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  Pharmaceutical Patents  

 

Title:  Methods of inhibiting ectopic calcification
United States Patent: 
7,419,950
Issued: 
September 2, 2008

Inventors:
 Giachelli; Cecilia M. (Mill Creek, WA), Steitz; Susie (Seattle, WA)
Assignee:
  University of Washington (Seattle, WA)
Appl. No.:
 10/376,383
Filed:
 February 26, 2003
 

Training Courses --Pharm/Biotech/etc.


Abstract

The invention provides a method of inhibiting ectopic calcification in an individual. The method consists of administering to the individual a therapeutically effective amount of osteopontin or a functional fragment thereof.

Description of the Invention

SUMMARY OF THE INVENTION

The invention provides a method of inhibiting ectopic calcification in an individual. The method consists of administering to the individual a therapeutically effective amount of osteopontin or a functional fragment thereof. The method can be used to inhibit ectopic calcification associated with a variety of conditions such as atherosclerosis, stenosis, restenosis, prosthetic valve replacement, angioplasty, renal failure, tissue injury, diabetes and aging.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to an effective method for the inhibition of ectopic calcification. Ectopic calcification commonly occurs in association with renal failure, cardiovascular disease, diabetes and the aging process. Ectopic calcification of the vasculature increases an individual's risk of myocardial infarction, ischemia, stroke, dissection after angioplasty and heart valve failure. Ectopic calcification of prosthetic implants, such as bioprosthetic heart valves, is the leading cause of implant failure. Therefore, the method will reduce disease and death associated with ectopic calcification.

The method is based on the discovery that osteopontin is able to effectively and specifically inhibit ectopic calcification. Therefore, ectopic calcification can be prevented or treated by administering a therapeutically effective amount of osteopontin or a functional fragment thereof to an individual, either systemically or at the predicted or known sites of ectopic calcification. As osteopontin is normally found in calcified tissues and at the sites of ectopic calcification, it can be administered with minimal toxic or immunogenic side effects.

As used herein, the term "ectopic calcification" is intended to mean the abnormal deposition of calcium crystals at sites other than bones and teeth. Ectopic calcification results in the accumulation of macroscopic hydroxyapatite deposits in the extracellular matrix.

Ectopic calcification can occur in a variety of tissues and organs and is associated with a number of clinical conditions. For example, ectopic calcification can be a consequence of inflammation or damage to the affected tissues or can result from a systemic mineral imbalance. Commonly, ectopic calcification occurs in vascular tissue, including arteries, veins, capillaries, valves and sinuses. Inflammation or damage to the blood vessels can occur, for example, as a result of environmental factors such as smoking and high-fat diet. Inflammation or damage can also occur as a result of trauma to the vessels that results from injury, vascular surgery, heart surgery or angioplasty. Vascular calcification is also associated with aging and with disease, including hypertension, atherosclerosis, diabetes, renal failure and subsequent dialysis, stenosis and restenosis.

Ectopic calcification also occurs in non-vascular tissues, such as tendons (Riley et al., Ann. Rheum. Dis. 55:109-115 (1996)), skin (Evans et al., Pediatric Dermatology 12:307-310 (1997)), sclera (Daicker et al., Opthalmologica 210:223-228 (1996) and myometrium (McCluggage et al., Int. J. Gynecol. Pathol. 15:82-84 (1996)), each of which is incorporated herein by reference. In diseases resulting in systemic mineral imbalance, such as renal failure and diabetes, ectopic calcification in visceral organs, including the lung, heart, kidney and stomach, is common (Hsu, Amer. J. Kidney Disease 4:641-649 (1997), incorporated herein by reference). Furthermore, ectopic calcification is a frequent complication of the implantation of biomaterials, prostheses and medical devices, including, for example, bioprosthetic heart valves (Vyavahare et al., Cardiovascular Pathology 6:219-229 (1997), incorporated herein by reference). The methods of the invention are applicable to ectopic calcification that occurs in association with all of these conditions.

The term "ectopic calcification" is not intended to refer to the calcification that normally occurs within the bone matrix during bone formation and growth. Ectopic calcification, as used herein, is also distinct from abnormal calcification that occurs in renal tubules and urine that results in the formation of primarily calcium oxalate-containing kidney stones.

As used herein, the term "inhibiting," in connection with inhibiting ectopic calcification, is intended to mean preventing, retarding, or reversing formation, growth or deposition of extracellular matrix hydroxyapatite crystal deposits.

