WO2011005510A2 - Peptide conjugates and uses thereof - Google Patents

Abstract

The invention features compositions including a peptide conjugated to a binding group having affinity for a biocompatible calcified substrate, their use in implantable materials, and for the treatment of orthopedic conditions.

Description

PEPTIDE CONJUGATES AND USES THEREOF

Background of the Invention

Bone has a remarkable capacity for growth, regeneration, and remodeling. This capacity is largely due to the induction of osteoblasts that are recruited to sites of new bone formation. The process of recruitment remains unclear, although the immediate environment of the cells is likely to play a role via cell-matrix-osteoinductive factor -cell interactions (see Reddi, A.H. Tissue Eng. 6:351 (2000); Rezania et al, J. Orthop. Res. 17:615 (1999); Rose et ah, Biochem. Biophys. Res. Commun. 292:1 (2002); Langer, R., and Vacanti, J.P. Science 260:920 (1993); Lutolf et al., Nat. Biotechnol. 21 :513 (2003);

Zandonella, C. Nature 421 :884 (2003); Dayoub et al., Tissue Eng. 9:347 (2003); Cancedda et al., Matrix Biol. 22:81 (2003); and Baylink et al., J. Bone Miner. Res. 8(Suppl. 2):S565 (1993)). The central and first step to successful tissue engineering is the ability of cells to adhere to an extracellular material followed by the ability of the cells to differentiate, leading to the production and organization of an extracellular matrix. Tremendous effort has centered on the improvement of cell adhesion with a variety of materials. However, the immediate limitation for many polymer materials is the absence of a chemically reactive pendent chain for the easy attachment of cells, drugs, cross-linkers, or biologically active moieties (Yang et al., Tissue Eng. 7:679 (2001)). Generally, cell adhesion is a series of interactive events comprising (1) initial cell attachment, (2) cell spreading, (3) organization of an actin cytoskeleton, and (4) formation of focal adhesions (LeBaron et al., Tissue Eng. 6:85 (2000)). The attachment of the cell to the extracellular matrix is known to be exquisitely controlled by various families of adhesion receptors, including the integrins, selectins, cadherins, and immunoglobulins (Ruoslahti et al., Science 238:491 (1987); Hutmacher et al., Int. J. Periodontics Restorative Dent. 21 :49 (2001); Stock et al., Annu. Rev. Med. 52:443 (2001); and

Muschler et al., Clin. Orthop. 395:66 (2002)). Numerous skeletal and connective-tissue related disorders have been treated with engineered implants designed to elicit specific, clinically-desirable responses from living cells and tissues in a patient's body. For example, it is desirable for osteoblasts to rapidly deposit mineralized matrix on the surface of (or in close apposition to) newly implanted prostheses. Anchorage-dependent cells (such as osteoblasts) must first adhere to a surface in order to perform subsequent cellular functions (e.g., proliferation, deposition of bone tissue, etc.). The swift deposition of bone stabilizes the prosthesis and minimizes motion-induced damage to surgically traumatized tissue at the implantation site. Because cell adhesion is needed for subsequent events, methods for promoting cell adhesion are of considerable interest. The effects on cell adhesion of peptides immobilized on the surfaces of substrates have been reported.

There is a need for the development of bone graft substitutes

incorporating cell adhesion peptides which are both localized and tightly bound to the surface of the bone graft material.

Summary of the Invention

The invention features compounds including a peptide conjugated to a binding group having affinity for a biocompatible calcified substrate, their use in implantable materials, and for the treatment of orthopedic conditions.

In a first aspect, the invention features an implantable composition for correcting bone defects including a calcified substrate bound to a peptide conjugate, or a salt thereof, wherein the peptide conjugate includes a peptide, or a bioactive fragment thereof, covalently tethered to a binding group including at least one phosphonate moiety, wherein the peptide conjugate exhibits enhanced affinity for the calcified substrate. In certain embodiments, the calcified substrate includes calcium phosphate particles (e.g., a calcium phosphate particle selected from hydroxyapatite particles, tetracalcium phosphate particles, calcium hydrogen phosphate particles, calcium polyphosphate particles, tricalciura phosphate particles, octacalcium phosphate particles, calcium fluorapatite particles, and mixtures thereof).

In certain embodiments, the peptide is a cell adhesion peptide, osteoinductive peptide, morphogenic protein stimulatory peptide, or

calmodulin. The peptide can be selected from, without limitaiton, BMP-2, BMP-7, DGF-5, parathyroid hormone, LIM mineralization peptide, agonists and partial agonists of BMP-2, and calmodulin, or any other peptide described herein.

The cell adhesion peptide can be derived from a binding domain of a cell adhesion protein of an extracellular matrix (e.g., fibronectin, vitronectin, laminin, elastin, fibrinogen, collagen type I, collagen type II, or collagen type V). For example, the cell adhesion peptide can include an amino acid sequence selected from arginine-glycine-aspartate (RGD) and tyrosine-isoleucine- glycine-serine-arginine (YIGSR). In particular embodiments, the cell adhesion peptide is an α2βl collagen mimetic peptide. Exemplary α2βl collagen mimetic peptides include, without limitation, peptides including an amino acid sequence selected from DGEA, GFOGER, GLOGER, GMOGER, GLSGER, GASGER, GAOGER, and GTPGPQGI AGQRG VV (P 15), or a bioactive fragment thereof.

In certain embodiments, the implantable composition further includes a hydrogel carrier. The hydrogel carrier can be any hydrogel carrier described herein (e.g., a the hydrogel carrier including (i) a polymer selected from sodium carboxymethylcellulose, hyaluronic acid, polyvinylalcohol,

polyvinylpyrrolidone, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, and ethylcellulose, (ii) water, and (iii) a dispersing agent selected from glycerin, polyethylene glycol, N-methyl pyrrolidone, and triacetin).

In one particular embodiment, the implantable composition of the invention includes a hydrogel carrier, the hydrogel carrier including (i) a polymer selected from sodium carboxymethylcellulose, hyaluronic acid, polyvinylalcohol, polyvinylpyrrolidone, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, and ethylcellulose, (ii) water, and (iii) a dispersing agent selected from glycerin, polyethylene glycol, N-methy] pyrrolidone, and triacetin, wherein the peptide is GTPGPQGIAGQRGVV (P 15), or a bioactive fragment thereof, and the calcified substrate includes particles of anorganic bone mineral.

The implantable compositions of the invention include those in which the binding group is covalently tethered to the peptide via a linker of formula I:

G^I-(Z¹)_o-(Y¹)_u-(Z²)_s-(R₁₀)-(Z³)_t-(Y²)_v-(Z⁴)_p-G² (I). In formula (I), G¹ is a bond between the peptide and the linker; G² is a bond between the linker and the binding group; Z¹, Z², Z³, and Z⁴ each,

independently, is selected from O, S, and NR₁₁; R_n is hydrogen or a Cj_-10 alkyl group; Y¹ and Y² are each, independently, selected from carbonyl,

thiocarbonyl, sulphonyl, or phosphoryl; o, p, s, t, u, and v are each,

independently, 0 or 1 ; and R₁₀ is a C_1-Jo alkyl, a linear or branched heteroalkyl of 1 to 10 atoms, a linear or branched C_2-10 alkene, a linear or branched C_2-I0 alkyne, a C_{2 6} heterocyclyl, Cg_₁₂ aryl, C₇_₁₄ alkaryl, C₃_₁₀ alkheterocyclyl, - (CH₂CI I₂O)_qCH₂CH₂- in which q is an integer of 1 to 4, or a chemical bond linking G¹-(Z¹)_O-(Y¹)_U-(Z²)_S- to -(Z³)_t-(Y²)_V-(Z⁴)_P-G². In particular

embodiments, the binding group is covalently tethered to the peptide via an amide, a phosphodiester, an ether, an ester, a sulfonamide, a urethane, or a carbamate bond.

The implantable compositions of the invention include those in which the binding group includes a bisphosphonate. The binding group can include 1 , 2, 3, 4, 5, or more bisphosphonate moieties.

In certain embodiments, the bisphosphonate-containing binding group is described by formula (II), or a salt thereof:

(H). In formula (II), n is 0, 1, 2, 3, 4, 5, 6, 7, or 8; Y is O, S, C(O), or N(R^a); X is H, halogen, NH₂, NHR^b, OR^b, heterocyclyl, or alkheterocyclyl; and each of R^a and R^b is, independently, selected from H, C₁^ alkyl, C₂^ alkenyl, C₂^ alkynyl, C₂_ ₆ heterocyclyl, C_6-^₂ aryl, C₇_₁₄ alkaryl, C₃^₁₀ alkheterocyclyl, and C_1--7 heteroalkyl.

The implantable compositions of the invention include those in which the binding group includes an amino methylene phosphonate. The binding group can include 1, 2, 3, 4, 5, or more an amino methylene phosphonate moieties.

