Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

• Kidney stones represent a widespread, painful medical problem that is believed to have increased over the last few decades, particularly among Caucasian males in the United States and other predominantly Caucasian-populated nations.
• the most common type of kidney stone, or renal stone is idiopathic in origin and generally calcareous.
• the calcium oxalate biomineralization which is believed to cause renal stones contributes significantly to the cost of health care in the United States.
• Problems associated with renal stones not only include the perception of pain in the afflicted patient, but also mechanical irritation and compromise of renal tissue, back-pressure from restricted urine flow, and risk of infection due to mechanical irritation or back-pressure or to the mere presence of a foreign body in the kidney.
• kidney stones lithotripsy, chemical irrigation for partial or complete dissolution, surgical interventions and other techniques. Lithotripsy alone is performed on about 500,000 residents of the United States every year, and the costs involved in this lithotripsy medical care and concomitant lost productivity are enormous. Certain citric acid and citrate derivative pharmaceutical compositions have been developed to solubilize the salts and dissolve calcareous formations. However, a need remains for a simple, noninvasive, outpatient approach for inhibiting the growth of kidney stones and discouraging recurrence on a maintenance basis
• the invention features novel phosphopeptides that are capable of inhibiting the formation and growth of calcium oxalate- and/or hydroxyapatite-containing crystals.
• Certain phosphopeptides are comprised of in the range of 10 - 20 amino acids am consist of the following consensus sequences: NNNNNNNTNNNNNNpNNN (SEQ ID NO: 6); NpNNNNNNONfNNNNNpNNN (SEQ ID NO: 7); NNNN P NNNNNNNNN P NNN (SEQ ID NO: 8), and NpNNN P NNNNNNNNN P NNN (SEQ ID NO: 9).
• the phosphorylated residue(s) may include phosphoserine, phosphotyrosine, or phosphothreonine.
• Th phosphorylated residues may be contiguous and adjacent to acidic amino acids.
• Other phosphopeptides are comprised of at least about 25% of acidic amino acids (such aspartic acid, glutamic acidor gamma carboxyglutamic acid), no more than about 10% basic amino acids (such as lysine or arginine) and at least about 15% phosphorylated amino acids, such as phosphoserine, phosphothreonine or phosphotyrosine.
• Still other phosphopeptides are phosphorylated forms of SHESTEQSD AIDS AEK (SEQ ID NO: 1), including SHESTEQSDAIDpSAEK (SEQ ID NO: 2), pSHESTEQSDAIDpSAEK (SEQ ID NO: 3) SHEpSTEQSDAIDpSAEK (SEQ ID NO: 4) and pSHEpSTEQSDAEDpSAEK (SEQ ID NO: 5).
• the invention features pharmaceutical preparations comprising a phosphopeptide and a pharmaceutically acceptable carrier.
• the invention features methods for treating of preventing kidney stone formation or growth, as well as other conditions that result from aberrant calcium oxalate monohydrate (COM) or hydroxyapatite (HA) containing crystals in a subject, comprising administering to the subject an effective amount of an inhibitory phosphopeptide.
• COM calcium oxalate monohydrate
• HA hydroxyapatite
• Figure 1 shows Scanning Electron Micrograph (SEM) images of calcium oxalate monohydrate (COM) crystals formed in the presence of : a) no peptide, b) 2 ⁇ g/ml of PO (SEQ ID NO. 1), c) 20 ⁇ g/ml PO (SEQ ID NO. I) 5 d) 2 ⁇ g/ml of Pl (SEQ ID NO. 2), e) 20 ⁇ g/ml of Pl (SEQ ID NO. 2), f) 2 ⁇ g/ml of P3 (SEQ ID NO. 5, and g) 20 ⁇ g/ml of P3 (SEQ ID NO. 5).
• SEM Scanning Electron Micrograph
• Pl inhibited crystal growth perpendicular to the ⁇ 100 ⁇ faces
• P3 inhibited crystal growth perpendicular to both ⁇ 100 ⁇ and ⁇ 120 ⁇ faces.
• the degree of inhibition of crystal growth increased with the number of phosphate groups on the peptide.