As used herein, the term "osteopontin" is intended to mean a molecule that is able to inhibit ectopic calcification and that is recognizably similar to one or more molecules known in the art as osteopontin. Osteopontin is characterized as a phosphorylated sialoprotein having a predicted molecular weight of about 34 kDa. Due to high negativity, post-translational modifications and alternatively spliced isoforms, osteopontin has been reported to have an apparent molecular weight of between about 44 and 85 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Giachelli et al., Trends Cardiovasc. Med 5:88-95 (1995)). All of the post-translationally modified forms and alternatively spliced isoforms of osteopontin are included within the definition of osteopontin as used herein.

Osteopontin has been identified in various species, including rat (Oldbert et al., Proc. Natl., Acad. Sci. USA 83:8819-8823 (1986)); mouse (Craig et al., J. Biol. Chem. 264:9682-9689(1989)); human (Kiefer et al., Nucleic Acids Res. 17:3306 (1989) and Young et al. Genomics 7:491-502 (1990)); pig (Wrana et al., Nucleic Acids Res. 17:10119 (1989)); cow (Kerr et al., Gene 108:237-243 (1991)); rabbit (Tezuka et al., Biochem. Biophys. Res. Commun. 186:911-917 (1992)); and chicken (Moore et al., Biochemistry 30:2502-2508 (1991)), each of which is incorporated herein by reference. Osteopontin from these species and osteopontin homologs from other vertebrates are included within the definition of osteopontin as used herein.

Osteopontin can be characterized by the presence of one or more domains that are conserved across known species. The conserved domains that characterize osteopontin include, for example, an N-terminal signal sequence, casein kinase II phosphorylation sites, an alternatively spliced domain, an Arg-Gly-Asp (RGD)-containing integrin-binding cell adhesion domain, an Asp-rich calcium binding domain, a calcium binding homology domain and two heparin binding homology domains (Giachelli et al., supra (1995)). Therefore, newly identified molecules that possess one or more of these characteristic features of osteopontin are also included within the definition of osteopontin.

Osteopontin is also known in the art as bone sialoprotein I, uropontin, secreted phosphoprotein I, 2ar, 2B7 and Eta 1 (Giachelli et al., supra (1995)). The molecules encompassed by all of these terms used in the art are included within the definition of osteopontin as used herein.

The nucleotide and deduced amino acid sequence for human osteopontin have been described by Kiefer et al., supra (1989), and are set forth herein as FIG. 1 (SEQ ID NOS: 1 and 2 (see Original Patent)). The term osteopontin is intended to include, for example, polypeptides having substantially the same amino acid sequence as shown as SEQ ID NO: 2 and encoded by substantially the same nucleotide sequence as shown as SEQ ID NO: 1.

Modifications of osteopontin and its functional fragments that either enhance or do not greatly affect the ability to inhibit ectopic calcification are also included within the term "osteopontin." Such modifications include, for example, additions, deletions or replacements of one or more amino acids from the native amino acid sequence of osteopontin with a structurally or chemically similar amino acid or amino acid analog. For example, the substitution of one or more phosphorylated amino acids, such as serine or threonine residues, by negatively charged amino acids, such as glutamic acid or aspartic acid, is contemplated. The substitution or addition of residues, such as kinase phosphorylation consensus sequences, that can be phosphorylated either in vivo or in vitro is also contemplated. Modifications of residues between the native sites of phosphorylation, such as to beneficially orient the phosphorylated residues to interact with hydroxyapatite or to reduce the distance between phosphorylation sites, is also contemplated. These modifications will either enhance or not significantly alter the structure, conformation or functional activity of the osteopontin or a functional fragment thereof.

Modifications that do not greatly affect the activity of osteopontin or its functional fragments can also include the addition or removal of sugar, phosphate or lipid groups as well as other chemical derivations known in the art. Additionally, osteopontin or its functional fragments can be modified by the addition of epitope tags or other sequences that aid in its purification and which do not greatly affect its activity.