In certain embodiments, the amino methylene phosphonate-containing binding group is described by formula (III), or a salt thereof:

In formula (III), Q is -CH(CH₂N(CH₂PO₃H₂)₂)-, -CH(N(CH₂PO₃H₂)₂)CH₂-, -CH(CH₂N(CH₂PO₃H₂)CH₂CH₂N(CH₂PO₃H₂)₂)-, or

-CH(N(CH₂PO₃H₂)CH₂CH₂N(CI I₂PO₃II₂)₂)CH₂-; m is 0 or 1; p is 0, 1, 2, 3, 4, or 5; Y is O, S, C(O), N(R^a), or is absent; and R^a is selected from H, C₁^ alkyl, C_2^1 alkenyl, C₂^ alkynyl, C₂_₆ heterocyclyl, C₆^₂ aryl, C₇_₁₄ alkaryl, C₃^₀ alkheterocyclyl, and C₁^₇ heteroalkyl.

The implantable compositions of the invention include those in which the peptide conjugate is a compound of any of formulas (IV)-(IX).

The implantable compositions of the invention include those in which the calcified substrate is on the surface of a dental implant, a vertebral implant, a bone rod, a bone plate, a bone screw, or any implantable device, paste, or gel described herein.

In a related aspect, the invention features a peptide conjugate, or a salt thereof, wherein the peptide conjugate includes an α2βl collagen mimetic peptide, or a bioactive fragment thereof, covalently tethered to a binding group including at least one phosphonate moiety, wherein the peptide conjugate exhibits enhanced affinity for a calcified substrate. Exemplary α2βl collagen mimetic peptides include, without limitation, peptides including an amino acid sequence selected from DGEA, GFOGER, GLOGER, GMOGER, GLSGER, GASGER, GAOGER, and GTPGPQGIAGQRGVV (P 15), or a bioactive fragment thereof. In certain embodiments, the binding group is covalently tethered to the C terminus of the α2βl collagen mimetic peptide. In still other embodiments, the binding group is covalently tethered to the N terminus of the α2βl collagen mimetic peptide.

The peptide conjugates of the invention include those in which the binding group is covalently tethered to the α2βl collagen mimetic peptide via a linker of formula I:

G'-CZVCYVCZVCRIQMZ'MYVCZVG² (I).

In formula (I), G¹ is a bond between the α2βl collagen mimetic peptide and the linker; G² is a bond between the linker and the binding group; Z¹, Z², Z³, and Z⁴ each, independently, is selected from O, S, and NRn; Rn is hydrogen or a C_1-10 alkyl group; Y and Y are each, independently, selected from carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; o, p, s, t, u, and v are each,

independently, 0 or 1; and R₁₀ is a Ci_-I0 alkyl, a linear or branched heteroalkyl of 1 to 10 atoms, a linear or branched C_2-1 o alkene, a linear or branched C_2-10 alkyne, a C₂_₆ heterocyclyl, C₆-_I2 aryl, C₇_₁₄ alkaryl, C₃_₁₀ alkheterocyclyl, - (CH₂CH₂O)_qCH₂CH₂- in which q is an integer of 1 to 4, or a chemical bond linking G¹-(Z¹)_O-(Y¹)_U-(Z²)_S- to -(Z³)_Γ(Y²)_V-(Z⁴)_P-^G2- ^In particular

embodiments, the binding group is covalently tethered to the α2βl collagen mimetic peptide via an amide, a phosphodiester, an ether, an ester, a

sulfonamide, a urethane, or a carbamate bond.

The peptide conjugates of the invention include those in which the binding group includes a bisphosphonate. The binding group can include 1 , 2, 3, 4, 5, or more bisphosphonate moieties. In certain embodiments, the bisphosphonate-containing binding group is described by formula (II), or a salt thereof:

In formula (II), n is 0, 1, 2, 3, 4, 5, 6, 7, or 8; Y is O, S, C(O), or N(R^a); X is H, halogen, NH₂, NHR^b, OR^b, heterocyclyl, or alkheterocyclyl; and each of R^a and R^b is, independently, selected from H, C₁^ alkyl, C₂^ alkenyl, C_2-4 alkynyl, C_{2 6} heterocyclyl, C₆^₁₂ aryl, C₇__]4 alkaryl, C₃^₀ alkheterocyclyl, and C₁.-_/ heteroalkyl.

The peptide conjugates of the invention include those in which the binding group includes an amino methylene phosphonate. The binding group can include 1, 2, 3, 4, 5, or more an amino methylene phosphonate moieties.

In certain embodiments, the amino methylene phosphonate-containing binding group is described by formula (III), or a salt thereof:

In formula (III), Q is -CH(CH₂N(CH₂PO₃H₂)₂)-, -CH(N(CH₂PO₃H₂)₂)CH₂-, -CH(CH₂N(CI I₂PO₃H₂)CH₂CH₂N(CH₂PO₃H₂)₂)-, or

-CH(N(CH₂PO₃H₂)CH₂CH₂N(CH₂PO₃H₂)₂)CH₂-; m is 0 or 1; p is 0, 1, 2, 3, 4, or 5; Y is O, S, C(O), N(R^a), or is absent; and R^a is selected from H, C₁^ alkyl, C_2^t alkenyl, C₂^ alkynyl, C₂_₆ heterocyclyl, C₆_₁₂ aryl, C₇__]4 alkaryl, C₃_|₀ alkheterocyclyl, and Cj_₇ heteroalkyl.

The invention further features a method for correcting a bone defect in a subject by implanting into the subject an implantable composition of the invention at the site of the bone defect. The bone defect can be any type of bone defect described herein.

By "α2βl collagen mimetic peptide" is meant a synthetic peptide of from 3 to 50 amino acid residues having affinity for α2βl integrin. α2βl collagen mimetic peptides include, without limitation, peptides including the peptide sequences of any of SEQ ID NOS. 1-20: Gly-Thr-Pro-Gly-Pro-Gln- Gly-Ile-Ala-Gly-Gln-Arg-Gly-Val-Val (SEQ ID NO. 1, also known as "P-15"), Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln -Arg (SEQ ID NO: 2), Gln-Gly-Ile-Ala-Gly- GIn (SEQ ID NO: 3), Gln-Gly-Ile-Ala-Gly-Gln-Arg (SEQ ID NO: 4), Phe-Gly- Ile-Ala-Gly-Phe (SEQ ID NO: 5), Gly-Ile-Ala-Gly-Gln (SEQ HD NO: 6), GIn- GIy- Ala-Ile- Ala-Gin (SEQ TD NO: 7), Phe-Gly-Ile-Ala-Gly-Phe (SEQ ID NO:8), Cys-Gly-Ile-Ala-Gly-Cys (SEQ ID NO:9), Glu-Gly-Ile-Ala-Gly-Lys (SEQ ID NO: 10), N- Acetyl He- Ala-Ala (SEQ ID NO:11), He- Ala- .beta. Ala (SEQ ID NO: 12), N-Acetyl Ile-Ala NMe (SEQ ID NO:13), Asp-Gly-Glu-Ala (SEQ ID NO: 14), Asp-Gly-Glu-Ala-Gly-Cys (SEQ ID NO: 15), Gly-Phe-Pro*- Gly-Glu-Arg (SEQ ID NO: 16, where Pro* = hydroxyproline), Gly-Leu-Pro*- Gly-Glu-Arg (SEQ ID NO: 17, where Pro* = hydroxyproline), Gly-Met-Pro*- Gly-Glu-Arg (SEQ ID NO: 18, where Pro* = hydroxyproline), Gly-Ala-Ser- Gly-Glu-Arg (SEQ ID NO: 19), Gly-Leu-Ser-Gly-Glu-Arg (SEQ ID NO:19), Gly-Ala-Pro*-Gly-Glu-Arg (SEQ ID NO:20, where Pro* = hydroxyproline), and any other α2βl collagen mimetic peptide s described in U.S. Patent No. 7,199,103, incorporated herein by reference.

As used herein, the term "bioactive fragment" refers to fragments of a cell adhesion peptide capable of binding to any anchorage dependent cells via cell surface molecules, such as integrins, displayed on the surface of the cells. Bioactive fragments include certain chemical modifications of a peptide described herein, such as the conversion of a C-terminus carboxylate group into a bisphononate moiety.

As used herein, the term "calcified substrate" refers to a substrate that includes calcium cations and phosphate or hydrogen phosphate anions. The peptide conjugates of the invention bind to calcified substrates.

As used herein, the term "cell adhesion peptide" refers to peptides of 3 to 100 amino acid residues in length (e.g., from 3 to 80, from 3 to 60, from 3 to 50, or from 3 to 40 amino acid residues in length) which are capable of binding to epithelial cells (e.g., endothelial cells) via cell surface molecules, such as integrins, displayed on the surface of epithelial cells. As used herein, the term "enhanced affinity" refers to an increase in the binding affinity of a peptide conjugate for a calcified substrate under physiological conditions in comparison to the binding affinity of the same peptide, but lacking a phosphonate moiety, for the same calcified substrate under the same conditions.

The term "morphogenic protein stimulatory peptide (MPSP)" refers to a peptide capable of stimulating the ability of a morphogenic protein to induce tissue formation from a progenitor cell. The MPSP may have a direct or indirect effect on enhancing morphogenic protein inducing activity. MPSP include, without limitation, insulin-like growth factor I (IGF-I), fibroblast growth factor (FGF), growth hormone (GH), insulin, LIM mineralization peptide (see U.S. patent No. 7,517,866), agonists and partial agonists of BMP-2 (see U.S. patent Publication No. 20050196425, incorporated herein by reference), parathyroid hormone (PTH), and IL-6.

