WO2011005510A2 - Conjugués peptidiques et leurs utilisations - Google Patents

Conjugués peptidiques et leurs utilisations Download PDF

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Publication number
WO2011005510A2
WO2011005510A2 PCT/US2010/039466 US2010039466W WO2011005510A2 WO 2011005510 A2 WO2011005510 A2 WO 2011005510A2 US 2010039466 W US2010039466 W US 2010039466W WO 2011005510 A2 WO2011005510 A2 WO 2011005510A2
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Prior art keywords
peptide
binding group
implantable composition
α2βl
bone
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PCT/US2010/039466
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English (en)
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WO2011005510A3 (fr
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James J. Benedict
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Cerapedics, Inc.
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Publication of WO2011005510A2 publication Critical patent/WO2011005510A2/fr
Publication of WO2011005510A3 publication Critical patent/WO2011005510A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/29—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/12—Phosphorus-containing materials, e.g. apatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • 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 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.
  • 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.
  • 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).
  • 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.
  • the peptide is a cell adhesion peptide, osteoinductive peptide, morphogenic protein stimulatory peptide, or
  • 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).
  • 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.
  • the cell adhesion peptide can include an amino acid sequence selected from arginine-glycine-aspartate (RGD) and tyrosine-isoleucine- glycine-serine-arginine (YIGSR).
  • the cell adhesion peptide is an ⁇ 2 ⁇ l collagen mimetic peptide.
  • Exemplary ⁇ 2 ⁇ l 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.
  • 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).
  • 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 hydrogel carrier including (i) a polymer selected from sodium carboxymethylcellulose, hyaluronic acid, polyvinylalcohol, polyvinylpyrrolidone, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose,
  • 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 -(Z 1 ) o -(Y 1 ) u -(Z 2 ) s -(R 10 )-(Z 3 ) t -(Y 2 ) v -(Z 4 ) p -G 2 (I).
  • G 1 is a bond between the peptide and the linker
  • G 2 is a bond between the linker and the binding group
  • Z 1 , Z 2 , Z 3 , and Z 4 each,
  • R n is hydrogen or a Cj -10 alkyl group
  • Y 1 and Y 2 are each, independently, selected from carbonyl
  • R 10 is a C 1-J o 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, C 7 _ 14 alkaryl, C 3 _ 10 alkheterocyclyl, - (CH 2 CI I 2 O) q CH 2 CH 2 - in which q is an integer of 1 to 4, or a chemical bond linking G 1 -(Z 1 ) O -(Y 1 ) U -(Z 2 ) S - to -(Z 3 ) t -(Y 2 ) V -(Z 4 ) P -G 2 .
  • R 10 is a C 1-J o alkyl, a linear or branched heteroalkyl of 1 to 10 atoms, a linear or branched C 2-10 alkene, a linear or
  • 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.
  • the bisphosphonate-containing binding group is described by formula (II), or a salt thereof: (H).
  • 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, NH 2 , NHR b , OR b , heterocyclyl, or alkheterocyclyl; and each of R a and R b is, independently, selected from H, C 1 ⁇ alkyl, C 2 ⁇ alkenyl, C 2 ⁇ alkynyl, C 2 _ 6 heterocyclyl, C 6- ⁇ 2 aryl, C 7 _ 14 alkaryl, C 3 ⁇ 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.
  • amino methylene phosphonate-containing binding group is described by formula (III), or a salt thereof:
  • Q is -CH(CH 2 N(CH 2 PO 3 H 2 ) 2 )-, -CH(N(CH 2 PO 3 H 2 ) 2 )CH 2 -, -CH(CH 2 N(CH 2 PO 3 H 2 )CH 2 CH 2 N(CH 2 PO 3 H 2 ) 2 )-, or
  • 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.
  • the invention features a peptide conjugate, or a salt thereof, wherein the peptide conjugate includes an ⁇ 2 ⁇ l 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 ⁇ 2 ⁇ l 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.
  • the binding group is covalently tethered to the C terminus of the ⁇ 2 ⁇ l collagen mimetic peptide. In still other embodiments, the binding group is covalently tethered to the N terminus of the ⁇ 2 ⁇ l collagen mimetic peptide.