As used herein, the term "functional fragment," in connection with osteopontin, is intended to mean a portion of osteopontin that maintains the ability of osteopontin to inhibit ectopic calcification. A functional fragment can be, for example, from about 6 to about 300 amino acids in length, for example, from about 7 to about 150 amino acids in length, more preferably from about 8 to about 50 amino acids in length. If desired, a functional fragment can include regions of osteopontin with activities that beneficially cooperate with the ability to inhibit ectopic calcification. For example, a functional fragment of osteopontin can include sequences that promote the ingrowth of cells, such as endothelial cells and macrophages, at the site of ectopic calcification. Similarly, a functional fragment of osteopontin can include sequences, such as the RGD-containing domain, that beneficially promote cell adhesion and survival at the site of ectopic calcification.

As used herein, the term "individual" is intended to mean a human or other mammal, exhibiting, or at risk of developing, ectopic calcification. Such an individual can have, or be at risk of developing, for example, ectopic calcification associated with conditions such as atherosclerosis, stenosis, restenosis, renal failure, diabetes, prosthesis implantation, tissue injury or age-related vascular disease. The prognostic and clinical indications of these conditions are known in the art. An individual treated by a method of the invention can also be a candidate for, or have undergone, vascular surgery, including prosthetic valve replacement or angioplasty. An individual treated by a method of the invention can have a systemic mineral imbalance associated with, for example, diabetes, renal failure or kidney dialysis.

As used herein, the term "substantially the amino acid sequence," in reference to an osteopontin amino acid sequence or functional fragment thereof is intended to mean a sequence that is recognizably homologous to an osteopontin amino acid sequence and that inhibits ectopic calcification. For example, a sequence that is substantially the same as an osteopontin sequence can have greater than about 70% homology with an osteopontin sequence, preferably greater than about 80% homology, more preferably greater than about 90% homology.

As used herein, the term "prosthetic device" refers to a synthetic or biologically derived substitute for a diseased, defective or missing part of the body. As used herein, the term "bioprosthetic device" refers to a partially or completely biologically derived prosthetic device. Prosthetic devices are susceptible to ectopic calcification leading to premature failure, which can be inhibited by the methods of the invention. A prosthetic device can be implanted or attached at various sites of the body including, for example, the ear, eye, maxillofacial region, cranium, limbs and heart.

The methods of the invention can advantageously be used to prevent ectopic calcification of prosthetic heart valves, such as an aortic or atrioventricular valve, with or without a stent. Replacement heart valves can be made of a variety of materials, including metals, polymers and biological tissues, or any combination of these materials. Bioprosthetic valves include xenografted replacement valves from mammals, such as sheep, bovine and porcine, as well as human valves. Bioprosthetic heart valves are commonly subjected to tissue fixation and can additionally be devitalized prior to implantation.

The invention provides a method of inhibiting ectopic calcification in an individual. The method consists of administering to the individual a therapeutically effective amount of osteopontin or a functional fragment thereof. The method is advantageous as it employs a molecule that normally occurs at the site of ectopic calcification as a therapeutic agent. Therefore, the method will result in minimal toxicity, immunogenicity and side effects.

Osteopontin can be prepared or obtained by methods known in the art including, for example, purification from an appropriate biological source or by chemical synthesis. An appropriate biological source of osteopontin can be tissues, biological fluids or cultured cells that contain or express osteopontin. The presence and abundance of osteopontin protein in a particular source can be determined, for example, using ELISA analysis (Min et al., Kidney Int. 53:189-93 (1998), incorporated herein by reference) or immunocytochemistry (O'Brien et al., Arterioscler. Thromb. 14:1648-1656 (1994), incorporated herein by reference).

Osteopontin has been determined to be present in or expressed by kidney cells, hypertrophic chondrocytes, odontoblasts, bone cells, bone marrow, inner ear and brain cells. Osteopontin is also found in biological fluids, including milk and urine. Osteopontin is also present in tumors, particularly metastatic tumors and is a component of kidney stones (Butler et al., In: Principles of Bone Biology, Bilezikian et al., eds., Academic Press, San Diego, pp. 167-181 (1996), incorporated herein by reference). Osteopontin is also produced by smooth muscle cells, macrophages and endothelial cells at the site of vascular lesions (O'Brien et al., Arterioscler. Thromb. 14:1648-1656 (1994), incorporated herein by reference). Therefore, osteopontin can be purified from any of these sources using biochemical purification methods known in the art.

Osteopontin can also be obtained from the secreted medium of cells of any of the above tissue lineages grown in culture. For example, osteopontin can be substantially purified from the conditioned medium of smooth muscle cell cultures as described by Liaw et al., Circ. Res. 74:214-224 (1994), incorporated herein by reference.

