Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds
  • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
  • A61K31/663—Compounds having two or more phosphorus acid groups or esters thereof, e.g. clodronic acid, pamidronic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
  • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
• • 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
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/548—Phosphates or phosphonates, e.g. bone-seeking
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0031—Rectum, anus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/18—Drugs for disorders of the endocrine system of the parathyroid hormones
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/635—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
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Embodiments disclosed herein relate generally to the modelling, treatment, reducing resistance to the treatment, prevention, and diagnosis of diseases/symptoms induced by bone-related diseases.
• Embodiments include methods of treating a bone related disease, comprising the steps of: providing to a subject at least one therapeutically effective dose of a compound disclosed herein.
• Healthy bone is a mix of 50-70% mineral, 20-40% organic matrix, 5-10% water, and 1- 5% lipids and is constantly being recycled into new bone in order to help healthy bone to maintain its rigidity and flexibility.
• monocytes receive several signals pushing them to differentiate into osteoclasts.
• Osteoblasts then express Receptor Activator of Nuclear Factor ⁇ B Ligand (RANKL) to the Receptor Activator of Nuclear Factor ⁇ B (RANK) surface receptor in monocytes, initiating the TRAF6 cascade, committing the monocytes to osteoclastogenesis.
• Mature osteoclasts then initiate healthy catabolic bone resorption.
• MSCs mesenchymal stem cells
• BMP-2/Runx2 and Wnt/p-catenin pathways a component of the bone matrix primarily composed of type I collagen, which mineralizes and becomes new bone.
• a first embodiment includes at least one compound of the formula X-Y-Z, or a pharmaceutically acceptable salt thereof, or a metabolite thereof, wherein X is at least one agent that modulates the activity of at least one of parathyroid hormone receptors; Z is at least one bone-targeting molecule; and Y is a linker that joins and/or links X and Z.
• X is at least one agent that enhances the activity of at least one of parathyroid hormone receptors.
• Z is at least one negatively charged oligopeptide or an equivalent thereof that binds to hydroxyapatite and/or raw bone.
• a second embodiment includes the compound according to the first embodiment, wherein X is at least one polypeptide having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% sequence identity to a full length parathyroid hormone related peptide (SEQ ID NO: 12), at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% sequence identity to a full length parathyroid hormone (SEQ ID NO: 13), and/or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%,
• Y is at least one polypeptide comprising at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% sequence identity to amino acid residues 35-40, 35-41, 35-42, 35-43, 35-44, 35-45, 35-46, 35-47, 35-48, 35-49, 35-50, 35-51, 35-52, 35-55, 35-84, 41-44, 41-45, 41-46, 41-47, 41-48, 41-49, 41- 50, and/or 41-84 of a full length parathyroid hormone related peptide or parathyroid hormone, and/or at least one Cathepsin K sensitive polypeptide; and
• Z is at least one polypeptide comprising about 4 or more, from about 4 to about 100, from about 4 to about 50, from 4 to about 20, from about 4 to about 15, from about 4 to about 10 acidic amino acid residues, polyphosphate, 2-aminohexanedioic (aminoadipic) acid or derivatives thereof, and/or alendronate or derivatives thereof.
• Z is at least one polypeptide comprising about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and/or 30 acidic amino acid residues, polyphosphate, 2- aminohexanedioic acid or derivatives thereof, and/or alendronate or derivatives thereof.
• Z is at least one negatively charged oligopeptide or an equivalent thereof that binds to hydroxyapatite and/or raw bone.
• X is at least one polypeptide that includes a polypeptide having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% sequence identity to a full length parathyroid hormone related peptide (SEQ ID NO: 12), at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% sequence identity to a full length parathyroid hormone (SEQ ID NO: 13), and/or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
• Z is at least one polypeptide that includes a polypeptide comprising about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and/or 30 acidic amino acid residues, polyphosphate, aminohexanedioic acid or derivatives thereof, and/or alendronate or derivatives thereof.
