WO2006020981A2

WO2006020981A2 - Methods for accelerating bone growth and repair

Info

Publication number: WO2006020981A2
Application number: PCT/US2005/029034
Authority: WO
Inventors: Kathleen E. Rodgers; Gere S. Dizerega
Original assignee: University Of Southern California
Priority date: 2004-08-12
Filing date: 2005-08-11
Publication date: 2006-02-23
Also published as: WO2006020981A3

Abstract

The present invention relates to methods and compositions for the repair, bone implantation, and/or prosthesis implantation.

Description

METHODS FOR ACCELERATING BONE GROWTH AND REPAIR

Cross Reference

This application claim the benefit of U.S. Provisional Patent Application Serial No. 60/601046 filed August 12, 2004, which is incorporated by reference herein in its entirety.

Field of the Invention

The present invention relates to methods and compositions for repair, bone implantation, and/or prosthesis implantation.

Background of the Invention

Living bone tissue is continuously being replenished by the processes of resorption and deposition of bone matrix and minerals. This temporally and spatially coupled process, termed bone remodeling, is accomplished largely by two cell populations, the osteoclasts and osteoblasts. (U.S. Patent No. 5,656,598, incorporated by reference herein in its entirety) The remodeling process is initiated when osteoclasts are recruited from the bone marrow or the circulation to the bone surface and remove a disk-shaped packet of bone. The bone matrix and mineral is subsequently replaced by a team of osteoblasts recruited to the resorbed bone surface from the bone marrow. Osteoblasts are derived from local mesenchymal (stromal) precursors which differentiate into osteoblasts.

New bone can be formed by three basic mechanisms: osteogenesis, osteoconduction and osteoinduction. (U.S. Patent No. 5,464,439 incorporated by reference herein in its entirety) In osteogenic transplantation, viable osteoblasts and peri-osteoblasts are moved from one body location to another where they establish centers of bone formation. Cancellous bone and marrow grafts provide such viable cells. TGF-beta has been shown to stimulate proliferation and matrix synthesis of osteoblastic cells (Centrella, et al. (1987) J. Biol. Chem. 262:2869-2874) and to inhibit the formation and activity of osteoclastic cells (Chenu, et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:683-5687; Kiebzak et al. (1988) J. Bone Min. Res. 3:439-446), and to stimulate local bone formation in vivo. (Joyce, et al. (1990) J. Cell. Biol. 110:2195-2207; Noda and CamiIIiere (1989) Endocrinology 124:2991-2294). Other factors reported to stimulate bone growth include bone morphogenetic proteins (WO 88/00205), insulin-like growth factor (IGF) (Endocrinol. Metab. 13:E367-72,1986), and parathyroid hormone (J. Bone & Min. Res. 1:377-381, 1986). For example, BMP-2 has been shown to be able to induce the formation of new bone tissue in vivo in a rat ectopic implant model, see U.S. Pat. No. 5,013,649; in mandibular defects in dogs, see Toriumi et al., Arch. Otolaryngol Head Neck Surg., 117:1101-1112 (1991); and in femoral segmental defects in sheep, see Gerhart et al., Trans Orthop Res Soc, 16:172 (1991). Other members of the BMP family have also been shown to have osteogenic activity, including BMP-4, -6 and -7 (see Wozney, Bone Morphogenetic Proteins and Their Gene Expression, in Cellular and Molecular Biology of Bone, pp. 131-167 (Academic Press, Inc. 1993)).

In the transplantation of large segments of cortical bone or allogenic banked bone, direct osteogenesis does not occur. Rather, osteoconduction occurs wherein the dead bone acts as a scaffold for the ingrowth of blood vessels, followed by the resorption of the implant and deposition of new bone. This process is very slow however, often requiring years to reunite a large segmental defect.