The term "'osteoinductive peptide" refers to peptides, such as the members of the Transforming Growth Factor-beta (TGF-beta) superfamily, which haλ'e osteoinductive properties. Osteoinductive peptides include, without limitation, peptides selected from BMP-I, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-1 1, BMP- 12, BMP- 13, BMP- 14, BMP- 15, BMP- 16, GDF-I, GDF-2, GDF-3, GDF-4, GDF-5,

GDF-6, GDF-7, GDF-8, GDF-9, GDF-10 and GDF-I l. Publications disclosing osteoinductive peptides include: OP-I and OP-2: U.S. Pat. No. 5,011,691, U.S. Pat. No. 5,266,683, Ozkaynak et al. (1990) EMBO J. 9: 2085-2093; OP-3: WO94/10203 (PCT US93/10520); BMP2, BMP3, BMP4: WO88/00205, Wozney et al. (1988) Science 242:1528-1534); BMP5 and BMP6: Celeste et al. (1991) PNAS 87: 9843-9847; Vgr-1 : Lyons et al. (1989) PNAS 86: 4554-4558; DPP: Padgett et al. (1987) Nature 325: 81-84; Vg-I : Weeks (1987) Cell 51 : 861-867; BMP-9: WO95/33830 (PCT/US95/07084); BMPlO: WO94/26893 (PCT/US94/05290); BMP-I l : WO94/26892 (PCT/US94/05288); BMP12: WO95/16035 (PCT/US94/14030); BMP-13: WO95/16035 (PCT/US94/14030); GDF-I : WO92/00382 (PCT/US91/04096) and Lee et al. (1991) PNAS 88: 4250-4254; GDF-8: WO94/21681 (PCT/US94/03019); GDF-9: WO94/15966 (PCT/US94/00685); GDF-IO: WO95/10539 (PCT/US94/11440); GDF-I l : WO96/01845 (PCT7US95/08543); BMP- 15: WO96/36710 (PCT/US96/06540); MP121 : WO96/01316 (PCT/EP95/02552); GDF-5 (CDMP-I, MP52):

WO94/15949 (PCT/US94/00657) and WO96/14335 (PCT/US94/12814) and WO93/16099 (PCT/EP93/00350); GDF-6 (CDMP-2, BMP13): WO95/01801 (PCT7US94/07762) and WO96/14335 and WO95/10635 (PCT/US94/14030); GDF-7 (CDMP-3, BMP- 12): WO95/10802 (PCT/US94/07799) and

WO95/10635 (PCT7US94/14030). Osteoinductive peptides further include fragments and variants of the peptides listed above having osteoinductive properties.

As used herein, the term "parathyroid hormone" refers to parathyroid hormone peptide, an important regulator of calcium and phosphorus

concentration in extracellular fluids, which is synthesized in vivo as a preprohormone and, after intracellular processing, is secreted as an 84 amino acid peptide, and bioactive fragments thereof. Parathyroid hormone peptides include, without limitation, hPTH(l-34), hPTH(7-31), hPTH(5-34), [NIe⁸'¹⁸, Tyr³⁴]hPTH (7-34)NH₂, [Tyr³⁴]hPTH (7-34)NH₂, and hPTH(5-36).

Except where called out separately, the terms "phosphonate" and "amino methylene phosphonate" encompass phosphonic acids and amino methylene phosphonic acids, respectively, as well as salts of phosphonic acids and amino methylene phosphonic acids. Suitable salts of the peptide conjugates of the invention include those in which one or more protons of a phosphonate group are replaced by a metal counterion (e.g., sodium, potassium, magnesium, zinc, and/or calcium), or an ammonium or

alkylammonium ion.

In the generic descriptions of compounds of this invention, the number of atoms of a particular type in a substituent group is generally given as a range. For example, an alkyl group containing from 1 to 10 carbon atoms. Reference to such a range is intended to include specific references to groups having each of the integer number of atoms within the specified range. For example, an alkyl group from 1 to 10 carbon atoms includes each of Ci, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, and C₁₀. Other numbers of atoms and other types of atoms are indicated in a similar manner.

By "C_1^1 alkyl" is meant a branched or unbranched hydrocarbon group having from 1 to 4 carbon atoms. A C_H alkyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl,

carboxyalkyl, and carboxyl groups. C₁^ alkyls include, without limitation, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclopropylmethyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, and cyclobutyl.

By "C_2^i alkenyl" is meant a branched or unbranched hydrocarbon group containing one or more double bonds and having from 2 to 4 carbon atoms. A C₂^ alkenyl may optionally include monocyclic or polycyclic rings, in which each ring desirably has from three to six members. The C₂^ alkenyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. C₂_₄ alkenyls include, without limitation, vinyl, allyl, 2-cyclopropyl-l-ethenyl, 1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl- 1-propenyl, and 2-methyl-2-propenyl.

By "C₂_₄ alkynyl" is meant a branched or unbranched hydrocarbon group containing one or more triple bonds and having from 2 to 4 carbon atoms. A C₂_₄ alkynyl may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has five or six members. The C₂-₄ alkynyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. C₂_₄ alkynyls include, Λvithout limitation, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl. By "C₂_₅ heterocyclyl" is meant a stable 5- to 7-membered monocyclic or 7- to 14-membered bicyclic heterocyclic ring which is saturated partially unsaturated or unsaturated (aromatic), and which consists of 2 to 6 carbon atoms and 1, 2, 3 or 4 heteroatoms independently selected from N, O, and S and including any bicyclic group in which any of the above-defined

heterocyclic rings is fused to a benzene ring. The heterocyclyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. The nitrogen and sulfur heteroatoms may optionally be oxidized. The heterocyclic ring may be covalently attached via any heteroatom or carbon atom which results in a stable structure, e.g., an imidazolinyl ring may be linked at either of the ring-carbon atom positions or at the nitrogen atom. A nitrogen atom in the heterocycle may optionally be quaternized. Preferably when the total number of S and O atoms in the heterocycle exceeds 1, then these heteroatoms are not adjacent to one another. Heterocycles include, without limitation, lH-indazole, 2-pyrrolidonyl, 2H,6H- 1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl, 4-piperidonyl, 4aH-carbazole, 4H- quinolizinyl, 6H-l,2,5-thiadiazinyl, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,

benzimidazalonyl, carbazolyl, 4aH-carbazolyl, b-carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl,

octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl,

oxazolidinylperimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl. phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,

pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, 4H- quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-l,2,5-thiadiazinyl, 1,2,3- thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,

thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5- triazolyl, 1,3,4-triazolyl, xanthenyl. Preferred 5 to 10 membered heterocycles include, but are not limited to, pyridinyl, pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, tetrazolyl, benzofuranyl, benzothiofuranyl, indolyl, benzimidazolyl, IH- indazolyl, oxazolidinyl, isoxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, quinolinyl, and isoquinolinyl. Preferred 5 to 6 membered heterocycles include, without limitation, pyridinyl, pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl, piperazinyl, piperidinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, and tetrazolyl.

By "C₆-_J2 aryl" is meant an aromatic group having a ring system comprised of carbon atoms with conjugated π electrons (e.g., phenyl). The aryl group has from 6 to 12 carbon atoms. Aryl groups may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has five or six members. The aryl group may be substituted or unsubstituted. Exemplary substituents include alkyl, hydroxy, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, fluoroalkyl, carboxyl, hydroxyalkyl, carboxyalkyl, amino, aminoalkyl, monosubstituted amino, disubstituted amino, and quaternary amino groups.

By "C₇_₁₄ alkaryl" is meant an alkyl substituted by an aryl group (e.g., benzyl, phenethyl, or 3,4-dichlorophenethyl) having from 7 to 14 carbon atoms.

By "C₃_]₀ alkheterocyclyl" is meant an alkyl substituted heterocyclic group having from 3 to 10 carbon atoms in addition to one or more hetcroatoms (e.g., 3-furanylmethyl, 2-furanylmethyl, 3-tetrahydrofuranylmethyl, or 2- tetrahydrofuranylmethyl).

By "Ci_₇ heteroalkyl" is meant a branched or unbranched alkyl, alkenyl, or alkynyl group having from 1 to 7 carbon atoms in addition to 1, 2, 3 or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. Heteroalkyls include, without limitation, tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides. A heteroalkyl may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has three to six members. The heteroalkyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, hydroxyalkyl, carboxyalkyl, and carboxyl groups. Examples of C]_₇ heteroalkyls include, without limitation, methoxymethyl and ethoxyethyl.

By "halide" is meant bromine, chlorine, iodine, or fluorine.

By "fluoroalkyl" is meant an alkyl group that is substituted with a fluorine atom.

By "perfluoroalkyl" is meant an alkyl group consisting of only carbon and fluorine atoms.

By "carboxyalkyl" is meant a chemical moiety with the formula

-(R)-COOH, wherein R is selected from C₁^₇ alkyl, C_{2 7} alkenyl, C₂_₇ alkynyl, C₂_₆ heterocyclyl, C₆-_J2 aryl, C₇__]4 alkaryl, C₃__I0 alkheterocyclyl, or C]_₇ heteroalkyl.

By "hydroxyalkyl" is meant a chemical moiety with the formula -(R)-

OH, wherein R is selected from Cj_₇ alkyl, C₂_₇ alkenyl, C₂_₇ alkynyl, C₂-_O heterocyclyl,

C₆-_I2 aryl, C₇_i₄ alkaryl, C₃__]0 alkheterocyclyl, or C|_₇ heteroalkyl.