• a third embodiment includes the compound according to any one the preceding embodiments, X is at least one polypeptide having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and/or 9; Y is at least one polypeptide comprising at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% sequence identity to amino acid residues 35-46 and/or 41-46 of a full length parathyroid hormone related peptide; and Z is at least one polypeptide comprising about 4 or more acidic amino acid residues, polyphosphate, and
• Z is at least one polypeptide comprising from about 4 to about 100, from about 4 to about 50, from 4 to about 20, from about 4 to about 15, from about 4 to about 10 acidic amino acid residues, polyphosphate, aminohexanedioic acid or derivatives thereof, and/or alendronate or derivatives thereof. In other embodiments, Z is at least one polypeptide comprising about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and/or 30 acidic amino acid residues, polyphosphate, aminohexanedioic acid or derivatives thereof, and/or alendronate or derivatives thereof.
• a fourth embodiment includes the compound according to any one of the first to the third embodiments, wherein X is at least one polypeptide having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%) identity to a full length abaloparatide or analogs thereof; Y is at least one polypeptide comprising at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% sequence identity to amino acid residues 35-46 of a full length parathyroid hormone related peptide; and Z is at least one polypeptide comprising about 4 or more, from about 4 to about 100, from about 4 to about 50, from 4 to about 20, from about
• Z is at least one polypeptide comprising about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and/or 30 acidic amino acid residues, polyphosphate, 2-aminohexanedioic acid or derivatives thereof, and/or alendronate or derivatives thereof.
• a fifth embodiment includes the compound according to any one of the first to the fourth embodiments, wherein Z is at least one polypeptide comprising about 6, 7, 8, 9, and/or 10 acidic amino acid residues.
• a sixth embodiment includes the compound according to any one of the first to the fifth embodiments, wherein the acidic amino acid residues comprise L- or D-aspartic acid, L- or D- glutamic acid, or a combination thereof.
• a seventh embodiment includes the compound according to any one of the first to the sixth embodiments, wherein the acidic amino acid residues further comprises branched amino acid, and/or branched chains of amino acids.
• An eighth embodiment includes the compound according to any one of the first to the seventh embodiments, wherein Y is at least one polypeptide comprising the formula of Gly-Gly- Pro-Nle, wherein Nle comprises norleucine, leucine, isoleucine, and/or an equivalent thereof.
• a ninth embodiment includes the compound according to any one of the first to the eighth embodiments, wherein the compound of the formula X-Y-Z is at least one polypeptide having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and/or 99% sequence identity to any one of SEQ ID NO: 10 and/or SEQ ID NO: 11.
• the compound of the formula X-Y-Z is at least one polypeptide that includes a polypeptide having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and/or 99% sequence identity to any one of SEQ ID NO: 10 and/or SEQ ID NO: 11.
• a tenth embodiment includes the compound according to any one of the first to the ninth embodiments, wherein the compound of the formula X-Y-Z is at least one polypeptide having SEQ ID NO: 10 and/or SEQ ID NO: 1 1. In some embodiments, the compound of the formula X- Y-Z is at least one polypeptide that includes a polypeptide having SEQ ID NO: 10 and/or SEQ ID NO: 1 1.
• An eleventh embodiment includes the compound according to any one of the first to the tenth embodiments, wherein X is at least one agonist of parathyroid hormone receptor 1.
• a twelfth embodiment includes the compound according to any one of the first to the eleventh embodiments, wherein X is at least one polypeptide having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99%), and/or 100% sequence homology to the first 13 amino acid residues of the full length parathyroid hormone related peptide (SEQ ID NO: 12), at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and/or 100% sequence homology to the first 13 amino acid residues of the full length parathyroid hormone (SEQ ID NO: 13), and/or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%,
• X is at least one polypeptide having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99%), and/or 100%) sequence homology to the amino acid residues 2, 3, 4, 6, 7, 9, 12 and/or 13 of the full length parathyroid hormone related peptide (SEQ ID NO: 12), at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%), 97%), 98%), 99%), and/or 100%) sequence homology to the amino acid residues 2, 3, 4, 6, 7, 9, 12 and/or 13 of the full length parathyroid hormone (SEQ ID NO: 13), and/or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
• a thirteenth embodiment includes a method of treating a bone-related disease, the method comprising the steps of: providing a subject at least one therapeutically effective dose of the compound of any one of the first to the twelfth embodiments, or a pharmaceutically acceptable salt or metabolite thereof.
• a fourteenth embodiment includes the method according to the thirteenth embodiment, wherein the subject comprises a human, an animal, a cell, and/or a tissue.