Osteoinduction is the phenotypic conversion of connective tissue into bone by an appropriate stimulus. As this concept implies, formation of bone can be induced at even non- skeletal sites. Osteoinduction is preferred over osteoconduction, as grafts of this type are typically incorporated into the host bone within a two-week period. In contrast, osteoconductive grafts have been found to be non-incorporated as long as one year after implantation. In order to provide an environment suitable for osteoinduction, a material should be selected which is not only capable of inducing osteogenesis throughout its volume, but is also biocompatible, non-inflammatory, and possesses the ability to be ultimately resorbed by the body and replaced with new, natural bone.

Among the pathological conditions associated with abnormal bone cell function are osteoporosis, osteoarthritis, Paget's disease, osteohalisteresis, osteomalacia, periodontal disease, bone loss resulting from multiple myeloma and other forms of cancer, bone loss resulting from side effects of other medical treatment (such as steroids), and age-related loss of bone mass. Inadequate organic matrix mass places an individual at risk of skeletal failure such that bone fractures can result from the minimal trauma of everyday life. Such fractures cause significant illness, or morbidity, inasmuch as there is insufficient repair or healing of the fractures. In certain pathologic conditions, osteoclast-mediated resorption is not regulated by osteoblasts but is driven by cancer cells, infecting organisms or the host's immune cells. In those disease conditions, resorption of bone far exceeds bone formation. Such accelerated osteoclastic activity leads to excessive release of calcium from the inorganic mineral in bone, with a concomitant net loss of skeletal mass, often with an attendant disturbance in calcium homeostasis in the form of elevated blood levels of calcium. (U.S. Patent No. 5,686,116, incorporated by reference herein in its entirety.)

Although methods for directing new bone formation are known, improved methods that provide for accelerated bone growth are needed. For example, currently approved therapeutic agents for osteoporosis are anti-resorptives. As such, they are not as effective in patients with established osteoporosis of either type (decreased bone density with fractures of the vertebrae and/or hip), or in patients with Type II osteoporosis. In addition, the most accepted preventive agent for osteoporosis currently in use is estrogen therapy, which is not an acceptable therapeutic agent for women with a history of breast cancer or endometrial cancer or for men with osteoporosis.

Similarly, successful implantation and function of bone implants depends on bonding of the adjacent bone to the implant. (U.S. Patent No. 5,686,116) Such bonding requires bone repair by the formation of new matrix components at the interface between the implant and the bone proximate to the implant. An estimated ten percent of bone and joint prosthetic devices that are placed in people fail to function due to non-bonding of the bone to an implant. The resulting disability often requires re-operation and re-implantation of the device. Furthermore, five to ten percent of all bone fractures are never repaired. Although many methods have been proposed to cure these non-healing bone fractures, none has yet proven to be satisfactory. Based on all of the above, there clearly exists a need in the art for improved methods that provide for accelerated bone growth.

We have previously shown that analogues and fragments of angiotensin II can be used to stimulate bone growth and repair (U.S. Patent Nos. 6,258,778 and 6,916,783, herein incorporated by reference in its entirety).

Although some of the above methods have met with limited success, there remains a need in the art for improved compositions and methods for enhancing bone repair, healing and augmentation, and for enhancing the attachment and fixation of bone implants.

Summary of the Invention

The present invention provides methods and compositions for enhancing bone repair, bone implantation, and/or prosthesis implantation. Detailed Description of the Preferred Embodiments

All references, patents and patent applications are hereby incorporated by reference in their entirety.

Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2^nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. EJ. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX).

As defined herein the phrase "enhancing bone repair" refers to increasing the rate of new bone formation via bone remodeling, osteogenesis, osteoconduction and/or osteoinduction. The methods for enhancing bone repair in a subject of the invention include those that stimulate bone formation and those that reverse bone loss. The methods can thus be used for (1) providing a subject with an amount of a substance sufficient to act prophylactically to prevent the development of a weakened and/or unhealthy state; or (2) providing a subject with a sufficient amount of a substance so as to alleviate or eliminate a disease state and/or the symptoms of a disease state, and a weakened and/or unhealthy state.