• Figures 2A and 2B show Scanning Electron Micrograph (SEM) images of calcium oxylate (COM) crystals formed in the presence of : A) 2 ⁇ g/ml of P2A (SEQ ID NO. 3) and B) 2 ⁇ g/ml P2B (SEQ ID NO. 4). Both phosphopeptides inhibited crystal growth perpendicular to the ⁇ 100 ⁇ faces. The degree of inhibition of crystal growth increased with the number of phosphate groups on the peptide (i.e. P2A and P2B more effectively inhibited crystal growth as compared to Pl, but less effectively inhibited crystal growth as compared to P3.)
• Figure 3 shows scanning confocal fluorescence microscopy images of AlexaFluor- 488-labeled peptides added to preformed COM crystals. Images shown are optical sections taken approximately half-way through the thickness of the crystals. Panels a, b and c are combined red (crystal) and green (peptide) green channel images; panels d, e and fare green-channel images converted to grey scale. Adsorption by PO (SEQ ID NO. 1) is shown in panels a and d. Adsorption by Pl (SEQ ID NO. 2) is shown in panels b and e. Adsorption by P3 (SEQ ID NO. 5) is shown in panels c and f.
• PO SEQ ID NO. 1
• Pl Adsorption by Pl
• P3 SEQ ID NO. 5
• Figure 4 provides molecular dynamic simulations of PO (SEQ ID NO. 1), Pl (SEQ ID NO. 2) and P3 (SEQ ID NO. 5) to ⁇ 100 ⁇ face of COM: peptide centre-of-mass calculations.
• the inset shows the conformation of P3 at 15 nsec.
• Figure 5 provides molecular dynamic simulations of PO (SEQ ID NO. 1), Pl (SEQ ID NO. 2) and P3 (SEQ ID NO. 5) to ⁇ 100 ⁇ face of COM: amino acid centre-of-mass calculations A. z-axis coordinate and B. root mean square deviation from initial position.
• the instant inventions are based at least in part on studies of the interaction between COM crystals and the synthetic peptide, PO (SEQ ID NO. 1).
• the peptide has been synthesized in forms containing 0, 1 , 2 or 3 phosphoserines. Scanning confocal microscopy of fluorescence-tagged peptides was used to determine the faces of COM with which the peptides interact. Scanning electron microscopy was used to characterize the effects of these peptides on COM growth habit (crystal size and shape). Finally molecular dynamics was used to simulate the interactions of the peptides with the ⁇ 100 ⁇ lattice plane of the COM crystal.
• Figures 1 and 2 show Scanning Electron Micrograph (SEM) images of calcium oxalate (COM) crystals formed in the presence of no peptide ( Figures Ia) and 2 or 20 g/ml of unphosphorylated SEQ ID NO. 1 ( Figures Ib and Ic) and certain phosphopeptides ( Figures ld-g and 2). Control crystals (no peptide) were penetration twins with ⁇ 100 ⁇ , ⁇ 010 ⁇ and ⁇ 121 ⁇ faces developed (Fig. Ia).
• Crystals grown in the presence of PO were very similar to controls (Fig. Ib and Ic). Those grown in the presence of Pl were of normal length ( ⁇ 001> directions) and thickness ( ⁇ 010> directions) but were decreased in width ( ⁇ 100>) (Fig. Id and Ie). Growth of COM in the presence of P2A and P2B was less than in the presence of Pl (Fig. 2A and 2B). Growth of COM in the presence of P3 had the most profound effects on growth habit. At 2 ⁇ g/ml, crystals were of normal length but reduced in width; no twin axis was apparent and interfacial edges (e.g., ⁇ 100 ⁇ / ⁇ 121 ⁇ were rounded ( Figure If)). At 20 ⁇ g/ml P3 crystals were reduced in length as well as width and ⁇ 100 ⁇ faces were not apparent (Figure Ig).
• Adsorption refers to a noncovalent attachment of an inhibitory phosphopeptide to a crystal, for example, through hydrogen bonding, van der Waal's forces, polar attraction, electrostatic forces (i.e., through ionic bonding), or the like.
• Amino acid is used herein to refer to natural or synthetic molecules including D or L optical isomers, analogs and petidomimetics.