• a fifteenth embodiment includes the method according to the thirteenth and/or fourteenth embodiments, wherein the bone-related disease comprises osteopenia, osteoporosis, rheumatoid arthritis, hematologic, autoimmunity, transplant rejection, osteomyelitis, and/or bone fracture.
• a sixteenth embodiment includes the method according to the thirteenth to the fifteenth embodiments, the effective dose of the compound according to any one of the first to the twelfth embodiments comprises from about 0.0001 nmol/kg/day to about 1000 nmol/kg/day, about 0.001 nmol/kg/day to about 1000 nmol/kg/day, from about 0.01 nmol/kg/day to about 1000 nmol/kg/day, from about 0.1 nmol/kg/day to about 1000 nmol/kg/day, from about 0.0001 nmol/kg/day to about 500 nmol/kg/day, about 0.001 nmol/kg/day to about 500 nmol/kg/day, from about 0.01 nmol/kg/day to about 500 nmol/kg/day, from about 1 nmol/kg/day to about 500 nmol/kg/day, from about 0.0001 nmol/kg/day to about 250 nmol/kg/day, from about 0.001 nmol/kg/day
• the effective dose of the compound according to any one of the first to the twelfth embodiments comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and/or 200 nmol/kg/day.
• the effective dose of the compound according to any one of the first to the twelfth embodiments comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and/or 200 pmol/kg/day.
• a seventeenth embodiment includes the method according to the thirteenth to the fifteenth embodiments, wherein the compound of any one of the first to the twelfth embodiments is administered orally, parenterally, rectally, transdermally, sublingually, and/or intranasally.
• An eighteenth embodiment includes a compound comprising: a compound of the formula X-Z; wherein: X is at least one peptide; and Z is at least one bone-targeting molecule; or a pharmaceutically acceptable salt thereof, or a metabolite thereof.
• a nineteenth embodiment includes the compound according to the eighteenth embodiment, wherein Z is at least one molecule that preferentially and/or selectively targets fractured bone.
• a twentieth embodiment includes the compound according to any one of eighteenth and nineteenth embodiments, wherein X is at least one peptide having fewer than 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40,
• a twenty first embodiment includes the compound according to any one of eighteenth to twentieth embodiments, wherein X is at least one peptide having more than 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10 amino acid residues.
• a twenty second embodiment includes the compound according to any one of eighteenth to twenty first embodiments, wherein X is at least one peptide comprising a hydrophilic peptide, a hydrophobic peptide, a neutral peptide, a cationic peptide, and/or an anionic peptide, and/or any combination thereof.
• a twenty third embodiment includes the compound according to any one of eighteenth to twenty second embodiments, wherein: X is at least one polypeptide comprising a sequence having at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and/or 90% sequence identity to any one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or analogs or metabolites thereof; and Z is at least one polypeptide comprising 4 or more acidic amino acid residues, polyphosphate, aminohexanedioic acid or derivatives thereof, alendronate or derivatives thereof, and/or bisphosphonate or derivatives thereof.
• a twenty fourth embodiment includes the compound according to any one of eighteenth to twenty third embodiments, wherein: X is at least one polypeptide comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and/or 99% sequence identity to any one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or analogs or metabolites thereof; and Z is at least one polypeptide comprising 4 or more acidic amino acid residues, polyphosphate, and/or bisphophonate or derivatives thereof.
• a twenty fifth embodiment includes the compound according to any one of eighteenth to twenty fourth embodiments, wherein X is at least one polypeptide comprising a sequence having at least 95%, 96%, 97%, 98%, and/or 99% sequence identity to any one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and/or SEQ ID NO: 19.
• a twenty sixth embodiment includes the compound according to any one of eighteenth to twenty fifth embodiments, wherein X is at least one polypeptide comprising a sequence having at least 100% sequence identity to any one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and/or SEQ ID NO: 19.
• a twenty seventh embodiment includes the compound according to any one of eighteenth to twenty sixth embodiments, wherein Z is at least one polypeptide comprising negatively charged amino acid residues.
• a twenty eighth embodiment includes the compound according to any one of eighteenth to twenty seventh embodiments, wherein Z is at least one polypeptide comprising 2, 3, 4, 5, ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, and/or 100 negatively charged amino acid residues.