The present invention fulfills the need for methods to promote bone repair in a subject suffering from bone fractures, defects, and disorders which result in weakened bones such as osteoporosis, osteoarthritis, Paget's disease, osteohalisteresis, osteomalacia, periodontal disease, bone loss resulting from multiple myeloma and other forms of cancer, bone loss resulting from side effects of other medical treatment (such as steroids), and age-related loss of bone mass. In addition, bony ingrowth into various prosthetic devices can be greatly enhanced so that such artificial parts are firmly and permanently anchored into the surrounding skeletal tissue through a natural osseous bridge.

In one aspect, the present invention provides polypeptides comprising or consisting of an amino acid sequence according to the general formula:

Rl-Val-R2-R3-His-Pro-Phe (SEQ ID NO: 10) wherein Rl is selected from the group consisting of Lys and Arg; R2 is selected from the group consisting of Tyr and homoSer; and

R3 is selected from the group consisting of He and Leu; wherein at least one of the following is true: Rl is Lys; R2 is homoSer; or R3 is Leu.

In the above formulas, the standard three-letter abbreviations for amino acid residues are employed, and "homoSer" refers to homoSerine.

Polypeptides according to this general formula can be used, for example, to enhance bone repair, and to enhance the attachment and fixation of bone and prosthetic implants.

In a preferred embodiment, the polypeptides comprise or consist of a sequence selected from the group consisting of:

Lysl-AIII: Lys-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 1);

HomoSer3-AIII: Arg-Val-HomoSer-Ile-His-Pro-Phe (SEQ ID NO:2); and

Leu4-AIII: Arg-Val-Tyr-Leu-His-Pro-Phe (SEQ ID NO:3).

The term "polypeptide" is used in its broadest sense to refer to a sequence of subunit amino acids, amino acid analogs, or peptidomimetics. The subunits are linked by peptide bonds, although the polypeptide can comprise further moieties that are not necessarily linked to the polypeptide by a peptide bond. For example, the polypeptide can further comprise moieties such as polyethylene glycol to increase half-life of the polypeptide.

The polypeptides described herein may be chemically synthesized or recombinantly expressed. Recombinant expression can be accomplished using standard methods in the art, generally involving the cloning of nucleic acid sequences capable of directing the expression of the polypeptides into an expression vector, which can be used to transfect or transduce a host cell in order to provide the cellular machinery to carry out expression of the polypeptides. Such expression vectors can comprise bacterial or viral expression vectors, and such host cells can be prokaryotic or eukaryotic.

Preferably, the polypeptides of the present invention are chemically synthesized. Synthetic polypeptides, prepared using the well-known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc (Na- amino protected Nα-t-butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of standard solid phase procedure, or base-labile Nα-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids. Both Fmoc and Boc Nα-amino protected amino acids can be obtained from Sigma, Cambridge Research Biochemical, or other chemical companies familiar to those skilled in the art. In addition, the polypeptides can be synthesized with other Nα-protecting groups that are familiar to those skilled in this art. Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, for example by using automated synthesizers.

The polypeptides of the invention can further be derivatized to provide enhanced half- life, for example, by linking to polyethylene glycol. The polypeptides of the invention may comprise L-amino acids, D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L-amino acids.

In addition, the polypeptides can have peptidomimetic bonds, such as ester bonds, to prepare polypeptides with novel properties. For example, a peptide may be generated that incorporates a reduced peptide bond, i.e., Ri-CH₂-NH-R₂, where Ri and R₂ are amino acid residues or sequences. A reduced peptide bond may be introduced as a dipeptide subunit. Such polypeptides are resistant to protease activity, and possess an extended half-live in vivo.

In a preferred embodiment, the polypeptides of the invention are substantially purified. As used herein, the term "substantially purified" means that the polypeptide has been substantially separated from its in vivo cellular environments, if produced recombinantly (ie: at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% purified). Thus, the polypeptide can be purified from host cells transfected to express the polypeptide using standard techniques. (See for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press.)) The polypeptide can thus be purified from prokaryotic or eukaryotic sources. In a further preferred embodiment, "substantially purified" means that the polypeptide is substantially free of gel agents, such as polyacrylamide and agarose, or of materials used for peptide synthesis (ie: at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% purified).