By "alkoxy" is meant a chemical substituent of the formula -OR, wherein R is selected from Ci_₇ alkyl, C₂_₇ alkenyl, C₂_₇ alkynyl, C₂_₆ heterocyclyl, C₆-_J2 aryl, C₇__]4 alkaryl, C₃_₁₀ alkheterocyclyl, or Ci_₇ heteroalkyl. By "aryloxy" is meant a chemical substituent of the formula -OR, wherein R is a C^₁₂ aryl group.

By "alkylthio" is meant a chemical substituent of the formula -SR, wherein R is selected from Ci_₇ alkyl, C₂-₇ alkenyl, C₂^₇ alkynyl, C₂_₆ heterocyclyl, C^₁₂ aryl, C₇_₁₄ alkaryl, C_{3 10} alkheterocyclyl, or Ci_₇ heteroalkyl.

By "arylthio" is meant a chemical substituent of the formula -SR, wherein R is a C^i₂ aryl group.

By "quaternary amino" is meant a chemical substituent of the formula -(R)-N(R')(R")(R'")⁺, wherein R, R', R", and R'" are each independently an alkyl, alkenyl, alkynyl, or aryl group. R may be an alkyl group linking the quaternary amino nitrogen atom, as a substituent, to another moiety. The nitrogen atom, N, is covalently attached to four carbon atoms of alkyl and/or aryl groups, resulting in a positive charge at the nitrogen atom.

Other features and advantages of the invention will be apparent from the following detailed description and the claims.

Detailed Description

The invention provides peptides conjugated to a binding group having affinity for a biocompatible calcified substrate, their use in implantable materials, and for the treatment of orthopedic conditions.

Each of the components of the invention is described in greater detail below.

Peptides

The methods and compositions of the invention feature a peptide conjugated to a binding group bearing a phosphonate moiety having affinity for a calcified substrate. The peptide can be, for example, a cell adhesion peptide, osteoinductive peptide, morphogenic protein stimulatory peptide, or

calmodulin (e.g., BMP-2, BMP-7, DGF-5, parathyroid hormone, LIM

mineralization peptide, agonists and partial agonists of BMP-2, and

calmodulin, or any other peptide described herein). Cell Adhesion Peptides

The molecules responsible for mediating cell-to-cell or cell-to-substrate adhesions, including the immunoglobulin family (see Albelda, S., Buck, C. FASEB J. 4:2868 (1990); and Edelman, G. M. Curr. Opin. Cell Biol. 5:869 (1988)), the cadherin family (Takeichi, M. Science 251 : 1451 (1991)), the selectin family (Lasky, L., Presta, L. G., and Erbe, D. V. in Cell Adhesion: Molecular Definition to Therapeutic Potential (Metcalf, B., Dalton, B., and Poste, G., ed) pp. 37-53, (1994) Plenum Publishing Corp., New York), and the integrin family (see Hynes, R. O. Cell 69:11 (1992); and Cheresh, D. A. Adv. MoI. Cell Biol. 6:225 (1993)) have been studied intensively.

Cell adhesion peptides can include any of the proteins of the

extracellular matrix which are known to play a role in cell adhesion, including fibronectin, vitronectin, laminin, elastin, fibrinogen, and collagens, such as types I, II, and V, as well as their bioactive fragments. Additionally, the cell adhesion peptides may be any peptide derived from any of the aforementioned proteins, including derivatives or fragments containing the binding domains of the above-described molecules. Exemplary peptides include those having integrin-binding motifs, such as the RGD (arginine-glycine-aspartate) motif, the YIGSR (tyrosine-isoleucine-glycine-serine-arginine) motif, and related peptides that are functional equivalents. For example, peptides containing RGD sequences (e.g., GRGDS) and WQPPRARI sequences are known to direct spreading and migrational properties of endothelial cells (see V.

Gauvreau et al., Bioconjug Chem. 16: 1088 (2005)). REDV tetrapeptide has been shown to support endothelial cell adhesion but not that of smooth muscle cells, fibroblasts, or platelets, and YIGSR pentapeptide has been shown to promote epithelial cell attachment, but not platelet adhesion (see Boateng et al., Am. J. Physiol. Cell Physiol. 288:30 (2005).. Another example of a cell- adhesive sequence is NGR tripeptide, which binds to CD 13 of endothelial cells (L. Holle et al., Oncol. Rep. 1 1 :613 (2004)).

Cell adhesion peptides that can be used in the implantable compositions and peptide conjugates of the invention include, without limitation, those mentioned above, and the peptides disclosed in U.S. patent No. 6,156,572; U.S. patent publication No. 2003/0087111; and U.S. patent publication No.

2006/0067909, each of which is incorporated herein by reference.

Alternatively, the cellular adhesion peptides can be obtained by screening peptide libraries for adhesion and selectivity to specific cell types (e.g. endothelial cells) or developed empirically via Phage display

technologies.

In certain embodiments, the cell adhesion peptide is an α2βl collagen mimetic peptide. α2βl collagen mimetic peptides

The integrin α2βl consists of two non-identical subunits, α2 and βl, members of the integrin family each with a single trans-membrane domain, and α2βl is known to bind to collagen via a specialised region of the α2-subunit. There are several known α2βl recognition sites within collagens. This knowledge arises from the use of collagen fragments derived from purified achains, hydrolysed into specific and reproducible peptides. α2βl collagen mimetic peptides that can be used in the implantable compositions and peptide conjugates of the invention include, without limitation, those described in PCT Publication Nos. WO/1999/050281; WO/2007/017671 ; and WO/2007/052067, each of which is incorporated herein by reference. α2βl collagen mimetic peptides include, without limitation, peptides including the peptide sequences of any of SEQ ID NOS. 1-20: Gly-Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln- Arg-Gly-Val-Val (SEQ ID NO. 1 , also known as "P- 15"), Gly-Pro-Gln-Gly- Ile-Ala-Gly-Gln -Arg (SEQ ID NO: 2), GIn-GIy-IIe-AIa-GIy-GIn (SEQ ID NO: 3), Gln-Gly-Ile-Ala-Gly-Gln-Arg (SEQ ID NO: 4), Phe-Gly-Ile-Ala-Gly-Phe (SEQ ID NO: 5), Gly-Ile-Ala-Gly-Gln (SEQ HD NO: 6), Gln-Gly-Ala-Ile-Ala- GIn (SEQ ID NO: 7), Phe-Gly-Ile-Ala-Gly-Phe (SEQ ID NO:8), Cys-Gly-Ile- Ala-Gly-Cys (SEQ ID NO:9), Glu-Gly-Ile-Ala-Gly-Lys (SEQ ID NO: 10), N- Acetyl Ile-Ala-Ala (SEQ ID NO:1 1), Ile-Ala-.beta.Ala (SEQ ID NO: 12), N- Acetyl He- Ala NMe (SEQ ID NO: 13), Asp-Gly-Glu-Ala (SEQ ID NO: 14), Asp-Gly-Glu-Ala-Gly-Cys (SEQ ID NO: 15), Gly-Phe-Pro* -GIy- GIu- Arg (SEQ ID NO: 16, where Pro* = hydroxyproline), Gly-Leu-Pro*-Gly-Glu-Arg (SEQ ID NO: 17, where Pro* = hydroxyproline), Gly-Met-Pro*-Gly-Glu-Arg (SEQ ID NO: 18, where Pro* = hydroxyproline), Gly-Ala-Ser-Gly-Glu-Arg (SEQ ID NO: 19), Gly-Leu-Ser-Gly-Glu-Arg (SEQ ID NO: 19), Gly-Ala-Pro*-Gly-Glu- Arg (SEQ ID NO:20, where Pro* = hydroxyproline), and any other α2βl collagen mimetic peptide s described in U.S. Patent No. 7,199,103,

incorporated herein by reference.

Binding Groups

The methods and compositions of the invention feature a peptide conjugated to a binding group bearing a phosphonatc moiety having affinity for a calcified substrate. Binding groups which can be used in the peptide conjugates of the invention include, without limitation, bisphosphonates and methylene phosphonates.

Bisphosphonates

Bisphosphonates are binding groups that include the moiety - C(PO₃H₂V. Examples of bisphosphonates that can be used in the peptide conjugates of the invention include, without limitation, 3 -amino- 1- hydroxypropylidene- -1-bisphosphonic acid ligand (pamidronate), alendronate, ibandronate. risedronate, tiludronate, zoledronate, and salts thereof.