The nucleotide sequences of osteopontin from a variety of species are known, as described previously. Therefore, osteopontin or its functional fragments can also be recombinantly expressed by appropriate host cells including, for example, bacterial, yeast, amphibian, avian and mammalian cells, using methods known in the art. Methods for recombinant expression and purification of peptides in various host organisms are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1992) and in Ansubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), both of which are incorporated herein by reference. Methods for the recombinant synthesis and purification of osteopontin and exemplary functional fragments therefrom are described, for example, in Smith et al., J. Biol. Chem. 271:28485-28491 (1996), incorporated herein by reference.

Following recombinant synthesis and purification, osteopontin and its functional fragments can be modified in a physiologically relevant manner by, for example, phosphorylation, acylation or glycosylation, using enzymatic methods known in the art. A kinase that can be used to phosphorylate osteopontin or its functional fragments at biologically relevant sites is casein kinase II, as described in Example IV. Other serine-threonine kinases known in the art, such as protein kinase C can also be used to phosphorylate osteopontin or its functional fragments.

The methods of the invention can be practiced using osteopontin or any of its functional fragments that possess the activity of inhibiting ectopic calcification. Fragments of osteopontin are selected, produced by methods known in the art and screened as described herein to determine their ability to inhibit ectopic calcification.

Fragments of osteopontin can be produced, for example, by enzymatic or chemical cleavage of osteopontin. Methods for enzymatic and chemical cleavage and for purification of the resultant protein fragments are well known in the art (see, for example, Deutscher, Methods in Enzymology, Vol. 182, "Guide to Protein Purification," San Diego: Academic Press, Inc. (1990), which is incorporated herein by reference). As an example, osteopontin contains a thrombin cleavage site between Arg169 and Ser170. Either the N-terminal cleavage fragment or the C-terminal cleavage fragment of osteopontin can be used in the methods of the invention.

Fragments of osteopontin can also be produced by chemical or recombinant synthesis of peptides that have substantially the sequence of osteopontin. For example, peptide libraries spanning overlapping sequences of osteopontin can be produced using methods known in the art and screened for their functional activity as described herein. Additionally, fragments corresponding to the N-terminal thrombin cleavage fragment or the C-terminal thrombin cleavage fragment of osteopontin can be recombinantly produced, as described by Smith et al., supra 271:28485-28491 (1996) and used in the methods of the invention.

As disclosed herein, osteopontin can inhibit ectopic calcification by directly adsorbing to and inhibiting apatite crystal growth and formation. Therefore, functional fragments of osteopontin can be selected based on their predicted ability to bind to calcium or calcium deposits. Regions that are contemplated to bind calcium include the aspartic acid rich sequence and the calcium binding homology domain. Therefore, a functional fragment of osteopontin can include, for example, substantially the sequence of the aspartic-rich calcium binding domain, DDMDDEDDDD (SEQ ID NO: 3) or include substantially the sequence of the calcium binding homology domain, DWDSRGKDSYET (SEQ ID NO: 4).

Additionally, as disclosed herein, phosphorylation can regulate the ability of osteopontin to inhibit ectopic calcification. Therefore, functional fragments of osteopontin can be selected by the presence of phosphorylation consensus sequences. A functional fragment of osteopontin can to chosen to include, for example, substantially the sequence of the casein kinase II phosphorylation consensus region, SGSSEEK (SEQ ID NO: 5), or the C-terminal heparin binding homology domain SKEEDKHLKFRISHELDSASSEVN (SEQ ID NO: 6), which contains three conserved sites of serine phosphorylation. A functional fragment of osteopontin can alternatively or additionally include the alternatively spliced domain, NAVSSEETNDFKQE (SEQ ID NO: 7), which contains two sites of serine phosphorylation. Additional sites of serine and threonine phosphorylation are described, for example, by Sorensen et al., Bioc. Biophys. Res. Comm. 198:200-205 (1994), incorporated herein by reference. A functional fragment of osteopontin can include one or several of these phosphorylated residues together with flanking amino acids.

Fragments of osteopontin having the ability to inhibit ectopic activity include regions of the molecule that are highly conserved among species. Regions within human osteopontin with high sequence conservation are presented, for example, in Giachelli et al., supra (1995). For example, a functional fragment can include the highly conserved sequence SDESHHSDESDE (SEQ ID NO: 8). A functional fragment of osteopontin can also include the conserved cell adhesion domain, DGRGDSVAYG (SEQ ID NO: 9) or the heparin binding homology domain RKKRSKKFRR (SEQ ID NO: 10).