The polypeptides may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifϊers, buffers etc.

In another aspect, the present invention provides pharmaceutical compositions, comprising one or more of the polypeptides disclosed herein, and a pharmaceutically acceptable carrier. Pharmaceutical compositions for carrying out the methods of the invention are described in more detail below. For administration, the polypeptides are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. The compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, dextran sulfate, heparin-containing gels, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include release-delaying material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.

The polypeptides may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions). The polypeptides of the invention may be applied in a variety of solutions, as described in more detail below.

The pharmaceutical compositions of the invention may further comprise one or more additional compounds that also promote bone repair, implantation, and/or prosthesis implantation, including but not limited to bone morphogenic protein-2, bone morphogenic protein-4, bone morphogenic protein-6, bone morphogenic protein-7, transforming growth factor-beta, insulin-like growth factor, and parathyroid hormone.

In another aspect, the present invention provides isolated nucleic acid sequences encoding a polypeptide of the present invention. Appropriate nucleic acid sequences according to this aspect of the invention will be apparent to one of skill in the art based on the polypeptide disclosure provided herein and the general level of skill in the art.

In another aspect, the present invention provides an expression vectors comprising DNA control sequences operably linked to the isolated nucleic acid molecules of the present invention, as disclosed above. "Control sequences" operably linked to the nucleic acid sequences of the invention are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites.

Such expression vectors can be of any type known in the art, including but not limited plasmid and viral-based expression vectors. In a further aspect, the present invention provides genetically engineered host cells comprising the expression vectors of the invention. Such host cells can be prokaryotic cells or eukaryotic cells, and can be either transiently or stably transfected, or can be transduced with viral vectors.

In another aspect, the invention provides improved biomedical devices, wherein the biomedical devices comprise one or more of the polypeptides of the present invention disposed on or in the biomedical device. As used herein, a "biomedical device" refers to a device to be implanted into a subject, for example, a human being, in order to bring about a desired result. Particularly preferred biomedical devices according to this aspect of the invention include, but are not limited to, bone implants, prosthetic implants, dental implants, orthopedic implants, scaffolds, and polymeric substrates as described below.

As used herein, "disposed on or in" means that the one or more polypeptides can be either directly or indirectly in contact with an outer surface, an inner surface, or embedded within the biomedical device. "Direct" contact refers to disposition of the polypeptides directly on or in the device, including but not limited to soaking a biomedical device in a solution containing the one or more polypeptides, spin coating or spraying a solution containing the one or more polypeptides onto the device, implanting any device that would deliver the polypeptide, and administering the polypeptide through a catheter directly on to the surface.

"Indirect" contact means that the one or more polypeptides do not directly contact the biomedical device. For example, the one or more polypeptides may be disposed in a matrix, such as a gel matrix or a viscous fluid, which is disposed on the biomedical device. Such matrices can be prepared to, for example, modify the binding and release properties of the one or more polypeptides as required.

In a further aspect, the present invention provides methods for promoting one or more of bone repair, bone implantation, and/or prosthesis implantation, comprising administering to a subject in need thereof an amount effective to promote bone repair, bone implantation, and/or prosthesis implantation of an active agent comprising or consisting of a polypeptide of the present invention, or pharmaceutical composition or biomedical device comprising such polypeptides of the invention, as described in the above aspects and embodiments. In this aspect, the subject is preferably a mammal, and even more preferably a human.

The active agent can be administered alone or in combination with other compounds that enhance bone repair, bone implantation, and/or prosthesis implantation, including but not limited to bone morphogenic protein-2, bone morphogenic protein-4, bone morphogenic protein-6, bone morphogenic protein-7, transforming growth factor-beta, insulin-like growth factor, and parathyroid hormone.

The active agents may be administered by any suitable route, including orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. Such vehicles may include a tantalum or hydroxyapatite scaffold as a vehicle with the compounds of the invention embedded therein. Alternatively, polymeric substrates can be used for compound delivery to the bone such as the polymeric substrates disclosed in U.S. Patent Nos. 5,443,515; 5,171,273; 5,607,474; 4,916,207; and 5,324,775; all references hereby incorporated in their entirety. The term parenteral as used herein includes topical (i.e.: placement in to the bone), subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally.