For example, the bisphosphonate binding group can be a compound of formula (II), or a salt thereof:

In formula II, n is 0, 1, 2, 3, 4, 5, 6, 7, or 8; Y is O, S, C(O), or N(R^a); X is H, halogen, NH₂, NHR^b, OR^b, heterocyclyl, or alkheterocyclyl; and each of R^a and R^b is, independently, selected from H, Ci_₄ alkyl, C₂^ alkenyl, C₂_₄ alkynyl, C₂^ heterocyclyl, C_6-.^ aryl, C₇_₁₄ alkaryl, C₃_i₀ alkheterocyclyl, and Ci_₇ heteroalkyl. Amino methylene phosphonates

Amino methylene phosphonates are binding groups that include the moiety

-NCH₂(PO₃H₂)-. Examples of amino methylene phosphonates that can be used in the peptide conjugates of the invention include, without limitation,

For example, the amino methylene phosphonate binding group can be a compound of formula (III), or a salt thereof:

Q is -CH(CH₂N(CH₂PO₃H₂)₂)-, -CH(N(CH₂PO₃H₂)₂)CH₂-,

-CH(CH₂N(CH₂Pθ₃H₂)CH₂CH₂N(CH₂Pθ₃H₂)₂)-₅ or

m is O or 1 ; p is O, 1 , 2, 3, 4, or 5; Y is O, S, C(O), N(R^a), or is absent; and R^a is selected from H, C₁^ alkyl, C₂-Z_t alkenyl, C₂^ alkynyl, C₂_₆ heterocyclyl, C₆^₂ aryl, C₇_₁₄ alkaryl, C₃_₁₀ alkheterocyclyl, and Ci_₇ heteroalkyl.

The binding groups can be attached to a peptide, or a bioactive fragment thereof, using any of the techniques described herein.

Linkers

The linker component of the invention is, at its simplest, a bond between a peptide and a binding group with affinity for a calcified substrate. The linker provides a linear, cyclic, or branched molecular skeleton having pendant groups covalently linking a peptide to a binding group.

Thus, the linking of a peptide to a binding group is achieved by covalent means, involving bond formation with one or more functional groups located on the peptide and the binding group. Examples of chemically reactive functional groups which may be employed for this purpose include, without limitation, amino, hydroxyl, sulfhydryl, carboxyl, carbonyl, carbohydrate groups, vicinal diols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl, imidazolyl, and phenolic groups.

The covalent linking of a peptide and a binding group may be effected using a linker which contains reactive moieties capable of reaction with such functional groups present in the peptide and the binding group. For example, a carboxyl group of the peptide may react with a hydroxyl group of the linker, or an activated derivative thereof, resulting in the formation of an ester linking the two.

Examples of moieties capable of reaction with sulfhydryl groups include α-haloacetyl compounds of the type XCH₂CO- (where X=Br, Cl or I), which show particular reactivity for sulfhydryl groups, but which can also be used to modify imidazolyl, thioether, phenol, and amino groups as described by Gurd, Methods Enzymol. 1 1 :532 (1967). N-Maleimide derivatives are also considered selective towards sulfhydryl groups, but may additionally be useful in coupling to amino groups under certain conditions. Reagents such as 2- iminothiolane (Traut et al., Biochemistry 12:3266 (1973)), which introduce a thiol group through conversion of an amino group, may be considered as sulfhydryl reagents if linking occurs through the formation of disulphide bridges.

Examples of reactive moieties capable of reaction with amino groups include, for example, alkylating and acylating agents. Representative alkylating agents include:

(i) α-haloacetyl compounds, which show specificity towards amino groups in the absence of reactive thiol groups and are of the type XCH₂CO- (where X=Cl, Br or I), for example, as described by Wong Biochemistry 24:5337 (1979);

(ii) N-maleimide derivatives, which may react with amino groups either through a Michael type reaction or through acylation by addition to the ring carbonyl group, for example, as described by Smyth et al., J. Λm. Chem. Soc. 82:4600 (1960) and Biochem. J. 91 :589 (1964);

(iii) aryl halides such as reactive nitrohaloaromatic compounds;

(iv) alkyl halides, as described, for example, by McKenzie et al., J. Protein Chem. 7:581 (1988);

(v) aldehydes and ketones capable of Schiff s base formation with amino groups, the adducts formed usually being stabilized through reduction to give a stable amine;

(vi) epoxide derivatives such as epichlorohydrin and bisoxiranes, which may react with amino, sulfhydryl, or phenolic hydroxyl groups;

(vii) chlorine-containing derivatives of s-triazines, which are very reactive towards nucleophiles such as amino, sufhydryl, and hydroxyl groups;

(viii) aziridines based on s-triazine compounds detailed above, e.g., as described by Ross, J. Adv. Cancer Res. 2:1 (1954), which react with nucleophiles such as amino groups by ring opening;

(ix) squaric acid diethyl esters as described by Tietze, Chem. Ber. 124:1215 (1991); and

(x) α-haloalkyl ethers, which are more reactive alkylating agents than normal alkyl halides because of the activation caused by the ether oxygen atom, as described by Benneche et al., Eur. J. Med. Chem. 28:463 (1993).

Representative amino-reactive acylating agents include:

(i) isocyanates and isothiocyanates, particularly aromatic derivatives, which form stable urea and thiourea derivatives respectively;

(ii) sulfonyl chlorides, which have been described by Herzig et al.,

Biopolymers 2:349 (1964);

(iii) acid halides;

(iv) active esters such as nitrophenylesters or N-hydroxysuccinimidyl esters; (v) acid anhydrides such as mixed, symmetrical, or N-carboxy anhydrides;

(vi) other useful reagents for amide bond formation, for example, as described by M. Bodansky, Principles of Peptide Synthesis, Springer- Verlag, 1984; (vii) acylazides, e.g. wherein the azide group is generated from a preformed hydrazide derivative using sodium nitrite, as described by Wetz et al., Anal. Biochem. 58:347 (1974); and

(viii) imidoesters, which form stable amidines on reaction with amino groups, for example, as described by Hunter and Ludwig, J. Am. Chem. Soc. 84:3491

(1962).

Aldehydes and ketones may be reacted with amines to form Schiff s bases, which may advantageously be stabilized through reductive amination.

Alkoxylamino moieties readily react with ketones and aldehydes to produce stable alkoxamines, for example, as described by Webb et al., in Bioconjugate Chem. 1 :96 (1990).

Examples of reactive moieties capable of reaction with carboxyl groups include diazo compounds such as diazoacetate esters and diazoacetamides, which react with high specificity to generate ester groups, for example, as described by Herriot, Adv. Protein Chem. 3:169 (1947). Carboxyl modifying reagents such as carbodiimides, which react through O-acylurea formation followed by amide bond formation, may also be employed.

It will be appreciated that functional groups in the peptide and/or the binding group may, if desired, be converted to other functional groups prior to reaction, for example, to confer additional reactivity or selectivity. Examples of methods useful for this purpose include conversion of amines to carboxyls using reagents such as dicarboxylic anhydrides; conversion of amines to thiols using reagents such as N-acetylhomocysteine thiolactone, S- acetylmercaptosuccinic anhydride, 2-iminothiolane, or thiol-containing succinimidyl derivatives; conversion of thiols to carboxyls using reagents such as α -haloacetates; conversion of thiols to amines using reagents such as ethylenimine or 2-bromoethylamine; conversion of carboxyls to amines using reagents such as carbodiimides followed by diamines; and conversion of alcohols to thiols using reagents such as tosyl chloride followed by

transesterification with thioacetate and hydrolysis to the thiol with sodium acetate.

So-called zero-length linkers, involving direct covalent joining of a reactive chemical group of the peptide with a reactive chemical group of the binding group without introducing additional linking material may, if desired, be used in accordance with the invention. For example, the C terminus carboxylic acid of the peptide can be directly converted into 1 -hydroxy bisphosphonate group, or the N terminus can be modified to form an amino methylene phosphonate group.

The zero-length linkers can also be utilized with a fragment of a peptide by, for example, direct conversion of carboxylic acid moiety to a

bisphosphonate group as shown in Scheme A. For example, the peptide Pl 5 contains Scheme A

Water +

a single carboxylic acid group at its C-terminus. This can be modified as described in Scheme A to produce a Pl 5 peptide fragment bearing a phosphonate group.

P 15 fragment bisphosphonate

Similar modifications can be made to nitrile groups (i.e., conversion to

-C(PO₃H₂)₂NH₂).

The zero-length linkers can also be utilized with a fragment of a peptide by, for example, direct conversion of amino group to an amino methylene phosphonate group using a Mannich-type reaction of the amine with orthophosphorous acid as described by Moedritzer et al., J. Org. Chem.

31 : 1603 (1966). For example, the N-terminus glycine can be chemically modified prior to its incorporation into the peptide chain to produce the Pl 5 fragment below bearing amino methylene phosphonate groups.

Pl 5 fragment amino methylene phosphonate

Most commonly, however, the linker will include two or more reactive moieties, as described above, connected by a spacer element. The presence of such a spacer permits bifunctional linkers to react with specific functional groups within the peptide and the binding group, resulting in a covalent linkage between the two. The reactive moieties in a linker may be the same

(homobifunctional linker) or different (heterobifunctional linker, or, where several dissimilar reactive moieties are present, heteromulti functional linker), providing a diversity of potential reagents that may bring about covalent attachment between the peptide and the binding group.

Spacer elements in the linker typically consist of linear or branched chains and may include a Ci_-I0 alkyl, a heteroalkyl of 1 to 10 atoms, a C_2-10 alkene, a C_2-10 alkyne, C₂_₆ heterocyclyl, Cβ-n aryl, C₇_₁₄ alkaryl, C₃_₁₀ alkheterocyclyl, or

-(CH₂CH₂O)_nCH₂CH₂-, in which n is 1 to 4.