• Antibody is used herein to refer to binding molecules including immunoglobulins and immunologically active portions thereof, i.e., molecules that contain an antigen binding site, including Fab, Fab', F(ab * ) 2 , scFv, Fv, dsFv, diabodies, minibodies, Fd fragments and single chain antibodies (SCAs).
• Constant amino acid substitution refers to a replacement of one amino acid with another having a similar side chain as defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic siA "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
• amino acids with basic side chains e.g., lysine, arginine, histidine
• acidic side chains e.g., aspartic acid, glutamic acid
• uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
• nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
• beta-branched side chains e.g., threonine, valine, isoleucine
• aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
• a predicted nonessential amino acid residue in a natural immunoglobulin can be preferably replaced with another amino acid residue from the same side chain family.
• mutations can be introduced randomly along all or part of a natural immunoglobulin coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity.
• Consisting essentially of is a transitional phrase that excludes from the scope of any succeeding recitation any other component, step or procedure, excepting those that are not essential to operability.
• Crystal or “crystalline,” as used herein refers to a plurality of atoms or molecules that are physically associated with each other in a regular manner and that involve alignment along one or more axes.
• “Degenerate” refers to codons that differ in at least one nucleotide from a reference nucleic acid, but encode the same amino acids as the reference nucleic acid. For example, codons specified by the triplets "UCU”, “UCC”, “UCA”, and “UCG” are degenerate with respect to each other since all four of these codons encode the amino acid serine.
• Ectopic calcification refers to aberrant deposition of calcium within the body. Ectopic calcification is inclusive of the deposition of calcium in renal tubules and urine that results in the formation of primarily calcium oxalate-containing kidney stones.
• Hybridization refers to the binding of complementary strands of nucleic acid (i.e., sense:antisense strands or probe :target-DNA) to each other through hydrogen bonds, similar to the bonds that naturally occur in chromosomal DNA. Stringency levels used to hybridize a given probe with target-DNA can be readily varied by those of skill in the art. "High stringency hybridization” refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65 0 C, for example, if a hybrid is not stable in 0.018M NaCl at 65 0 C, it will not be stable under high stringency conditions, as contemplated herein.
• High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5 x Denhart's solution, 5 x SSPE, 0.2% SDS at 42 0 C, followed by washing in 0.1 x SSPE, and 0.1% SDS at 65 0 C.
• Inhibit or “inhibition,” means preventing, retarding, or reversing formation, growth or deposition of a crystal.
• Nucleic acid is used herein to refer to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single and double-stranded polynucleotides. "Percent identity” or “percent similarity” indicates the degree of sameness between two molecules, e.g. peptides or nucleic acids.
• the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
• the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
• the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
• the percent identity between the two sequences is a function of the number of • identical positions shared by the sequences and the percent homology between two sequences is a function of the number of conserved positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
• the comparison of sequences and determination of percent identity and/or homology between two sequences can be accomplished using a mathematical algorithm.
• the percent identity between two amino acid sequences is determined using the Needleman and
• the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available on the world wide web with the extension gcg.com), using a NWSgapdna CMP matrix and a gap weight of 40, 50, 60, 70; or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
• a particularly preferred set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5.
• the percent identity and/or homology between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
• treating refers to a reduction in the severity and/or frequency of symptoms
• An inhibitory phosphopeptide as referenced throughout the specification and claims is intended to refer to a peptide (i.e. a complex of at least two amino acids) that comprises at least one phosphate group and is capable of inhibiting the formation or growth of calcium oxalate or hydroxyapatite crystals.
• Appropriate inhibitory phosphopeptides may consist of the consensus sequences NNNNTSINNNNNNNNpNNN (SEQ ID NO: 6); N P NNNNNNNNNN P NNN (SEQ ID NO: 7); NNNN P NNNNNNNNN P NNN (SEQ ID NO: 8), or N P NNN P NNNNNNNNN P NNN (SEQ ID NO: 9).
• the amino acids (N) forming all or a part of a peptide may be any of the twenty conventional, naturally occurring amino acids, i.e., alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (T), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y).
• a phosphopeptide may comprise, 1, 2, 3, 4, 5, 6, or more phosphorylated residues.