The active agents of the present invention can also be incorporated into a coating on the surface of a prosthetic device. Such coatings may be composed of a polymer that allows slow diffusion of the active agents at a rate sufficient to enhance bone attachment for a suitable period of time. Suitable coatings include, but are not limited to, hydroxyapatite, methacrylate and tricalcium phosphate. Further, the polymeric coatings can be applied only to the sites on the prosthetic device where bony ingrowth is desired. Bone grafts can be coated with or soaked or immersed in a rinse or gel prior to implantation so as to impregnate the graft with the active agents. Topical or local administration of the active agents to either the site of implantation or the implant itself is preferred, as it diminishes drug exposure for tissues and organs not requiring treatment.

The active agents may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions). The compounds of the invention may be applied in a variety of solutions. Suitable solutions for use in accordance with the invention are sterile, dissolve sufficient amounts of the peptide, and are not harmful for the proposed application. In this regard, the compounds of the present invention are very stable but are hydrolyzed by strong acids and bases. The compounds of the present invention are soluble in organic solvents and in aqueous solutions at pH 5-8.

Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose. The dosage regimen for use of the active agents in the methods of the invention is based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods. Dosage levels of the order of between 0.1 ng/kg and 10 mg/kg body weight of the active agents are useful for all methods of use disclosed herein.

As an example, the dosage regimen for accelerating bone repair with the active agents wherein the polypeptides are embedded in a scaffold used for bone, such as tantalum or hydroxyapatite, would be based upon the volume of bone to be filled and not upon the weight of the subject being treated. Following determination of the specific dose volume to be administered to the subject, calculated based on the defect, individual doses are prepared. The final delivered drug product will be mixed under aseptic conditions at a ratio of: 1 part (20%) of the active agents (in a concentration of between about .001 μg/ml to about 5 mg/ml) to 4 parts (80%) diluent.

The treatment regime will also vary depending on the disease being treated, based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. For example, the active agents are administered to an osteoporosis patient for up to 30 days. The therapy is administered for 1 to 6 times per day at dosages as described above.

In a preferred embodiment, the active agent is administered subcutaneously. A suitable subcutaneous dose of active ingredient of active agent is preferably between about 0.1 ng/kg and about 10 mg/kg administered twice daily for a time sufficient to carry out the methods of the invention. In a more preferred embodiment, the concentration of active agent is between about 100 ng/kg body weight and about 10.0 mg/kg body weight. In a most preferred embodiment, the concentration of active agent is between about 10 μg/kg body weight and about 10.0 mg/kg body weight. This dosage regimen maximizes the therapeutic benefits of the subject invention while minimizing the amount of active agent needed. Such an application minimizes costs as well as possible deleterious side effects.

For subcutaneous administration, the active ingredient may comprise from 0.0001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1% to 1% of the formulation. In another preferred embodiment of the present invention, the active agent is administered topically at the site of bone loss or repair. Suitable topical doses and active ingredient concentration in the formulation are as described for subcutaneous administration.

In another preferred embodiment, the active agent is administered at the desired site of bone repair, such as via capsule delivery in gingival tissue at sites of bone resorption or in a scaffold surgically implanted at the site of a non-union bone fracture. Suitable doses and active ingredient concentration in the formulation are as described for subcutaneous administration.

In one embodiment, ex vivo methods are presented for enhancing bone repair via isolation of osteoblastic cell populations from a subject, contacting the isolated cell population with the active agent, and subsequent re-infusion of the osteoblastic cell population into the subject. Methods for the isolation of osteoblastic cell population have been described. (Hiruma et al., 1997; Lamparter et al., 1998) In a preferred embodiment, human bone cells are cultured from outgrowths of trabecular bone fragments of the femoral head from patients undergoing hip replacement due to fracture, and treated by several consecutive collagenase digestion periods, using 1 mg/ml type II collagenase solution (Worthington Diagnostic Systems) for 20 minutes per digestion period.