In some instances, the linker is described by formula I: G¹-(Z¹)_o-(Y¹)_u-(Z²)_s-(R₁₀)-(Z³)_t-(Y²)_v-(Z⁴)_p-G² (1)

In formula III, G is a bond between the peptide and the linker, G is a bond between the linker and the binding group, each of Z\ Z², Z³, and Z⁴ is, independently, selected from O, S, and NRn; Rn is hydrogen or a C_1-4 alkyl

1 0

group; each of Y and Y is, independently, selected from carbonyl,

thiocarbonyl, sulphonyl, or phosphoryl group; o, p, s, t, u, and v are each independently 0 or 1 ; and R₁₀ is a Cj_-10 alkyl, a linear or branched heteroalkyl of 1 to 10 atoms, C_2-10 alkene, a C_2-10 alkyne, a C₂_₆ heterocyclyl, C₆_₁₂ aryl, C₇_ i₄ alkaryl, C₃-_I0 alkheterocyclyl,

-(CH₂CH₂O)_qCH₂CH₂- in which q is an integer of 1 to 4, or a chemical bond linking 0'-(Z¹V(Y¹V(ZV to -(Z³)_r(Y²)_v-(Z⁴)_p-G².

Peptide Conjugates

The peptide conjugates can be compound of any of formulas (I V)-(IX), or a salt thereof:

^-PO₃H₂

Peptide-(CO)— L—Y-(CH₂)_p (Q)_m-N

^-PO₃H₂ (VI);

^PO₃H₂

Peptide-N(R^C)—L— Y-(CH₂)_p (Q)₁₁₁-N

-PO₃H₂ (VII);

^PO₃H₂

Peptide'— N

"PO₃H₂ (IX); In formulas (IV)-(IX), n, p, m, Q, X, L, and Y are as described in formulas (I), (II), and (III); Peptide-(CO) is a peptide having a phosphonate moiety covalently tethered to the C-terminus of the peptide; Peptide-N(R^C) is a peptide having a phosphonate moiety covalently tethered to the N-terminus of the peptide; R^c is selected from H, C_1-^ alkyl, C₂-₄ alkenyl, C₂^ alkynyl, C₂_₆ heterocyclyl, C₆^₂ aryl, C₇_₁₄ alkaryl, C₃_₁₀ alkheterocyclyl, and C]_₇ heteroalkyl; and Peptide^f is a bioactive fragment of a peptide in which the C- terminus carboxylate has been converted into a bisphosphonate group or the N- terminus amine has been converted into the amino methylene phosphonate group -N(CH₂PO₃H₂^. In any of formulas (IV)-(VII) the peptide can be linked to the phosphonate moiety via an amide, a phosphodiester, an ether, an ester, a sulfonamide, a urethane, or a carbamate bond. In certain embodiments of formulas (IV)-(IX), the peptide is a cell adhesion peptide, osteoinductive peptide, morphogenic protein stimulatory peptide, calmodulin, or an α2βl collagen mimetic peptide (e.g., an α2βl collagen mimetic peptide comprising an amino acid sequence selected from DGEΛ, GFOGER, GLOGER,

GMOGER, GLSGER, GASGER, GAOGER, and GTPGPQGIAGQRGVV (P 15), or a bioactive fragment thereof).

Calcified Substrates

The peptide conjugates of the invention are able to bind to a calcified substrate. The calcified substrate can be, for example, selected from calcium phosphate materials, such as mineralized bone, deorganified bone mineral, anorganic bone mineral, or a mixture thereof.

The calcium phosphate may be any biocompatible, calcium phosphate material known in the art. The calcium phosphate material may be produced by any one of a variety of methods and using any suitable starting components. For example, the calcium phosphate material may include amorphous, apatitic calcium phosphate. Calcium phosphate material may be produced by solid- state acid-base reaction of crystalline calcium phosphate reactants to form crystalline hydroxyapatite solids. Other methods of making calcium phosphate materials are known in the art, some of which are described below. Crystalline Hydroxyapatite

Alternatively, the calcium phosphate material can be crystalline hydroxyapatite (HA). Crystalline HA is described, for example, in U.S. Patent Nos. Re. 33,221 and Re. 33,161. These patents teach preparation of calcium phosphate remineralization compositions and of a finely crystalline, non- ceramic, gradually resorbable hydroxyapatite carrier material based on the same calcium phosphate composition. A similar calcium phosphate system, which consists of tetracalcium phosphate (TTCP) and monocalcium phosphate (MCP) or its monohydrate form (MCPM), is described in U.S. Patent Nos. 5,053,212 and 5,129,905. This calcium phosphate material is produced by solid-state acid-base reaction of crystalline calcium phosphate reactants to form crystalline hydroxyapatite solids. Carbonate substituted crystalline HA materials (commonly referred to as dahllite) may be prepared (see U.S. Patent No. 5,962,028). These HA materials (commonly referred to as carbonated hydroxyapatite) can be formed by combining the reactants with an aqueous liquid to provide a substantially uniform mixture, shaping the mixture as appropriate, and allowing the mixture to harden in the presence of water. During hardening, the mixture crystallizes into a solid and essentially monolithic apatitic structure.

The reactants will generally include a phosphate source, e.g., phosphoric acid or phosphate salts, an alkali earth metal, particularly calcium, optionally crystalline nuclei, particularly hydroxyapatite or calcium phosphate crystals, calcium carbonate, and a physiologically acceptable lubricant. The dry ingredients may be pre-prepared as a mixture and subsequently combined with aqueous liquid ingredients under conditions where substantially uniform mixing occurs.

Peptide-Bound Anorganic Bone Mineral

The peptide conjugates can be coated onto ABM particles have a mean particle diameter of 300 microns, and nearly all will fall within a range between 200 microns to 425 microns. However, a particle size range between 50 microns to 2000 microns may also be used.

Anorganic bone mineral (ABM) may also include a synthetic alloplast matrix or some other type of xenograft or allograft mineralized matrix that might not fit the definition of "anorganic." The alloplast could be a calcium phosphate material or it could be one of several other inorganic materials that have been used previously in bone graft substitute formulations, e.g., calcium carbonates, calcium sulphates, calcium silicates, used in a mixture that includes calcium phosphate and that could function as biocompatible, osteoconductive matrices. The anorganic bone mineral, synthetic alloplast matrix, and xenograft or allograft mineralized matrix are collectively referred to as the calcified substrate. Implantable Compositions

The implantable compositions of the invention can be used, or example, as a bone graft substitute, or as a coating for device implanted into a bony tissue.

Pastes and Gels

The peptide conjugate can be attached to a calcified substrate and suspended in a biocompatible polysaccharide gel to improve the delivery of the material to a site in vivo. Polysaccharides that may be utilized include, for example, any suitable polysaccharide within the following classes of polysaccharides: celluloses/starch, chitin and chitosan, hyaluronic acid, alginates, carrageenans, agar, and agarose. Certain specific polysaccharides that can be used include agar methyl cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, oxidized cellulose, chitin, chitosan, alginic acid, sodium alginate, and xanthan gum.

The pastes and gels will typically include a solvent to control the viscosity of the material. The solvent may be an aqueous alcohol or alcohol ester, including for example, water, glycerol, triacetin, isopropyl alcohol, ethanol, and propylene glycol, or mixtures of these. The paste or gel can include others components, such as surfactants, stabilizers, pH buffers, and other additives (e.g., growth factors, antibiotics, analgesics, etc.). For example, a suitable gel or paste can be prepared using water, glycerin and sodium carboxymethylcellulose. Implantable Devices

The peptide conjugate can be attached to a calcified substrate and coated onto an implantable device prior to implantation. Alternatively, an implantable device having a surface formed from bone, or another hardened calcified substrate, can be coated with a peptide conjugate to form an implantable composition of the invention. Therapy

The compositions of the invention can be used in the preparation of bone graft substitutes which are implanted into a subject. Because the compositions of the invention include a cell adhesion peptide, osteoinductive peptide, morphogenic protein stimulatory peptide, or calmodulin, the compositions promote rapid ossification of the implant.

The compositions of the invention can be useful for repairing a variety of orthopedic conditions. For example, the compositions may be injected into the vertebral body for prevention or treatment of spinal fractures, injected into long bone or flat bone fractures to augment the fracture repair or to stabilize the fractured fragments, or injected into intact osteoporotic bones to improve bone strength. The compositions can be useful in the augmentation of a bone-screw or bone-implant interface. Additionally, the compositions can be useful as bone filler in areas of the skeleton where bone may be deficient. Examples of situations where such deficiencies may exist include post-trauma with segmental bone loss, post-bone tumor surgery where bone has been excised, and after total joint arthroplasty (e.g., impaction grafting and so on). The compositions may be formulated as a paste prior to implantation to hold and fix artificial joint components in patients undergoing joint arthroplasty, as a strut to stabilize the anterior column of the spine after excision surgery, as a structural support for segmented bone (e.g., to assemble bone segments and support screws, external plates, and related internal fixation hardware), and as a bone graft substitute in spinal fusions.

The compositions of the invention can be used to coat prosthetic bone implants. For example, where the prosthetic bone implant has a porous surface, the composition may be applied to the surface to promote bone growth therein (i.e., bone ingrowth). The composition may also be applied to a prosthetic bone implant to enhance fixation within the bone.