  • the peptide conjugates of the invention include those in which the binding group is covalently tethered to the ⁇ 2 ⁇ l collagen mimetic peptide via a linker of formula I:
  • G 1 is a bond between the ⁇ 2 ⁇ l collagen mimetic peptide and the linker
  • G 2 is a bond between the linker and the binding group
  • Z 1 , Z 2 , Z 3 , and Z 4 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,
  • R 10 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 C 2 _ 6 heterocyclyl, C 6 - I2 aryl, C 7 _ 14 alkaryl, C 3 _ 10 alkheterocyclyl, - (CH 2 CH 2 O) q CH 2 CH 2 - in which q is an integer of 1 to 4, or a chemical bond linking G 1 -(Z 1 ) O -(Y 1 ) U -(Z 2 ) S - to -(Z 3 ) ⁇ (Y 2 ) V -(Z 4 ) P - G2 -
  • R 10 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
  • the binding group is covalently tethered to the ⁇ 2 ⁇ l collagen mimetic peptide via an amide, a phosphodiester, an ether, an ester, a
  • 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.
  • the bisphosphonate-containing binding group is described by formula (II), or a salt thereof:
  • n 0, 1, 2, 3, 4, 5, 6, 7, or 8; Y is O, S, C(O), or N(R a ); X is H, halogen, NH 2 , NHR b , OR b , heterocyclyl, or alkheterocyclyl; and each of R a and R b is, independently, selected from H, C 1 ⁇ alkyl, C 2 ⁇ alkenyl, C 2-4 alkynyl, C 2 6 heterocyclyl, C 6 ⁇ 12 aryl, C 7 _ ]4 alkaryl, C 3 ⁇ 0 alkheterocyclyl, and C 1 .- / 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.
  • amino methylene phosphonate-containing binding group is described by formula (III), or a salt thereof:
  • Q is -CH(CH 2 N(CH 2 PO 3 H 2 ) 2 )-, -CH(N(CH 2 PO 3 H 2 ) 2 )CH 2 -, -CH(CH 2 N(CI I 2 PO 3 H 2 )CH 2 CH 2 N(CH 2 PO 3 H 2 ) 2 )-, or
  • 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.
  • ⁇ 2 ⁇ l collagen mimetic peptide is meant a synthetic peptide of from 3 to 50 amino acid residues having affinity for ⁇ 2 ⁇ l integrin.
  • ⁇ 2 ⁇ l 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.
  • 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
  • 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.
  • 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.
  • 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.
  • epithelial cells e.g., endothelial cells
  • 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.
  • morphogenic protein stimulatory peptide 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.
  • osteoinductive peptide refers to peptides, such as the members of the Transforming Growth Factor-beta (TGF-beta) superfamily, which ha ⁇ '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.
  • Osteoinductive peptides further include fragments and variants of the peptides listed above having osteoinductive properties.
  • parathyroid hormone refers to parathyroid hormone peptide, an important regulator of calcium and phosphorus
  • Parathyroid hormone peptides include, without limitation, hPTH(l-34), hPTH(7-31), hPTH(5-34), [NIe 8 ' 18 , Tyr 34 ]hPTH (7-34)NH 2 , [Tyr 34 ]hPTH (7-34)NH 2 , and hPTH(5-36).
  • 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
  • the number of atoms of a particular type in a substituent group is generally given as a range.
  • 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.
  • an alkyl group from 1 to 10 carbon atoms includes each of Ci, C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , and C 10 .
  • Other numbers of atoms and other types of atoms are indicated in a similar manner.
  • 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.
  • substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl,
  • C 1 ⁇ alkyls include, without limitation, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclopropylmethyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, and cyclobutyl.
  • 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 C 2 ⁇ alkenyl may optionally include monocyclic or polycyclic rings, in which each ring desirably has from three to six members.
  • the C 2 ⁇ alkenyl group may be substituted or unsubstituted.
  • substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
  • C 2 _ 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.
  • C 2 _ 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 C 2 _ 4 alkynyl may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has five or six members.
  • the C 2 - 4 alkynyl group may be substituted or unsubstituted.
  • substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
  • C 2 _ 4 alkynyls include, ⁇ vithout limitation, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl.
  • C 2 _ 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.
  • 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,
  • 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,
  • 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.
  • C 6 - J2 aryl is meant an aromatic group having a ring system comprised of carbon atoms with conjugated ⁇ 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.
  • substituents include alkyl, hydroxy, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, fluoroalkyl, carboxyl, hydroxyalkyl, carboxyalkyl, amino, aminoalkyl, monosubstituted amino, disubstituted amino, and quaternary amino groups.
  • C 7 _ 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.
  • aryl group e.g., benzyl, phenethyl, or 3,4-dichlorophenethyl
  • C 3 _] 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).
  • 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.
  • 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.
  • halide is meant bromine, chlorine, iodine, or fluorine.
  • fluoroalkyl is meant an alkyl group that is substituted with a fluorine atom.
  • perfluoroalkyl an alkyl group consisting of only carbon and fluorine atoms.
  • R is selected from C 1 ⁇ 7 alkyl, C 2 7 alkenyl, C 2 _ 7 alkynyl, C 2 _ 6 heterocyclyl, C 6 - J2 aryl, C 7 _ ]4 alkaryl, C 3 _ I0 alkheterocyclyl, or C]_ 7 heteroalkyl.
  • hydroxyalkyl is meant a chemical moiety with the formula -(R)-
  • R is selected from Cj_ 7 alkyl, C 2 _ 7 alkenyl, C 2 _ 7 alkynyl, C 2 - O heterocyclyl,
  • alkoxy is meant a chemical substituent of the formula -OR, wherein R is selected from Ci_ 7 alkyl, C 2 _ 7 alkenyl, C 2 _ 7 alkynyl, C 2 _ 6 heterocyclyl, C 6 - J2 aryl, C 7 _ ]4 alkaryl, C 3 _ 10 alkheterocyclyl, or Ci_ 7 heteroalkyl.
  • aryloxy is meant a chemical substituent of the formula -OR, wherein R is a C ⁇ 12 aryl group.
  • alkylthio is meant a chemical substituent of the formula -SR, wherein R is selected from Ci_ 7 alkyl, C 2 - 7 alkenyl, C 2 ⁇ 7 alkynyl, C 2 _ 6 heterocyclyl, C ⁇ 12 aryl, C 7 _ 14 alkaryl, C 3 10 alkheterocyclyl, or Ci_ 7 heteroalkyl.
  • arylthio is meant a chemical substituent of the formula -SR, wherein R is a C ⁇ i 2 aryl group.
  • 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.
  • 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.
  • 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
  • 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.
  • Cell adhesion peptides can include any of the proteins of 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.
  • RGD arginine-glycine-aspartate
  • YIGSR tyrosine-isoleucine-glycine-serine-arginine
  • peptides containing RGD sequences e.g., GRGDS
  • WQPPRARI sequences are known to direct spreading and migrational properties of endothelial cells (see V.
  • 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.
  • 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
  • the cell adhesion peptide is an ⁇ 2 ⁇ l collagen mimetic peptide. ⁇ 2 ⁇ l collagen mimetic peptides
  • the integrin ⁇ 2 ⁇ l consists of two non-identical subunits, ⁇ 2 and ⁇ l, members of the integrin family each with a single trans-membrane domain, and ⁇ 2 ⁇ l is known to bind to collagen via a specialised region of the ⁇ 2-subunit.
  • ⁇ 2 ⁇ l recognition sites within collagens There are several known ⁇ 2 ⁇ l recognition sites within collagens. This knowledge arises from the use of collagen fragments derived from purified achains, hydrolysed into specific and reproducible peptides.
  • ⁇ 2 ⁇ l 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.
  • ⁇ 2 ⁇ l 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.
  • 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
  • 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 are binding groups that include the moiety - C(PO 3 H 2 V.
  • 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.
  • the bisphosphonate binding group can be a compound of formula (II), or a salt thereof:
  • Amino methylene phosphonates are binding groups that include the moiety
  • amino methylene phosphonates that can be used in the peptide conjugates of the invention include, without limitation,
  • amino methylene phosphonate binding group can be a compound of formula (III), or a salt thereof:
  • Q is -CH(CH 2 N(CH 2 PO 3 H 2 ) 2 )-, -CH(N(CH 2 PO 3 H 2 ) 2 )CH 2 -,
  • the binding groups can be attached to a peptide, or a bioactive fragment thereof, using any of the techniques described herein.