If desired, such as to optimize their functional activity, selectivity, stability or bioavailability, osteopontin or a functional fragment thereof can be modified to include D-stereoisomers, non-naturally occurring amino acids, and amino acid analogs and mimetics. Examples of modified amino acids are presented in Sawyer, Peptide Based Drug Design, ACS, Washington (1995) and Gross and Meienhofer, The Peptides: Analysis, Synthesis. Biology, Academic Press, Inc., New York (1983), both of which are incorporated herein by reference.

If desired, one or more phosphorylated serine or threonine residues can be substituted by negatively charged amino acids, such as glutamic acid or aspartic acid. Such a modification can be advantageously made to reduce the susceptibility of osteopontin or a functional fragment to inactivation by phosphatases.

The ability of osteopontin or a fragment selected and prepared as described above to inhibit ectopic calcification can be assayed by a variety of in vitro and in vivo assays known in the art or described herein. For example, as described in Example I, cultured vascular cells, such as bovine aortic smooth muscle cells, form calcified deposits in a time-dependent manner when treated with calcification medium containing .beta.-glycerophosphate. Additionally, as described in Example III, human vascular smooth muscle cells form calcified deposits in the presence of elevated levels of inorganic phosphate. Other culture systems for assaying the efficacy of osteopontin or a functional fragment thereof in inhibiting ectopic calcification can be determined by those skilled in the art. For example, osteopontin can be assayed using cells or tissues derived from other sites in the body where ectopic calcification occurs including, for example, viscera, skin, and endothelial cells.

The amount or extent of calcification prior to and following administering osteopontin or a functional fragment can be determined using such culture systems, either qualitatively by a visual or histochemical assessment, or by more quantitative methods. For example, calcified deposits can be detected visually as opaque areas by light microscopy and as black areas by von Kossa staining. The amount or extent of calcification can also be quantitatively assessed by the method described by Jono et al., Arterioscler. Thromb. Vasc. Biol. 17:1135-1142 (1997), incorporated herein by reference, or by using a commercially available calorimetric kit such as the Calcium Kit available from Sigma. Alternatively, the amount or extent of calcification can also be quantitatively assessed using known methods of atomic absorption spectroscopy.

As described in Examples I and III, the calcified deposits observed in cultured vascular smooth muscle cells, as assessed by histochemical, ultrastructural and electron diffraction analysis, can resemble the apatite deposits present at sites of ectopic calcification. Therefore, the ability of osteopontin or a functional fragment thereof to inhibit the deposition of calcium by cultured cells, in comparison with a vehicle or protein control, is an accurate indicator of its ability to inhibit ectopic calcification in an individual.

The ability of osteopontin or a functional fragment thereof to inhibit ectopic calcification can also be tested in animal models known in the art to be reliable indicators of the corresponding human pathology. For example, ectopic calcification can be induced by the subcutaneous or circulatory implantation of bioprosthetic valves, such as porcine, sheep or bovine valves, into animals as described, for example, in Vyavahare et al., supra (1997). A reduction in the amount or rate of valve calcification by administration of osteopontin or a functional fragment can be detected, and is a measure of the functional activity of the preparation.

Similarly, animal models that are reliable indicators of human atherosclerosis, renal failure, hyperphosphatemia, diabetes, age-related vascular calcification and other conditions associated with ectopic calcification are known in the art. For example, topical and systemic calciphylaxis, calcinosis and calcergy, which are experimental models of ectopic calcification are described, for example, in Bargmann, J. Rheumatology 22:5-6 (1995), Lian et al., Calcified Tissue International, 35:555-561 (1983) and Boivin et al., Cell and Tissue Res. 247:525-532 (1987). An experimental model of calcification of the vessel wall is described, for example, by Yamaguchi et al., Exp. Path. 25:185-190 (1984).

A preferred animal model for examining ectopic calcification and the effect of osteopontin preparations is an osteopontin-deficient mouse, described by Liaw et al., J. Clin. Invest. 101:1468-1478 (1998), incorporated herein by reference, in which, as described in Example V, ectopic calcification is enhanced compared to wild-type control animals.