• a twenty ninth embodiment includes the compound according to any one of eighteenth to twenty eighth embodiments, wherein Z is at least one mono-, bi-, tri-, tetra-, penta-, hexa- bisphosphonate, and/or multiple-bisphosphonate.
• a thirtieth embodiment includes the compound according to any one of eighteenth to twenty ninth embodiments, wherein Z is at least one acidic amino acid residues comprising L- or D-aspartic acid, L- or D-glutamic acid, or a combination thereof.
• a thirty first embodiment includes the compound according to any one of eighteenth to thirtieth embodiments, wherein Z is at least one acidic amino acid residues comprising branched amino acid and/or branched chains of amino acids, or a combination thereof.
• a thirty second embodiment includes the compound according to any one of eighteenth to thirty first embodiments, wherein Z is at least one acidic amino acid residues comprising branched amino acid and/or branched chains of amino acids, or a combination thereof.
• a thirty third embodiment includes the compound according to any one of eighteenth to thirty second embodiments, where the compound of the formula X-Z comprises a sequence having at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and/or 90% sequence identity to any one of SEQ ID NOs: 23-81, or analogs or metabolites thereof.
• a thirty fourth embodiment includes the compound according to any one of eighteenth to thirty third embodiments, where the compound of the formula X-Z comprises a sequence having
• a thirty fifth embodiment includes the compound according to any one of eighteenth to thirty fourth embodiments, where the compound of the formula X-Z comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 23-81, or analogs or metabolites thereof.
• a thirty sixth embodiment includes the compound according to any one of eighteenth to thirty fifth embodiments, further comprising a linking moiety Y, wherein Y is a linker that joins and/or links both X and Z.
• the present disclosure includes a compound for the targeted treatment bone fractures, the compound comprising a formula of X-Y-Z, wherein X is an active anabolic peptide or the effective fragment thereof that is an agonist of parathyroid hormone receptor 1 (PTHR1); Y is a linker; and Z is a negatively charged oligopeptide or an equivalent thereof that binds to hydroxyapatite and/or raw bone.
• PTHR1 parathyroid hormone receptor 1
• Y is a linker
• Z is a negatively charged oligopeptide or an equivalent thereof that binds to hydroxyapatite and/or raw bone.
• PTH and PTHrP could have agonistic activity of PTHR1 and be used in place of PTH or PTHrP as long as their affinity to PTHR1 is within the range of therapeutic value.
• Present disclosure also provides a peptide of SEQ ID NO: 3 with 2-methylalanyl at residue 29 and aminated at residue 34 to treat bone fracture.
• the aforementioned active anabolic peptide or the effective fragment thereof is selected from the group consisting of parathyroid hormone related peptide (PTHrP), parathyroid hormone (PTH), Abaloparatide and agonists thereof.
• PTHrP parathyroid hormone related peptide
• PTH parathyroid hormone
• Abaloparatide Abaloparatide
• the active anabolic peptide or the effective fragment thereof has SEQ ID NOS: 1-9, with SEQ ID NO: 3 having 2-methylalanyl at residue 29 and aminated at residue 34.
• the aforementioned compound has the sequence of SEQ ID NO: 10.
• the active anabolic peptide or the effective fragment thereof is an variant of SEQ ID NOS: 1-3, wherein at least residues of 2, 3, 4, 6, 7, 9, 12, 13 of SEQ ID NOS: 1-3 are conserved.
• the active anabolic peptide or the effective fragment thereof is n variant of SEQ ID NOS: 1-3, whereinl, 5, 10, 11, or 14-34 may be substituted with conservative amino acid.
• the aforementioned active anabolic peptide or the effective fragment thereof that is an agonist of PTHRl has a therapeutic affinity range to PTHRl between about IC 5 o 0.46 nM and about IC50 135 nM.
• the aforementioned linker Y is at least some extension of any active anabolic peptide or the effective fragment thereof.
• the linker Y may be at least one non-releasable fragment selected from various lengths of native PTHrP residues 35- 173, or the combination thereof.
• the linker Y may be at least one non-releasable fragment selected from various lengths of native PTH residues 35-84, or the combination thereof.
• the aforementioned linker Y is selected from the group consisting of various lengths of native PTHrP residues 35-173, or the combination thereof, and various lengths of native PTH residues 35-84, or the combination thereof.