• the phosphorylated residue(s) (-- Np--) may include phosphoserine, phosphotyrosine, or phosphothreonine.
• the phosphorylated residues may be contiguous and adjacent to acidic amino acids.
• Phosphopeptides that are comprised of at least about 25% of acidic amino acids (such as aspartic acid or glutamic acid), no more than about 10% basic amino acids (such as lysine or arginine) and at least about 15% phosphorylated amino acids, such as phosphoserine and phosphothreonine appear to better mold themselves to crystal faces and thus may be more effective inhibitors of crystal growth.
• nonpolar amino acids such as valine and leucine do not seem to affect activity.
• Certain inhibitory phosphopeptides are phosphorylated forms of
• SHESTEQSDAIDSAEK SEQ TD NO: 1
• SHESTEQSDAIDpSAEK SEQ ID NO: 2
• pSHESTEQSDAIDpSAEK SEQ ID NO: 3
• SHEpSTEQSDAIDpSAEK SEQ ID NO: 4
• pSHEpSTEQSDAIDpSAEK SEQ ID NO: 5
• Any amino acid in the above sequences may be replaced by an isomer or analog of a conventional amino acid (e.g., a D-amino acid), non-protein amino acids post- translationally modified amino acids enzymatically modified amino acid, a construct or structure designed to mimic an amino acid (e.g., an ⁇ , ⁇ -disubstituted amino acid, N-alkyl amino acid, lactic acid, ⁇ -alanine, naphthylalanine, 3-pyridylalanine, 4-hydroxyproline, O- phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and nor-leucine).
• a conventional amino acid e.g., a D-amino acid
• non-protein amino acids post- translationally modified amino acids enzymatically modified amino acid e.g., a construct or structure designed to mimic an amino acid (e.g., an ⁇ , ⁇
• Phosphopeptide compounds herein also include compounds wherein the naturally occurring amide --CONH-- linkage is replaced at one or more sites within the peptide backbone with a non-conventional linkage such as an N-substituted amide, ester, thioamide, retropeptide (--NHCO-), retrothioamide (--NHCS-), sulfonamido (--SO 2 NH-), and/or peptoid (N-substituted glycine) linkage.
• phosphopeptide molecules herein include pseudopeptides and peptidomimetics.
• the phosphopeptides of this invention can be (a) naturally occurring, (b) produced by chemical synthesis, (c) produced by recombinant DNA technology, (d) produced by biochemical or enzymatic fragmentation of larger molecules, (e) produced by methods resulting from a combination of methods (a) through (d) listed above, or (f) produced by any other means for producing peptides.
• Preferred inhibitory phosphopeptides include, for example, polypeptides having substantially the same amino acid sequence as any one of SEQ ID NOs: 2-5.
• a phosphopeptide disclosed herein may be modified to include an addition, deletion or replacement of one or more amino acids. Suitable replacements can include isolated conservative amino acid substititutions, such as replacement of a leucine with isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
• an inhibitory phosphopeptide can be modified, for example, 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.
• phosphopeptides should consist of at least 5 amino acids, but preferably less than 20 amino acids is preferred for use as a drug. Based on the instant disclosure of particular inhibitory phosphopeptides, one of skill in the art could empirically determine optimally sized phosphopeptides, which may for example comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acid residues.
• Phosphopeptides with modified amino and/or carboxy termini are also envisioned.
• Amino terminus modifications include methylation (e.g., --NHCH 3 or -N(CHa) 2 ), acetylation (e.g., with acetic acid or a halogenated derivative thereof such as ⁇ -chloroacetic acid, ⁇ -bromoacetic acid, or ⁇ -iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO-- or sulfonyl functionality defined by R-SO 2 -, where R is selected from alkyl, aryl, heteroaryl, alkyl aryl, and the like, and similar groups.
• the N-terminus may be acetylated.
• An N-terminal glycine may be acetylated to yield N-acetylglycine (AcG).
• Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints.
• C-terminal functional groups of the compounds of the present invention include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.
• Heterocyclic groups preferably contain one or more nitrogen, oxygen, and/or sulfur heteroatoms.
• groups include the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g.