Medical imaging techniques known in the art, such as magnetic resonance imaging, X-ray imaging, computed tomography and ultrasonography, can be used to assess the efficacy of osteopontin or a functional fragment thereof in inhibiting ectopic calcification in either a human or an animal. For example, the presence and extent of calcium deposits within vessels can be determined by the intravascular ultrasound imaging method described by Fitzgerald et al., Circulation 86:64-70 (1994), incorporated herein by reference. A decrease in the amount or extent of ectopic calcification can readily be identified and is indicative of the therapeutic efficacy of osteopontin or a functional fragment thereof.

Osteopontin or its functional fragments, assayed for their functional activity as described above, are administered to an individual in a therapeutically effective amount to inhibit ectopic calcification. Appropriate formulations, dosages and routes of delivery for administering osteopontin or a functional fragment are well known to those skilled in the art and can be determined for human patients, for example, from animal models as described previously. The dosage of osteopontin or a functional fragment thereof required to be therapeutically effective can depend, for example, on such factors as the extent of calcification, the site of calcification, the route and form of administration, the bio-active half-life of the molecule being administered, the weight and condition of the individual, and previous or concurrent therapies. The appropriate amount considered to be a therapeutically effective dose for a particular application of the method can be determined by those skilled in the art, using the guidance provided herein. One skilled in the art will recognize that the condition of the patient needs to be monitored throughout the course of therapy and that the amount of the composition that is administered can be adjusted accordingly.

For treating humans, a therapeutically effective amount of osteopontin or its functional fragments can be, for example, between about 10 .mu.g/kg to 500 mg/kg body weight, for example, between about 0.1 mg/kg to 100 mg/kg, or between about 1 mg/kg to 50 mg/kg, depending on the treatment regimen. For example, if osteopontin or a functional fragment is administered several times a day, or once a day, or once every several days, a lower dose would be needed than if osteopontin or a functional fragment were administered only once, or once a week, or once every several weeks. Similarly, formulations that allow for timed-release of osteopontin would provide for the continuous release of a smaller amount of osteopontin than would be administered as a single bolus dose.

Osteopontin or a functional fragment can be delivered systemically, such as intravenously or intraarterially, to inhibit ectopic calcification throughout the body. Osteopontin or a functional fragment can also be administered locally at a site known to contain or predicted to develop ectopic calcification. Such a site can be, for example, an atherosclerotic plaque, a segment of artery undergoing angioplasty or the site of prosthetic implantation. Appropriate sites for administration of osteopontin and its functional fragments can be determined by those skilled in the art depending on the clinical indications of the individual being treated and whether or not the individual is concurrently undergoing invasive surgery.

Administration of osteopontin or a functional fragment can be achieved using various formulations of osteopontin. If desired, osteopontin can be administered as a solution or suspension together with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can be, for example, water, sodium phosphate buffer, phosphate buffered saline, normal saline or Ringer's solution or other physiologically buffered saline, or other solvent or vehicle such as a glycol, glycerol, an oil such as olive oil or an injectable organic ester.

A pharmaceutically acceptable carrier can additionally contain physiologically acceptable compounds that act, for example, to stabilize or increase the absorption of osteopontin or a functional fragment. Such physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextrans; antioxidants such as ascorbic acid or glutathione; chelating agents such as EDTA, which disrupts microbial membranes; divalent metal ions such as calcium or magnesium; low molecular weight proteins; lipids or liposomes; or other stabilizers or excipients. Osteopontin can also be formulated with a material such as a biodegradable polymer or a micropump that provides for controlled slow release of the molecule. Additionally, osteopontin can be formulated with a molecule, such as a phosphatase inhibitor, that reduces or inhibits dephosphorylation of osteopontin.

Osteopontin or a functional fragment can also be expressed from cells that have been genetically modified to express the protein. Expression of osteopontin from a genetically modified cell provides the advantage that sustained localized or systemic expression of the protein can occur, thus obviating the need for repeated administrations.

Methods for recombinantly expressing proteins in a variety of mammalian cells for therapeutic purposes are known in the art and are described, for example, in Lee et al., Transfusion Medicine II 9:91-113 (1995), which is incorporated herein by reference. Types of cells that are particularly amenable to genetic manipulation include, for example, hematopoietic stem cells, hepatocytes, vascular endothelial cells, keratinocytes, myoblasts, fibroblasts and lymphocytes.