Alternatively, the effects of the active agents on matrix component synthesis in bone cells are determined in organ culture and in isolated cells by measuring the levels of two matrix proteins, as described in U.S. Patent No. 5,686,116 incorporated by reference herein in its entirety. Type I collagen is the major matrix protein of bone. Collagen is almost entirely composed of proline, OH-proline, alanine and glycine. Thus, new-bone collagen synthesis can be determined by measuring the uptake of ³H-Pro or by following the appearance of ³H- OH-Pro, which is formed by conversion of proline subsequent to its incorporation into collagen. Osteocalcin is a bone-specific matrix protein which is thought to be a cell signal for attracting osteoclasts to bone to initiate bone breakdown, and is also thought to slow or impede formation of newly mineralizing bone. Price et al., Proc. Natl. Acad. Sci. USA, 79:7734-7738 (1982). Therefore, decreased osteocalcin synthesis is associated with bone repair.

In vivo bone repair can be assessed, for example, by administration by injection of the active agent into the subcutaneous tissue over the right calvarium of mice. Control vehicle is PBS supplemented with 1% BSA. Heparin is administered at a dose of 50 units/ml. The animals are sacrificed on day 14 and bone growth is measured by histomorphometry. Bone samples for quantitation are cleaned from adjacent tissues and fixed in 10% buffered formalin for 24-48 hours, decalcified in 14% EDTA for 1-3 weeks, processed through graded alcohols and embedded in paraffin wax. Three micron sections of the calvaria and femurs are prepared. Representative sections are selected for histomorphometric assessment of the effects of the active agent on bone formation and bone resorption. Sections are measured by using a camera attachment to directly trace the microscopic image onto a digitizing plate. Bone changes are measured on sections collected 200 mu m apart, over 4 adjacent 1 x 1 mm fields on both the injected and noninjected sides of the calvaria. New bone is identified by its woven, rather than lamellar structure, and osteoclasts and osteoblasts are identified by their distinctive morphology. Histomorphometry software (Osteomeasure, Osteometrix, Inc., Atlanta) is used to process digitizer input to determine cell counts and feature areas or perimeters.

Alternatively, in vivo activity can be assessed as follows. The active agents of the invention are used to potentiate osteoblast function in intact animals. In a preferred embodiment, a model for abnormal osteoblast activity, weanling Sprague-Dawley rats, are placed on a phosphate and vitamin D-defϊcient diet as per the manufacturer's instructions (the diet, #80039, Teklad, Madison, Wis.) The animals on the diet are also kept in the dark to prevent de novo vitamin D synthesis. Animals placed under such conditions show abnormal bone formation and a marked deficiency in total bone mass. One group of the weanling rats on the diet is treated with active agent at between about 0.1 ng/kg and 10 mg/kg, given as a subcutaneous injection, every other day for 21 days. One group on the diet remains untreated and served as the control. Littermate controls not on the diet and not treated with active agent supply blood samples at the time of sacrifice of the animals on the diet for determination of alkaline phosphatase activity. Upon sacrifice, the long bones are removed from the animals on the diet for subsequent analyses.

Serum alkaline phosphatase activity is used as a reliable indicator of osteoblast activity, such that increased levels of alkaline phosphatase activity are evidence of the abnormal bone turnover in the experimental animals. Serum alkaline phosphatase activity is determined by measuring the hydrolysis of p-nitrophenyl phosphate by serum samples according to the method of Lowry et al., J. Biol. Chem., 207:19-37 (1954). Markedly elevated serum alkaline phosphatase activity indicates abnormal osteoblast activity. By contrast, normalization of bone cell function is evidenced by a decreased level of serum alkaline phosphate activity and increased bone mass (tested via determination of the ash weight of bones) relative to control animals. In a further embodiment, dental and orthopedic implants can be coated with active agents. In general, implant devices are coated with the active agents dissolved at a concentration in the range of 0.1 ng/ml to 10 mg/ml in phosphate-buffered saline (PBS) containing 2 mg/ml serum albumin. The porous end of an implant is dipped in the solution and is air dried (or lyophilized) or implanted immediately into the bony site. The viscosity of the coating solution is increased, if desired, by adding hyaluronate at a final concentration of 0.1 mg/ml to 100 mg/ml or by adding other pharmaceutically acceptable excipients. Alternatively, the solution containing the active agent is mixed with collagen gel or human collagen (e.g. Zyderm Registered TM Collagen Implant, Collagen Corp., Palo Alto, Calif.) to a final collagen concentration of 2 mg/ml to 100 mg/ml to form a paste or gel, which is then used to coat the porous end of the implant device. The coated implant device is placed into the bony site immediately or is air dried and rehydrated with PBS prior to implanting, with the objective of maximizing new bone formation into the implant while minimizing the ingrowth of soft tissue into the implant site.