The compositions of the invention can be used as a remodeling implant or prosthetic bone replacement, for example in orthopedic surgery, including hip revisions, replacement of bone loss, e.g. in traumatology, remodeling in maxillofacial surgery or filling periodontal defects and tooth extraction sockets, including ridge augmentation. The compositions of the invention may thus be used for correcting any number of bone deficiencies at a bone repair site.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compounds claimed herein are performed, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

Example 1. Synthesis of (4- Amino- 1 -hydroxy butylidene) Bisphosphonic Acid (amino-BPA).

(4-Amino- 1 -hydroxybutylidene) bisphosphonic acid was synthesized as shown in Scheme 1.

Scheme 1 NH₂(CH₂)₃COOH + H-

To a double neck round bottom flask equipped with a stir bar was added 4- aminobutyric acid (20.0Og, 0.1939mol), phosphorous acid (16.0Og,

0.195 lmol), and methanesulfonic acid (80.0OmL, 1.233mol). The round bottom flask was equipped with a 10OmL graduated addition funnel sealed with a rubber septum and purged with nitrogen and placed in an oil bath at 65 "C. Phosphorous trichloride (PCl₃) (35.0OmL, 0.4012mol) was added over 20 minutes to the solution via the addition funnel with stirring under nitrogen. The reaction was stirred for 16 hours, cooled to room temperature, quenched by pouring the mixture into 300 mL of 0 ⁰C deionized ultra- filtered water, and the resulting mixture was stirred vigorously for 10 minutes. The mixture was then refluxed for 5 hours at 110 ⁰C, and then cooled to room temperature. The pH was adjusted to 1.8, by slow addition of about 90 grams of sodium hydroxide pellets, making sure to minimize the exothermic reaction. The entire reaction mixture was placed in the refrigerator for 2 hours. The product was collected by filtration, rinsed twice with cold water (50 mL), rinsed twice with absolute ethanol (50 mL), and then dried under vacuum. Yield: 34g (white crystals); degrades at ca. 210 ^°C. Elemental Analysis: Calculated: C=19.38; H=5.22; N=5.62; 0=44.98; P=24.90; Found: C=17.80; H=5.49; N-5.24; 0=47.50; P= not determined.

Example 2. Synthesis of (4-Amino-l-hydroxybutylidene) Bisphosphonic Acid Monosodium Salt.

The procedure outlined in Example 1 was followed with the exception of adjusting the pH to 4.8 using a 50% solution of sodium hydroxide, again making sure to minimize the exothermic reaction. The reaction mixture was stored in a refrigerator for 2 hours, the resulting precipitate was collected by filtration, rinsed twice with cold DI H₂O (5OmL), rinsed twice with absolute ethanol (5OmL), and then dried under vacuum to yield white crystals (Yield: 34g, degrades at ca. 210 ^°C).

Example 3. Conjugation of amino-BPA with the Pl 5 peptide.

Pl 5 peptide was conjugated to (4-Amino-l-hydroxybutylidene) bisphosphonic acid as shown in Scheme 2.

Scheme 2

To a 25 mL schlenk tube, equipped with a stir bar, was added phosphate buffered saline solution (5 mL), amino-BPA (0.0178g), and EDAC coupling agent (0.011 Ig). The tube was sealed and gently purged with high purity nitrogen for 30min. To a second 25 mL schlenk tube, equipped with a stir bar, was added PBS buffer solution (5mL) and P15-peptide (lOOmg). The second tube was sealed and gently purged for 30min. Both solutions were allowed to stir at room temperature to allow for complete dissolution. The peptide solution was then transferred to the amino-BPA solution via cannula. The reaction mixture was stirred at 35 °C for 48 hours in the dark. The product was then purified using dialysis tubing (MWCO ~ 1000 Daltons). The remaining solution was then collected and dried under vacuum.

Example 4. Conjugation of hydroxybisphosphonate to Pl 5 peptide.

The terminal amine function of 4-aminobutane-l -hydroxy- 1,1- bisphosphonate was reacted with the C-terminal carboxylate group of the P- 15 peptide in a condensation reaction using previously described methodology (see Bioconjugate Techniques: G. T. Hermanson, Academic Press, (1995). Synthetic protocol based on the discussion on p-174).

The 4-aminobutane- 1 -hydroxy- 1 , 1 -bisphosphonate reagent was prepared as previously described in Journal of Organic Chemistry 60:8310 (1995). P-15 peptide was synthesized using traditional Merrifield resin techniques. In a 15 mL plastic centrifuge tube was mixed 1.0 mL of a 10 mg/ml PBS solution (7.1 μmoles) of the P-15 peptide with 1.0 ml of a 23 mg/ml PBS solution (71 μmoles) of 4-aminobutane- 1 -hydroxy- 1,1 -bisphosphonate. To this solution was added 0.5 ml of a 45 mg/ml NHS solution (210 μmoles) and 0.5 ml of a 31 mg/ml EDC solution (81 μmoles), both prepared in PBS. The reaction mixture was allowed to stand at room temperature for 65 minutes and was monitored by HPLC.

The HPLC profile indicated that a considerable amount of the P-15 had reacted in the first hour at room temperature. A residual P-15 peak was visible having a retention time of 8.8 minutes but a much bigger peak was present with a retention time of 10.0 minutes. Also a considerable amount of material was present in the void peak.

Fractions were collected from several preparative runs and those making up the peak having a retention time of 10.0 minutes were combined. The mobile phase solvent from those fractions was evaporated and the residue analyzed for protein using the BCA assay (see Pierce Applications Handbook and Catalog, Pierce Biotechnology Inc. Rockford, IL). Considerable protein was present in the HPLC peak having a retention time of 10.0 minutes. Mass spectroscopic and infrared analysis of the material making up that peak was inconsistent with it being the desired compound (i.e. inconsistent with a structure containing both the P-15 and hydroxybisphosphonate moieties). This material was observed to bind rabbit P-15 polyclonal antibody, as evaluated in an ELISA assay, and had a surprisingly high affinity for ABM granules.

Protein analysis of the void volume material showed the presence of a considerable amount of protein. Void peak fractions from several HPLC runs were combined and the mobile phase solvent was evaporated. The residue was redissolved and dialyzed overnight against PBS and overnight again against deionized water using a 1000 mw cutoff membrane. The dialyzed sample was collected and lyophilized. A sample was submitted for infrared and Cl 8 reverse phase HPLC analyses. The infrared spectrum was consistent with the void peak including a condensation product of P- 15 and the bisphosphonate, 4-ABHBP. The retention characteristics on the C 18 reverse phase HPLC column were consistent with the bisphosphonate group imparting so much polarity to the adduct that it no longer exhibits a significant hydrophobic interaction with the reverse phase column packing material. We suspect that the P- 15/bisphosphonate adduct elutes in the void peak because of this increase in polarity in comparison to unmodified P- 15 peptide.

The void volume material was observed to bind rabbit P- 15 polyclonal antibody, as evaluated in an ELISA assay, although the affinity of the antibody for the void volume material appeared to be diminished relative to unmodified P-15.

These experimental observations demonstrate that we were successful in synthesizing the coupled P-15/bis phosphonate adduct.

The P- 15/bis phosphonate adduct can have good affinity for synthetic calcium phosphates, such as hydroxy apatite and anorganic bone mineral. By increasing the amount of P-15 residing on the surface of the calcium phosphate, an increase in the osteogenic activity of this integrin attachment factor may be observed.

Other Embodiments

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. This application claims priority to U.S. Provisional Application No. 61/219,165, filed on June 22, 2009, which is incorporated herein by reference in its entirety.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

What is claimed is:

Claims

1. An implantable composition for correcting bone defects comprising a calcified substrate bound to a peptide conjugate, or a salt thereof, wherein said peptide conjugate comprises a peptide covalently tethered to a binding group comprising at least one phosphonate moiety, wherein said peptide conjugate exhibits enhanced affinity for said calcified substrate.

2. The implantable composition of claim 1, further comprising a hydrogel carrier.

3. The implantable composition of claim 2, wherein said hydrogel carrier comprises (i) a polymer selected from sodium carboxymethylcellulose, hyaluronic acid, polyvinylalcohol, polyvinylpyrrolidone, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, and ethylcellulose, (ii) water, and (iii) a dispersing agent selected from glycerin, polyethylene glycol, N-methyl pyrrolidone, and triacetin.

4. The implantable composition of any of claims 1-3, wherein said calcified substrate comprises calcium phosphate particles.

5. The implantable composition of claim 4, wherein said calcified substrate is a calcium phosphate particle selected from hydroxyapatite particles, tetracalcium phosphate particles, calcium hydrogen phosphate, calcium polyphosphate particles, tricalcium phosphate particles, octacalcium phosphate particles, calcium fluorapatite particles, and mixtures thereof.

6. The implantable composition of any of claims 1-5, wherein said peptide is a cell adhesion peptide, osteoinductive peptide, morphogenic protein stimulatory peptide, or calmodulin.

7. The implantable composition of claim 6, wherein said peptide is selected from BMP-2, BMP-7, DGF-5, parathyroid hormone, LIM

mineralization peptide, agonists and partial agonists of BMP-2, and

calmodulin.

8. The implantable composition of claim 6, wherein said peptide is a cell adhesion peptide.

9. The implantable composition of claim 8, wherein said cell adhesion peptide is derived from a binding domain of a cell adhesion protein of an extracellular matrix.