• morpholino oxazolyl
• piperazinyl e.g., 1-piperazinyl
• piperidyl e.g., 1- piperidyl, piperidino
• pyranyl pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g. thiomorpholino), and triazolyl.
• These heterocyclic groups can be substituted or unsubstituted. Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.
• the phosphopeptide compounds of the invention may also serve as structural models for non-peptidic compounds with similar biological activity.
• Those of skill in the art recognize that a variety of techniques are available for constructing compounds with the same or similar desired biological activity as the lead peptide compound, but with more favorable activity than the lead with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis [See, Morgan and Gainor (1989) Ann. Rep. Med. Chem. 24:243- 252]. These techniques include replacing the peptide backbone with a backbone composed of phosphonates, amidates, carbamates, sulfonamides, secondary amines, and N- methylamino acids.
• inhibitory phosphopeptide can be prepared or obtained by methods known in the art including, for example, purification from an appropriate biological source or by chemical synthesis. In addition to synthesis, inhibitory phosphopeptides can be produced, for example, by enzymatic or chemical cleavage of larger sequences. 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).
• inhibitory phosphopeptides can be modified in a physiologically relevant manner by, for example, further phosphorylation, acylation or glycosylation, using enzymatic methods known in the art.
• a kinase that can be used to phosphorylate an inhibitory phosphopeptide at biologically relevant sites is casein kinase II.
• Other serine-threonine kinases known in the art, such as protein kinase C can also be used to phosphorylate.
• An inhibitory phosphopeptide 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.
• SEQ ID NO: 10 TCT CAC GAA TCT ACC GAA CAG TCT GAC GCT ATC GAC TCT GCT GAA AAA), as shown below.
• S H E S T E Q S D A I D S A E K SEQ ID NO: 10
• nucleic acids encoding phosphopeptides that, by virtue of the degeneracy of the genetic code, do not necessarily hybridize to the invention nucleic acids under specified hybridization conditions.
• Preferred nucleic acids encoding the phosphopeptides are comprised of nucleotides that encode substantially the same amino acid sequence as set forth in SEQ ID NOs: 2-5.
• Peptide libraries spanning overlapping sequences can be produced using methods known in the art and screened for their functional activity as described herein. 4.
• an inhibitory phosphopeptide selected and prepared as described above to inhibit COM or HA See e.g., Pampena, D. et al. s (2004) Biochem. J. 378: 1083-1087
• crystal formation or growth can be assayed by a variety of in vitro and in vivo assays known in the art or described herein.
• cultured vascular cells such as bovine aortic smooth muscle cells, form calcified deposits in a time-dependent manner when treated with calcification medium containing ⁇ -glycerophosphate.
• human vascular smooth muscle cells form calcified deposits in the presence of elevated levels of inorganic phosphate.
• an inhibitory phosphopeptide 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 ectopic calcification prior to and following administering an inhibitory phosphopeptide can be determined using such culture systems, either qualitatively by a visual or histochemical assessment, or by more quantitative methods.
• 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 ectopic calcification can also be quantitatively assessed by the method described by Jono et al., Arterioscler.
• ectopic calcification can also be quantitatively assessed using known methods of atomic absorption spectroscopy.
• the ability of an inhibitory phosphopeptide to inhibit ectopic calcification can also be tested in animal models known in the art to be reliable indicators of the corresponding human pathology.
• ectopic calcification can be induced by the subcutaneous or circulatory implantation of bioprosthetic valves, such as porcine or bovine valves, into animals. A reduction in the amount or rate of valve calcification by administration of an inhibitory phosphopeptide can be detected, and is a measure of the functional activity of the preparation.
• Medical imaging techniques known in the art can be used to assess the efficacy of an inhibitory phosphopeptide in inhibiting ectopic calcification in either a human or an animal.
• 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 an inhibitory phosphopeptide.
• inhibitory phosphopeptides 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 the inhibitory phosphopeptide to be administered.
• 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.
• Inhibitory phosphopeptides can also be formulated with a material such as a biodegradable polymer or a micropump that provides for controlled slow release of the molecule.
• inhibitory phosphohopeptides can be formulated with a molecule, such as a phosphatase inhibitor, that -reduces or inhibits dephosphorylation of the inhibitory phosphopeptide.