The present invention fulfills the need for methods to enhance bone repair in a subject suffering from a wide variety of disorders and injuries including, but not limited to bone fractures, defects, and disorders which result in weakened bones such as osteoporosis, osteoarthritis, Paget's disease, osteohalisteresis, osteomalacia, periodontal disease; bone loss resulting from multiple myeloma and other foπns of cancer; bone loss resulting from side effects of other medical treatment (such as steroids); and age-related loss of bone mass.

Example 1. Bone Healing

Sprague Dawley rats underwent intramuscular anesthesia with ketamine/rompum and were prepared for sterile surgery by shaving the surgical site and scrubbing with Betadine scrub followed by 70% ethanol. The rat was then placed on a sterile field in a lateral decubitis position facing the surgeon. The shaved legs were then covered with Betadine solution and draped aseptically. A skin incision was performed parallel to the long axis of the right medial diaphysis. The muscle was separated along fascial planes to expose the tibia. A defect of 1.3 mm in diameter was then drilled from the lateral side of the midshaft cortex so that the defect extended from one cortical side to the other, through the bone marrow. Sterile saline (0.9% NaCl) for injection was then used to clean the surgical area of tissue debris and bone fragments. Either vehicle (acid insoluble collagen or 20% carboxymethyl cellulose)or test peptide in vehicle was placed in the bone defect to fill the defect with polymer (approximately 0.1 ml ). The incision was closed with 3-0 Vicryl suture using continuous mattress suture. The animals were allowed to recover from anesthesia, given Bupronex for analgesia and allowed free movement, until euthanasia 7 days later.

In addition to All, the peptides tested were the following: Peptide 1 2 3 4 5 6 7 8

Lys IAIII (SEQ ID 1) K V Y I H P F

HomoSer3 AIII (SEQ ~ R V HomoSer I H P F

ID 2)

NorLeu3 All (SEQ ID D R NorLeu Y I H P F

4)

Ala4 AIII (SEQ ID 5) R V Y A H P F

AIII (SEQ ID 6) R V Y I H P F

Lys2 All (SEQ ID 7) D K V Y I H P F

HomoSer4 All (SEQ D R V HomoSer I H P F

ID 8)

NorLeu2 AIII (SEQ ID - R NorLeu Y I H P F

Q\

Leu4 AIII (SEQ ID 3) ~ R V Y L H P F

By gross observation, the defects that received peptides were more completely filled with new tissue that had begun to calcify. Microscopic evaluation of the tissue sections confirmed the gross observations. The vehicle-treated defects were filled with fibroproliferative tissue that was well vascularized. In the majority of vehicle treated defects, there was no callus formation and little osteoid formation. Occasionally, an osteoblast was observed at the site of injury.

In all of the All-treated animals, there was extensive fibroproliferative and osteoblastic activity and new blood vessel growth. With All treatment, callus can be seen external to the cortex as well as within the marrow space. The callus, within the marrow space, is composed of richly vascular fibroblastic tissue with peripheral areas of new bone formation. The new bone is characterized by innumerable, highly active osteoblasts surrounding islands of osteoid formation.