• the aforementioned linker Y is a hydrolysable substrate sensitive to at least one abundant moiety produced in an osteoclast during bone remodeling.
• the abundant moiety may be Cathepsin K.
• the aforementioned linker Y is a hydrolysable substrate comprising a sequence of Gly-Gly-Pro-Nle, wherein Nle is norleucine, leucine, isoleucine or any equivalent hydrophobic modification thereof.
• the aforementioned hydrolysable substrate comprises disulfide bonds that are sensitive to Glutathione.
• the aforementioned linker Y is a releasable ester.
• the aforementioned compound further comprises at least one spacer comprising polyethylene glycol (PEG),
• the aforementioned negatively charged oligopeptide comprising at least 4 acidic amino acid residues and no more than 20 acidic amino acid residues.
• the aforementioned negatively charged oligopeptide comprising acidic amino acid residues selected from the group consisting of aspartic acid, glutamic acid, D- aspartic, D-glutamic acid, and the combination thereof.
• the negatively charged oligopeptide is a linear acidic amino acid chain.
• the negatively charged oligopeptide comprises least two branched acidic amino acid chains, wherein the branched acidic amino acid chains are connected by at least one Lysine.
• Z is at least one bisphosphonate. In other embodiment, Z is polyphosphate.
• Z is a collagen mimetic peptide that intercalates imperfect collagen fibrils at bone fracture site.
• collagen mimetic peptide may have a structure of [Gly- Pro-Hyp] 9 -OH.
• Z is aminohexanedioic acid (alpha-armnoadipic acid) or its derivatives with more than one carbon between the backbone and the acid.
• Z may be 2-aniinomalonic acid.
• This disclosure further provides a method of treating bone fracture by administering a therapeutic amount of any aforementioned compound to a patient suffering from the bone fracture.
• FIG. 1 Graph illustrating bone density data collected from microCT. A selection of 100 CT frames (slices) from each fracture callus were analyzed. The stack of frames were chosen from the area of the fracture where the callus was the largest. [0075] FIG. 2. Graph illustrating trabecular spacing data collected from microCT. Narrower spacing is associated higher density bone and can be indicative of progressed healing. A selection of 100 CT frames (slices) from each fracture callus were analyzed.
• FIG. 3 Graph illustrating bone density data collected from microCT. A selection of 100 CT frames (slices) from each fracture callus were analyzed. The stack of frames were chosen from the area of the fracture where the callus was the largest.
• FIG. 4 Graph illustrating alkaline phosphatase (ALP) expression ratio for both targeted and non-targeted PTHrP in MC3T3E1 cells.
• the ALP ratio is calculated by (Treated ALP expression/Vehicle control ALP expression). The expression was standardized to GAPDH.
• FIG. 5 Graph illustrating osteopontin (OPN) expression ratio for both targeted and non- targeted PTHrP in MC3T3E1 cells.
• the OPN ratio is calculated by (Treated OPN expression/Vehicle control OPN expression).
• FIG. 6. Graph illustrating gene expression ratio of several key bone markers for both targeted and non-targeted PTHrP in MC3T3E1 cells.
• Activity markers include alkaline phosphatase (ALP), Collagen I-alpha (Coll -alpha), osteocalcin(OC), osteoprotegerin (OPG), osteopontin (OPN, and Osterix (OSX).
• ALP alkaline phosphatase
• Collagen I-alpha Coll -alpha
• osteoprotegerin OPG
• osteopontin osteopontin
• OSX Osterix
• FIG. 7 Graph illustrating the effects of PTHrPDIO (targeted) or PTHrP (free) on bone volume after treatment.
• FIG. 8 Graph illustrating the effects of abaloparatide D10 (targeted), abaloparatide (not targeted) and saline on bone volume after treatment.
• FIG. 9 Graph illustrating the effects of PTHrP targeted with polyphosphate or saline on bone volume after treatment.
• FIG. 10 Graph illustrating the effects of PTHrP targeted with a single alendronate, tri- branched alendronate or free PTHrP on bone volume after treatment.
• FIG. 11 Chemical formula illustrating linear polymers of acidic amino acids of varying carbon chain length.