A nucleic acid encoding osteopontin or a functional fragment can be operatively linked to a promoter sequence, which can provide constitutive or, if desired, inducible expression of appropriate levels of the encoded protein. Suitable promoter sequences for a particular application of the method can be determined by those skilled in the art and will depend, for example, on the cell type and the desired osteopontin expression level.

The nucleic acid encoding osteopontin or a functional fragment thereof can be inserted into a mammalian expression vector and introduced into cells by a variety of methods known in the art (see, for example, Sambrook et al., supra (1989); and Ausubel et al., supra (1994)). Such methods include, for example, transfection, lipofection, electroporation and infection with recombinant vectors. Infection with viral vectors such as retrovirus, adenovirus or adenovirus-associated vectors is particularly useful for genetically modifying a cell. A nucleic acid molecule also can be introduced into a cell using known methods that do not require the initial introduction of the nucleic acid sequence into a vector.

In one embodiment of the invention, a prosthetic device can be contacted with osteopontin or a functional fragment thereof. Contacting a prosthetic device with osteopontin or a functional fragment will effectively prevent or reduce ectopic calcification of the prosthetic device, preventing failure of the device and the need for premature replacement. The prosthetic device can be contacted with osteopontin or a functional fragment either prior to, during or following implantation into an individual, as needed.

Osteopontin or a functional fragment can contact a prosthetic device by attaching the molecule either covalently or non-covalently to the prosthetic device. An appropriate attachment method for a particular application of the method can be determined by those skilled in the art. Those skilled in the art know that an appropriate attachment method is compatible with implantation of the prosthetic device in humans and, accordingly, will not cause unacceptable toxicity or immunological rejection. Additionally, an appropriate attachment method will enhance or not significantly reduce the ability of osteopontin or a functional fragment thereof to inhibit ectopic calcification of the prosthetic device and the surrounding tissue.

Methods for covalently attaching proteins to polymers, metals and tissues are known in the art. For example, osteopontin can be attached to the prosthetic device using chemical cross-linking. Chemical cross-linking agents include, for example, glutaraldehyde and other aldehydes. Cross-linking agents that link osteopontin or a functional fragment thereof to a prosthetic device through either a reactive amino acid group, a carbohydrate moiety, or an added synthetic moiety are known in the art. Such agents and methods are described, for example, in Hermason, Bioconjugate Techniques, Academic Press, San Diego (1996), which is incorporated herein by reference. These methods can be used to contact a prosthetic device with a therapeutically effective amount of osteopontin or a functional fragment thereof.

Osteopontin can also be attached non-covalently to the prosthetic device by, for example, adsorption to the surface of the prosthetic device. A solution or suspension containing osteopontin or a functional fragment thereof, together with a pharmaceutically acceptable carrier, if desired, can be coated onto the prosthetic device in a therapeutically effective amount.

To provide sustained delivery of osteopontin or a functional fragment, a prosthetic device can also be contacted with osteopontin or a functional fragment thereof produced by cells attached to the prosthetic device. Such cells can be seeded onto the prosthetic device and expanded either ex vivo or in vivo. Appropriate cells include cells that normally produce and secrete osteopontin including, for example, macrophages, smooth muscle cells or endothelial cells. Additionally, cells that have been genetically modified to produce osteopontin or a functional fragment thereof including, for example, endothelial cells and fibroblasts, can be attached to the prosthetic device. The cells that are attached to the prosthetic device are preferably either derived from the individual receiving the prosthetic implant, or from an immunologically matched individual to reduce the likelihood of rejection of the implant.

The ability of osteopontin or a functional fragment that contacts a prosthetic device to inhibit ectopic calcification can be determined by various methods known in the art. One such method is to implant the prosthetic device into animals and measure calcium deposition, as described in Example V, in response to administration of osteopontin or a functional fragment thereof. Either a decrease in the rate or the amount of calcium deposition at the site of the explant is indicative of the therapeutic efficacy of the composition.
 

Claim 1 of 2 Claims

1. A method of inhibiting ectopic calcification in an individual, comprising administering to said individual a therapeutically effective amount of osteopontin or a functional fragment thereof, wherein said osteopontin or functional fragment is produced by endothelial cells or macrophages attached to a prosthetic device at a site of ectopic calcification, and wherein said amount of osteopontin or functional fragment is sufficient to inhibit ectopic calcification in said individual.

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