 1 ⁇ g BMP-2 BBP + 1 ⁇ g BMP-2. combination when compared to low dose BMP-2 (1 ⁇ g) only (p ⁇ 0.05). Manual palpation and histology at 8th week revealed higher rate of fusion with the same combination with a nearly significant difference
  • Table 6 shows the proportions of subjects in each group judged to be "fused" by three independent clinical evaluators.
  • the critical comparison was between BMP-2 low dose group versus BBP low dose + BMP-2 low dose and BBP high dose + BMP-2 low dose groups.
  • the fusion rate assessed by manual palpation was higher in both combination groups when compared to BMP-2 low dose group (FIGS. 15 and 16).
  • FIG. 17 is a histological section of rat spinal region 8 weeks after treatment of a combination of BBP and rhBMP-2 showing thick fusion mass between L4 and L5 transverse process with cortical-matured bone.
  • H & E stain. Original magnification 8.4 X The thickness of the fusion mass tended to be thicker and the maturity of the bone tended to be more mature in the specimens of the fused combination groups when compared to the specimens of the fused low dose BMP-2 group (FIGS. 17 and 18).
  • FIG. 17 is a histological section of rat spinal region 8 weeks after treatment of a combination of BBP and rhBMP-2 showing thick fusion mass between L4 and L5 transverse process with cortical-matured bone.
  • H & E stain. Original magnification 8.4 X The thickness of the fusion mass tended to be thicker and the maturity of the bone tended to be more mature in the specimens of the fused combination groups when compared to the specimens of the
  • Table 6 also shows the proportion of subjects in each group judged to be fused by an independent histologist. The results confirmed the results of both the manual palpation and radiology at the 8th week demonstrating the similar fusion rates. The thickness of the fusion mass tended to be thicker and the maturity of the bone tended to be more mature in the specimens of the fused combination groups when compared to the specimens of the fused low dose BMP-2 group (FIGS. 17 and 18).
  • Specific growth factor binding agents such as BBP
  • BBP can be compounded into carriers used in fusion procedures to decrease the dosage of BMP and possibly decrease the side effects which are most likely dose-related. This may also decrease costs and improve clinical outcomes.

Abstract

La présente invention porte sur l'application clinique de BBP, seul ou en combinaison avec d'autres facteurs de croissance, en vue d'une utilisation dans des applications de guérison osseuse, telles qu'une chirurgie spinale. Des applications supplémentaires comprennent l'utilisation dans des prothèses implantables orthopédiques et l'implantation dans d'autres sites chirurgicaux (par exemple, une reconstruction chirurgicale, une ostéopénie régionale, etc.) où de l'os est souhaité.
PCT/US2007/026315 2004-01-28 2007-12-26 Applications chirurgicales pour une protéine de liaison à bmp WO2008079400A2 (fr)

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AU2007338627A AU2007338627B2 (en) 2006-12-22 2007-12-26 Surgical applications for BMP binding protein
US12/448,497 US8415302B2 (en) 2004-01-28 2007-12-26 Surgical applications for BMP binding protein
CA002673600A CA2673600A1 (fr) 2006-12-22 2007-12-26 Applications chirurgicales pour une proteine de liaison a bmp
US13/857,711 US9050300B2 (en) 2004-01-28 2013-04-05 Surgical applications for BMP binding protein
US14/731,852 US9610320B2 (en) 2004-01-28 2015-06-05 Surgical applications for BMP binding protein

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US8193312B2 (en) 2004-01-28 2012-06-05 The Regents Of The University Of California Bone morphogenic protein binding peptide
US8415302B2 (en) 2004-01-28 2013-04-09 The Regents Of The University Of California Surgical applications for BMP binding protein
US9050300B2 (en) 2004-01-28 2015-06-09 The Regents Of The University Of California Surgical applications for BMP binding protein
US8975231B2 (en) 2004-01-28 2015-03-10 The Regents Of The University Of California Bone morphogenic protein binding peptide
US8753660B2 (en) 2004-11-29 2014-06-17 The Regents Of The University Of California Activating extraction of demineralized bone matrix
EP2227242A2 (fr) * 2007-11-16 2010-09-15 The Regents of the University of California Peptide de liaison à une protéine morphogénique de l'os
EP2926826A1 (fr) * 2007-11-16 2015-10-07 The Regents of The University of California Peptide de liaison de la protéine morphogénétique osseuse
EP2227242A4 (fr) * 2007-11-16 2011-08-10 Univ California Peptide de liaison à une protéine morphogénique de l'os
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US9072709B2 (en) 2009-06-23 2015-07-07 The Regents Of The University Of California Enhancement of bone morphogenic protein (BMP) retention with BMP binding peptide (BBP)
EP2445512A2 (fr) * 2009-06-23 2012-05-02 The Regents of the University of California Amélioration de la rétention des protéines morphogénétiques osseuses (bmp)
US9694047B2 (en) 2009-06-23 2017-07-04 The Regents Of The University Of California Enhancement of bone morphogenic protein (BMP) retention with BMP binding peptide (BBP)
WO2011005298A2 (fr) 2009-06-23 2011-01-13 The Regents Of The University Of California Amélioration de la rétention des protéines morphogénétiques osseuses (bmp)
WO2023280615A1 (fr) * 2021-07-05 2023-01-12 Universiteit Maastricht Moyens et méthodes pour le traitement de maladies liées au dépôt de cristaux de calcium

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AU2007338627A1 (en) 2008-07-03
AU2007338627B2 (en) 2013-08-15
JP5466017B2 (ja) 2014-04-09
JP2010513535A (ja) 2010-04-30

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