  • 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.
  • 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.
  • functional groups located on the peptide and the binding group.
  • chemically reactive functional groups 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.
  • a linker which contains reactive moieties capable of reaction with such functional groups present in the peptide and the binding group.
  • 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.
  • 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.
  • reactive moieties capable of reaction with amino groups include, for example, alkylating and acylating agents.
  • Representative alkylating agents include:
  • 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. ⁇ m. Chem. Soc. 82:4600 (1960) and Biochem. J. 91 :589 (1964);
  • epoxide derivatives such as epichlorohydrin and bisoxiranes, which may react with amino, sulfhydryl, or phenolic hydroxyl groups;
  • Representative amino-reactive acylating agents include:
  • active esters such as nitrophenylesters or N-hydroxysuccinimidyl esters
  • acid anhydrides such as mixed, symmetrical, or N-carboxy anhydrides
  • 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).
  • 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.
  • 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.
  • 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 ⁇ -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
  • 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.
  • 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
  • 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.
  • 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.
  • 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
  • heterofunctional 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, C 2 _ 6 heterocyclyl, C ⁇ -n aryl, C 7 _ 14 alkaryl, C 3 _ 10 alkheterocyclyl, or
  • n 1 to 4.
  • the linker is described by formula I: G 1 -(Z 1 ) o -(Y 1 ) u -(Z 2 ) s -(R 10 )-(Z 3 ) t -(Y 2 ) v -(Z 4 ) p -G 2 (1)
  • G is a bond between the peptide and the linker, G is a bond between the linker and the binding group, each of Z ⁇ Z 2 , Z 3 , and Z 4 is, independently, selected from O, S, and NRn; Rn is hydrogen or a C 1-4 alkyl
  • each of Y and Y is, independently, selected from carbonyl
  • R 10 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 C 2 _ 6 heterocyclyl, C 6 _ 12 aryl, C 7 _ i 4 alkaryl, C 3 - I0 alkheterocyclyl,
  • the peptide conjugates can be compound of any of formulas (I V)-(IX), or a salt thereof:
  • 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, C 2 - 4 alkenyl, C 2 ⁇ alkynyl, C 2 _ 6 heterocyclyl, C 6 ⁇ 2 aryl, C 7 _ 14 alkaryl, C 3 _ 10 alkheterocyclyl, and C]_ 7 heteroalkyl
  • Peptide f is a bioactive fragment of a peptide in which the C- terminus
  • 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.
  • the peptide is a cell adhesion peptide, osteoinductive peptide, morphogenic protein stimulatory peptide, calmodulin, or an ⁇ 2 ⁇ l collagen mimetic peptide (e.g., an ⁇ 2 ⁇ l collagen mimetic peptide comprising an amino acid sequence selected from DGE ⁇ , GFOGER, GLOGER,
  • GMOGER GLSGER
  • GASGER GAOGER
  • GTPGPQGIAGQRGVV GTPGPQGIAGQRGVV
  • 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.
  • 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
  • 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.
  • 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
  • a physiologically acceptable lubricant e.g., 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.
  • 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 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.
  • 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.
  • 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.).
  • a suitable gel or paste can be prepared using water, glycerin and sodium carboxymethylcellulose.
  • the peptide conjugate can be attached to a calcified substrate and coated onto an implantable device prior to implantation.
  • 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.
  • 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.
  • compositions of the invention can be useful for repairing a variety of orthopedic conditions.
  • 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.
  • 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).
  • 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.
  • compositions of the invention can be used to coat prosthetic bone implants.
  • 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.
  • 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.
  • Example 2 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 H 2 O (5OmL), rinsed twice with absolute ethanol (5OmL), and then dried under vacuum to yield white crystals (Yield: 34g, degrades at ca. 210 ° C).