10. The implantable composition of claim 8, wherein said cell adhesion protein is selected from fibronectin, vitronectin, laminin, elastin, fibrinogen, collagen type I, collagen type II, and collagen type V.

1 1. The implantable composition of claim 8, wherein said cell adhesion peptide comprises an amino acid sequence selected from arginine-glycine- aspartate (RGD) and tyrosine-isoleucine-glycine-serine-arginine (YIGSR).

12. The implantable composition of claim 8, wherein said cell adhesion peptide is an α2βl collagen mimetic peptide.

13. The implantable composition of claim 12,wherein said cell adhesion peptide is an α2βl collagen mimetic peptide comprising an amino acid sequence selected from DGEA, GFOGER, GLOGER, GMOGER, GLSGER, GASGER, GAOGER, and GTPGPQGIAGQRGVV (P 15), or a bioactive fragment thereof.

14. The implantable composition of claim 1, further comprising a hydrogel carrier, said hydrogel carrier comprising (i) a polymer selected from sodium carboxymethylcellulose, hyaluronic acid, polyvinylalcohol, polyvinylpyrrolidone, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, and ethylcellulose, (ii) water, and (iii) a dispersing agent selected from glycerin, polyethylene glycol, N-methyl pyrrolidone, and triacetin, wherein said peptide is GTPGPQGIAGQRGVV (P 15), or a bioactive fragment thereof, and said calcified substrate comprises particles of anorganic bone mineral.

15. The implantable composition of claim 1, wherein said binding group is covalently tethered to said peptide via a linker of formula I:

G¹-(Z¹)_o-(Y¹)_u-(Z²)_s-(R₁₀)-(Z³)_t-(Y²)_v-(Z⁴)_p-G² (I) wherein,

G¹ is a bond between said peptide and said linker;

G² is a bond between said linker and said binding group;

Z¹, Z², Z³, and Z⁴ each, independently, is selected from O, S, and NR_n;

Ri i is hydrogen or a Cj_-1O alkyl group;

Y¹ and Y² are each, independently, selected from carbonyl,

thiocarbonyl, sulphonyl, or phosphoryl;

o, p, s, t, u, and v are each, independently, 0 or 1 ; and

Rio is a C]_-10 alkyl, a linear or branched heteroalkyl of 1 to 10 atoms, a linear or branched C₂. io alkene, a linear or branched C_2-10 alkyne, a C_{2 6} heterocyclyl,

C₆ i₂ aryl, C_{7 )4} alkaryl, C₃_i₀ alkheterocyclyl, -(CH₂CH₂O)_qCH₂CH₂- in which q is an integer of 1 to 4, or a chemical bond linking G ' -(Z¹V

(Y¹V(Z²V to -(Z³HY²V(ZVG².

16. The implantable composition of claim 15, wherein said binding group is covalently tethered to said peptide via an amide, a phosphodiester, an ether, an ester, a sulfonamide, a urethane, or a carbamate bond.

17. The implantable composition of any of claims 1-16, wherein said binding group comprises a bisphosphonate.

18. The implantable composition of claim 17, wherein said binding group is described by formula (II), or a salt thereof:

wherein,

n is O, 1, 2, 3, 4, 5, 6, 7, or 8;

Y is O, S, C(O), or N(R^a);

X is H, halogen, NH₂, NHR^b, OR^b, heterocyclyl, or alkheterocyclyl; and each of R^a and R^b is, independently, selected from H, C₁^ alkyl, C₂_₄ alkenyl, C₂^ alkynyl, C₂-_O heterocyclyl, C₆^₁₂ aryl, C₇_₁₄ alkaryl, C₃__{] 0} alkheterocyclyl, and C]_₇ heteroalkyl.

19. The implantable composition of any of claims 1-16, wherein said binding group comprises an amino methylene phosphonate.

20. The implantable composition of claim 19, wherein said binding group is described by formula (III), or a salt thereof:

wherein,

Q is -CH(CH₂N(CH₂PO₃H₂)₂)-, -CH(N(CH₂PO₃H₂)₂)CH₂-,

-CH(CH₂N(CH₂PO₃H₂)CH₂CH₂N(CI I₂PO₃I I₂)₂)-, or

-CH(N(CH₂PO₃H₂)CH₂CH₂N(CH₂PO₃H₂)₂)CH₂-;

m is 0 or 1 ; p is 0, 1, 2, 3, 4, or 5;

Y is O, S, C(O), N(R^a), or is absent; and R^a is selected from H, Cι_₄ alkyl, C₂-_A alkenyl, C₂_₄ alkynyl, C₂^ heterocyclyl, C₆-_O aryl, C₇_i₄ alkaryl, C₃_₁₀ alkheterocyclyl, and Cj_₇ heteroalkyl.

21. The implantable composition of any of claims 1-20, wherein said calcified substrate is on the surface of a dental implant, a vertebral implant, a bone rod, a bone plate, or a bone screw.

22. A peptide conjugate, or a salt thereof, wherein said peptide conjugate comprises an α2βl collagen mimetic peptide, or a bioactive fragment thereof, covalently tethered to a binding group comprising at least one phosphonate moiety, wherein said peptide conjugate exhibits enhanced affinity for a calcified substrate.

23. The peptide conjugate of claim 22, wherein said α2βl collagen mimetic peptide comprises an amino acid sequence selected from DGEA,

GFOGER, GLOGER, GMOGER, GLSGER, GASGER, GAOGER, and GTPGPQGIAGQRGVV (P 15), or a bioactive fragment thereof.

24. The peptide conjugate of claim 22, wherein said binding group is covalently tethered to the C terminus of said α2βl collagen mimetic peptide.

25. The peptide conjugate of claim 22, wherein said binding group is covalently tethered to the N terminus of said α2βl collagen mimetic peptide.

26. The peptide conjugate of any of claims 22-25, wherein said binding group is covalently tethered to said α2βl collagen mimetic peptide via a linker of formula I:

G¹-(Z¹)_O-(Y¹)_U-(Z²)_S-(R₁₀)-(Z³)_Γ(Y²)_V-(ZVG² (I) wherein, G¹ is a bond between said α2βl collagen mimetic peptide and said linker;

G is a bond between said linker and said binding group;

Z¹, Z², Z³, and Z⁴ each, independently, is selected from O, S, and NR₁₁; R₁₁ is hydrogen or a C_1-10 alkyl group;

Y and Y are each, independently, selected from carbonyl,

thiocarbonyl, sulphonyl, or phosphoryl;

o, p, s, t, u, and v are each, independently, 0 or 1 ; and

R₁₀ is a C_1-10 alkyl, a linear or branched heteroalkyl of 1 to 10 atoms, a linear or branched C_{2-I 0} alkene, a linear or branched C_2-10 alkyne, a C₂_₆ heterocyclyl, C₆^₁₂ aryl, C₇_i₄ alkaryl, C₃__]0 alkheterocyclyl, - (CH₂CH₂O)_qCH₂CH₂- in which q is an integer of 1 to 4, or a chemical bond linking 0'-(Z¹V(Y¹V(ZV to -(Z³)_t-(Y²)_v-(Z⁴)_p-G².

27. The peptide conjugate of claim 26, wherein said binding group is covalently tethered to said α2βl collagen mimetic peptide via an amide, a phosphodiester, an ether, an ester, a sulfonamide, a urethane, or a carbamate bond.

28. The peptide conjugate of any of claims 22-25, wherein said binding group comprises a bisphosphonate.

29. The peptide conjugate of claim 28, wherein said binding group is described by formula (II), or a salt thereof:

wherein,

n is O, 1, 2, 3, 4, 5, 6, 7, or 8;

Y is O, S, C(O), or N(R^a);

X is H, halogen, NH₂, NHR^b, OR^b, heterocyclyl, or alkheterocyclyl; and each of R^a and R^b is, independently, selected from H, C₁^ alkyl, C₂-₄ alkenyl, C₂^ alkynyl, C₂-^ heterocyclyl, C₆^₁₂ aryl, C₇_₁₄ alkaryl, C_3-.^ alkheterocyclyl, and C]_₇ heteroalkyl.

30. The peptide conjugate of any of claims 22-25, wherein said binding group comprises an amino methylene phosphonate.

31. The peptide conjugate of claim 30, wherein said binding group is described by formula (III), or a salt thereof:

wherein,

Q is -CH(CH₂N(CH₂PO₃H₂)₂)-, -CH(N(CH₂PO₃H₂)₂)CH₂-,

-CH(CH₂N(CH₂PO₃H₂)CH₂CH₂N(CH₂PO₃H₂)₂)-, or

-CH(N(CH₂PO₃H₂)CH₂CH₂N(CH₂PO₃H₂)₂)CH₂-;

m is 0 or 1 ; p is 0, 1, 2, 3, 4, or 5;

Y is O, S, C(O), N(R^a), or is absent; and

R^a is selected from H, C ₁-₄ alkyl, C_2^4 alkenyl, C₂^ alkynyl, C₂_₆ heterocyclyl, Cβ_i₂ aryl, C₇_i₄ alkaryl, C₃_₁₀ alkheterocyclyl, and C]_₇ heteroalkyl.

32. A method for correcting a bone defect in a subject, said method comprising implanting into said subject an implantable composition of any of claims 1-21 at the site of said bone defect.