Defects filled with peptide 2A (Lys '-AIII) resulted in a large amount of fibroproliferative activity, callus formation and deposition of osteoid. In three of 5 tibia, the newly healing tissue appeared to displace the old bone. Similarly, defects filled with peptide 41A (HomoSer³-AIII) had extensive callus formation,, osteoid formation and fibroproliferative activity. Defects filled with peptide 2B (Lys²-AII) resulted in a large amount of fibroproliferative activity, and callus formation. Defects filled with peptide 5A (Leu4-AIII) were filled with some fibroproliferative activity and osteoid formation. Bones treated with this peptide had extensive callus formation. Thus, peptides 2 A, 4 IA, and 5 A were more potent and effective in stimulating new bone formation than other peptides tested as disclosed herein, as well as others disclosed in US Patent Nos. 6,258,778 and 6,916,783. Furthermore, these peptides are not anticipated to be pressors, as All is Through these and other similar experiments, Peptide 41A was the most potent and effective peptide tested in stimulating new bone formation.

The remaining peptides (39B [NorLeu³-AII], 22A [Ala⁴-AIII], A(I -9), 4 IB [HomoSer⁴-AII], 39A (NorLeu²-AIII) and AIII) had reduced activity compared with All, peptide 2A, 2B, 5 A and peptide 4 IA, but the osteoid development was superior to vehicle control.

It is to be understood that the invention is not to be limited to the exact details of operation, or to the exact compounds, compositions, methods, procedures or embodiments shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art, and the invention is therefore to be limited only by the full scope of the appended claims.

Claims

We claim:

1. A polypeptide consisting of an amino acid sequence according to the general formula: Rl-Val-R2-R3-His-Pro-Phe (SEQ IDNO:10) wherein Rl is selected from the group consisting of Lys and Arg;

R2 is selected from the group consisting of Tyr and homoSer; and

2. The polypeptide of claim 1, wherein the polypeptide consists of the amino acid sequence

Lys-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 1).

3. The polypeptide of claim 1, wherein the polypeptide consists of the amino acid sequence Arg-Val-HomoSer-Ile-His-Pro-Phe (SEQ ID NO:2).

4. The polypeptide of claim 1, wherein the polypeptide consists of the amino acid sequence Arg-Val-Tyr-Leu-His-Pro-Phe (SEQ ID NO:3).

5. A pharmaceutical composition, comprising the polypeptide of claim 1 and a pharmaceutically active carrier.

6. An isolated nucleic acid encoding the polypeptide of claim 1.

7. An expression vector comprising the isolated nucleic acid of claim 6.

8. A recombinant host cell, comprising the expression vector of claim 7.

9. A biomedical device, comprising one or more of the polypeptides according to claim 1.

10. The biomedical device of claim 9, wherein the biomedical device is selected from the group consisting of bone implants, prosthetic implants, dental implants, orthopedic implants, scaffolds, and polymeric substrates.

11. A method for promoting one or more of bone repair, bone implantation, and prosthesis implantation, comprising administering to a subject in need thereof an amount effective to promote bone repair, bone implantation, and/or prosthesis implantation of an active agent comprising a polypeptide consisting of an amino acid sequence according to the general formula:

Rl-Val-R2-R3-His-Pro-Phe wherein Rl is selected from the group consisting of Lys and Arg;

R2 is selected from the group consisting of Tyr and homoSer; and

12. The method of claim 11, wherein the polypeptide consists of the amino acid sequence Lys-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 1).

13. The method of claim 11, wherein the polypeptide consists of the amino acid sequence Arg-Val-HomoSer-Ile-His-Pro-Phe (SEQ ID NO:2).

14. The method of claim 11, wherein the polypeptide consists of the amino acid sequence Arg-Val-Tyr-Leu-His-Pro-Phe (SEQ ID NO:3).

15. The method of claim 11, wherein the method is used to promote bone repair associated with one or more disorders selected from the group consisting of osteoporosis, osteoarthritis, Paget's disease, osteohalisteresis, osteomalacia, periodontal disease, bone loss resulting from cancer, bone loss resulting from steroid treatment, and age-related loss of bone mass.