  • 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 ⁇ moles) of the P-15 peptide with 1.0 ml of a 23 mg/ml PBS solution (71 ⁇ moles) of 4-aminobutane- 1 -hydroxy- 1,1 -bisphosphonate.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention porte sur des compositions comprenant un peptide conjugué à un groupe de liaison ayant une affinité pour un substrat calcifié biocompatible, sur leur utilisation dans des matériaux implantables, et pour le traitement d'états pathologiques.
PCT/US2010/039466 2009-06-22 2010-06-22 Conjugués peptidiques et leurs utilisations WO2011005510A2 (fr)

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US8337879B2 (en) 2003-09-23 2012-12-25 Orthocon, Inc. Absorbable implants and methods for their use in hemostasis and in the treatment of osseous defects
WO2014032099A1 (fr) * 2012-08-28 2014-03-06 The Sydney Children's Hospitals Network (Randwick And Westmead) (Incorporating The Royal Alexandra Hospital For Children) Composition et procédé pour la croissance osseuse
AU2013207900B2 (en) * 2012-01-12 2017-12-07 Cambridge Enterprise Limited Compounds and methods for the enhanced degradation of targeted proteins and other polypeptides by an E3 ubiquitin ligase
WO2018022553A1 (fr) 2016-07-25 2018-02-01 Cerapedics, Inc. Particules de phosphate de calcium recouvertes de peptide
US9938264B2 (en) 2015-11-02 2018-04-10 Yale University Proteolysis targeting chimera compounds and methods of preparing and using same
US9988376B2 (en) 2013-07-03 2018-06-05 Glaxosmithkline Intellectual Property Development Limited Benzothiophene derivatives as estrogen receptor inhibitors
US9993514B2 (en) 2013-07-03 2018-06-12 Glaxosmithkline Intellectual Property Development Limited Compounds
EP3530294A1 (fr) * 2018-02-22 2019-08-28 DSM IP Assets B.V. Procédés de revêtement de particules inorganiques avec un peptide ou une protéine utiles pour améliorer l'activité cellulaire liée à la croissance osseuse
US11235086B2 (en) 2018-02-22 2022-02-01 Cerapedics, Inc. Processes for coating inorganic particles with a peptide or protein useful for improving cellular activity related to bone growth
CN115403654A (zh) * 2022-05-19 2022-11-29 首都医科大学 熊果酰-Asp-Gly-Glu-Ala、其合成、活性和应用

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AU2013207900B2 (en) * 2012-01-12 2017-12-07 Cambridge Enterprise Limited Compounds and methods for the enhanced degradation of targeted proteins and other polypeptides by an E3 ubiquitin ligase
US10730862B2 (en) 2012-01-12 2020-08-04 Yale University Compounds and methods for the enhanced degradation of targeted proteins and other polypeptides by an E3 ubiquitin ligase
WO2014032099A1 (fr) * 2012-08-28 2014-03-06 The Sydney Children's Hospitals Network (Randwick And Westmead) (Incorporating The Royal Alexandra Hospital For Children) Composition et procédé pour la croissance osseuse
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US9938264B2 (en) 2015-11-02 2018-04-10 Yale University Proteolysis targeting chimera compounds and methods of preparing and using same
EP3487512A4 (fr) * 2016-07-25 2020-06-17 Cerapedics, Inc. Particules de phosphate de calcium recouvertes de peptide
WO2018022553A1 (fr) 2016-07-25 2018-02-01 Cerapedics, Inc. Particules de phosphate de calcium recouvertes de peptide
AU2017301511B2 (en) * 2016-07-25 2023-05-18 Cerapedics, Inc. Peptide-coated calcium phosphate particles
EP3530294A1 (fr) * 2018-02-22 2019-08-28 DSM IP Assets B.V. Procédés de revêtement de particules inorganiques avec un peptide ou une protéine utiles pour améliorer l'activité cellulaire liée à la croissance osseuse
US11235086B2 (en) 2018-02-22 2022-02-01 Cerapedics, Inc. Processes for coating inorganic particles with a peptide or protein useful for improving cellular activity related to bone growth
CN115403654A (zh) * 2022-05-19 2022-11-29 首都医科大学 熊果酰-Asp-Gly-Glu-Ala、其合成、活性和应用

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