• Inhibitory phosph ⁇ peptides can also be expressed from cells that have been genetically modified to express the protein. Expression of an inhibitory phosphopeptide 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 may be used to administer an inhibitory phosphopeptide. These 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 which may be used in conjunction with the methods provided herein include, for example, hematopoietic stem cells, hepatocytes, vascular endothelial cells, keratinocytes; myoblasts, fibroblasts and lymphocytes.
• a nucleic acid encoding an inhibitory phosphopeptide can be operatively linked to a promoter sequence, which can provide constitutive or, if desired, inducible expression of appropriate levels of the encoded inhibitory phosphopeptide.
• a promoter sequence which can provide constitutive or, if desired, inducible expression of appropriate levels of the encoded inhibitory phosphopeptide.
• 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 inhibitory phosphopeptide expression level.
• the nucleic acid encoding an inhibitory phosphopeptide 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., 1989; and Ausubel et al., 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.
• a prosthetic device can be contacted with an inhibitory phosphopeptide. Contacting a prosthetic device with an inhibitory phosphopeptide 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 an inhibitory phosphopeptide either prior to, during or following implantation into an individual, as needed.
• An inhibitory phosphopeptide 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 an inhibitory phosphopeptide to inhibit ectopic calcification of the prosthetic device and the surrounding tissue.
• an inhibitory phosphopeptide 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 an inhibitory phosphopeptide 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
• An inhibitory phosphopeptide 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 an inhibitory phosphopeptide, together with a pharmaceutically acceptable carrier, if desired, can be coated onto the prosthetic device in a therapeutically effective amount.
• a prosthetic device can also be contacted with an inhibitory phosphopeptide 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 an inhibitory phosphopeptide including, for example, macrophages, smooth muscle cells or endothelial cells. Additionally, cells that have been genetically modified to produce an inhibitory phosphopeptide 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 an inhibitory phosphopeptide 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, in response to administration of an inhibitory phosphopeptide. 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.
• ectopic calcification can result from inflammation or damage to the affected tissues or can result from a systemic mineral imbalance.
• 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)).
• 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 provided herein are applicable to ectopic calcification that occurs in association with all of these conditions, as well as, for example, heart or circulatory diseases such as Arteriosclerosis, Atherosclerosis, Valve Calcifications, Calcific Aortic Stenosis, Vascular Thrombosis; Dental Diseases such as Dental Calculus (dental pulp stones), calcification of the dentinal papilla, and Salivary Gland Stones, Kidney and Bladder Stones, Gall Stones, Gout, Pancreatic Duct Stones, Adrenal Calcification, Liver Cysts, Chronic Calculous Prostatitis, Prostate Calcification, Calcification in Hemodialysis Patients, Polycystic Kidney Disease, Glomerulopathies; Eye Diseases such as Corneal Calcifications, Cataracts, Ear Diseases such as Otosclerosis, Thyroglossal cysts, Thyroid Cysts, Ovarian Cysts, Skin diseases such as Calcinosis Cutis, Skin
• 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 ovine, 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. Inhibitory phosphopeptides can be administered to an individual in a therapeutically effective amount to inhibit ectopic calcification.
• Appropriate formulations, dosages and routes of delivery for administering an inhibitory phosphopeptide 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 an inhibitory phosphopeptide 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.
• a therapeutically effective amount of an inhibitory phosphopeptide is to be administered, which is any amount deemed nontoxic but sufficient to inhibit biomineralization. If an inhibitory phosphopeptide is administered several times a day, or once a day, or once every several days, a lower dose would be needed than if an inhibitory phosphopeptide were administered only once, or once a week, or once every several weeks. Similarly, formulations that allow for timed-release of an inhibitory phosphopeptide would provide for the continuous release of a smaller amount than would be administered as a single bolus dose.
• Inhibitory phosphopeptides can be delivered systemically, such as intravenously or intraarterially, to inhibit ectopic calcification throughout the body. Inhibitory phosphopeptides 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 an inhibitory phosphopeptide 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.
• Peptides The following peptides were synthesized manually using Fmoc chemistry as previously described (Pampena, D. A. et al., (2004) Biochem J. 378: 1083-1087) and studied in all experiments:
• P2A pSHESTEQSDAIDpSAEK (SEQ ID NO. 3) - ⁇ 2 phosphate groups
• P2B SHEpSTEQSDAIDpSAEK (SEQ ID NO. 4) ⁇ 2 phosphate groups
• P3 pSHEpSTEQSDAIDpSAEK (SEQ ID NO. 5) - ⁇ 3 phosphate groups
• Residue "pS" indicates phosphoserine.
• Fluorescence labeling and solution preparation of polypeptides Peptides were labeled with the Alexa fluorochrome according to the manufacturer's recommendations. Briefly, 5 ⁇ l of AlexaFluor-488 carboxylic acid (AlexFluor-488 Invitrogen Corp) in dimethylformamide (10 ⁇ g/ ⁇ l; HPLC grade, 99.9% was added to 200 ⁇ l of peptide in phosphate-buffered saline (2.5 mg/ml) and 20 ⁇ l of 1 M disodium carbonate (Na 2 COa), pH ⁇ 8.3, and incubated for 1 h at room temperature (23°C).
• Unconjugated label was removed by extensive dialysis against Tris-buffered saline, pH 7.4, using 1-kDa dialysis tubing (Spectra/Por 3, SPECTRUM LABORATORIES, Collinso Dominques, CA). After freeze- drying, the samples were stored at -20°C. All chemicals were supplied by SIGMA- ALDRICH LTD. (Oakville, Canada) except Na 2 CO 3 , which was purchased from MERCK KgaA ⁇ Damrstadt, Germany). Amino acid analysis (Alberta Peptide Institute; University of Alberta, Edmonton, Canada) was carried out using norleucine as an internal standard to determine the yield of labeled peptides. The masses obtained were used to prepare aqueous stock solutions peptides. In addition, aqueous stock solutions of 2 mg/ml and 20 ⁇ g/ml unlabelled peptide were prepared.
• Crystallization experiments Crystallization of COM was initiated using the method previously described (Grohe, Bernd et al., (2006) J. Crystal Growth 295: 148-157). Final concentrations were 1 mM calcium nitrate, 1 mM sodium oxalate, 10 mM sodium acetate and 150 mM sodium chloride. For scanning electron microscopy, 1-ml aliquots of these solutions were added to wells of tissue-culture plates (24-well), FALCON, Becton Dickinson; Franklin Lakes, NJ) containing freshly cleaved mica disks (diameter: 9.5 mm, V-I grade, SPI SUPPLIES, Toronto, Canada).
• Scanning confocal microscopy Scanning confocal interference microscopy (SCIM; (Grohe, W.K. et al. (2006) J.Crystal Growth 295:148-157) was used to image the crystal- glass interface.
• Conventional scanning confocal microscopy SCM was used to make optical sections at higher levels of the crystal and to image fluorescence-labeled peptides. In both cases, a 63X oil-immersion objective and a 90/10 mirror as a beam-splitter were used. All procedures were carried out in the dark to avoid the effects of scattered light on crystal imaging and to prevent the fluorochrome from bleaching.
• Atomic scale simulations were performed using the GROMACS suite. The coordinates for the COM ⁇ 100 ⁇ face were taken from previously obtained experimental results. The .topology for oxalate was generated using PRODRG. Extended conformations were used as the initial peptide structure. For each simulation, peptides were oriented parallel to the crystal surface where the center of mass difference between the crystal slab and the peptide was approximately 4 nm in the direction perpendicular to the surface. The crystal slab was placed at the center of the periodic cell and constructed to be approximately 0.7 nm thick with the Ca 2+ dense layers of the ⁇ 100 ⁇ face exposed on each side.
• Three-dimensional periodic boundary conditions were defined with the size of the periodic cell being 8.7 nm x 6.2 nm in the plane of the surface and 10 nm perpendicular to the surface.
• the system was solvated with simple point charge (SPC) water and CF counter-ions were added to maintain a system net charge of zero.
• Energy minimization was performed without constraints using the steepest descent integrator for 1000 steps with an initial step size of 0.1 A.

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