• FIG. 12 Chemical formula illustrating branched polymers of acidic amino acids of varying carbon chain length.
• FIG. 13 Chemical formula illustrating non-amino-acid-based bone targeting ligands. Top represents bisphosphonates. Bottom represents the polyphosphate targeting ligand.
• FIG. 14A Bar graph illustrating the fold difference of relative counts between fractured femur and healthy femur using various targeting ligands radiolabeled with 125 I.
• FIG. 14B Bar graph illustrating the counts per minute of the fractured femur compared to the healthy femur using various targeting ligands radiolabeled with 125 I.
• FIG. 15 Near infrared image illustrating the targeting of LS288 conjugated to a linear polymer of 10 L-aspartic acids 10 days post osteotomy on the right femur. Some off target signal is visible along the back due to the injection site.
• FIG. 16 Near infrared image illustrating the targeting of LS288 conjugated to a linear polymer of 10 L-aspartic acids at 3 (bottom row), 6 (middle row), and 10 days (top row) post osteotomy on the right femur.
• a femur on the left represents the fractured femur while a femur on the right represents the healthy femur.
• FIG. 17A Bar graph illustrating the relative distribution of the radiolabeled conjugated peptide, PreptinDlO, as a percent of the total counts that was found in each of the individual organs. The counts are standardized per gram of tissue weight.
• FIG. 17B Bar graph illustrating the fold difference of relative counts between fractured femur and healthy femur using radiolabeled 125 I PreptinDlO.
• FIG. 18A Bar graph illustrating the fold difference of relative counts between fractured femur and healthy (non-fractured) femur using various radiolabeled 125 I peptides conjugated with L-AsplO, D-AsplO, L-GlulO, or D-GlulO.
• FIG. 18B Bar graph illustrating the fold difference of relative counts between fractured femur and healthy (non-fractured) femur using various radiolabeled 125 I peptides conjugated with L-Asp20, L-Glu20, or D-Glu20.
• FIG. 18C Bar graph illustrating the fold difference of relative counts between fractured femur and healthy (non-fractured) femur using various radiolabeled 125 I peptides conjugated with branched L- Asp 10, branched D- Asp 10, branched L-Asp4, or branched L-Asp8.
• FIG. 19A Bar graph illustrating the fold difference of relative counts between fractured femur and healthy (non-fractured) femur using various conjugated peptides radiolabeled with
• FIG. 19B Bar graph illustrating the fold difference of relative counts between fractured femur and healthy (non-fractured) femur using various conjugated peptides radiolabeled with
• FIG. 20A Bar graph illustrating the relative distribution of the radiolabeled I peptides (e.g., Ck2.3C and PACAPC) conjugated with L-AsplO (i.e., a liner polymer of 10 L-aspartic acids) as a percent of the total counts that was found in each of the individual organs.
• L-AsplO i.e., a liner polymer of 10 L-aspartic acids
• FIG. 20B Bar graph illustrating the relative distribution of the radiolabeled 125 I peptides (e.g., ODPC, P4C, Ck2.3C, and PACAPC) conjugated with D-AsplO (i.e., a liner polymer of 10 D-aspartic acids) as a percent of the total counts that was found in each of the individual organs.
• D-AsplO i.e., a liner polymer of 10 D-aspartic acids
• FIG. 20C Bar graph illustrating the relative distribution of the radiolabeled 125 I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, and F109C) conjugated with L-Asp20 (i.e., a liner polymer of 20 L-aspartic acids) as a percent of the total counts that was found in each of the individual organs. The counts are standardized per gram of tissue weight.
• the radiolabeled 125 I peptides e.g., ODPC, P4C, Ck2.3C, CTCC, and F109C
• L-Asp20 i.e., a liner polymer of 20 L-aspartic acids
• FIG. 21A Bar graph illustrating the relative distribution of the radiolabeled 125 I peptides (e.g., P4C, Ck2.3C, and CTCC) conjugated with L-GlulO (i.e., a liner polymer of 10 L- glutamic acids) as a percent of the total counts that was found in each of the individual organs. The counts are standardized per gram of tissue weight.
• L-GlulO i.e., a liner polymer of 10 L- glutamic acids
• FIG. 21B Bar graph illustrating the relative distribution of the radiolabeled 125 I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, and F109C) conjugated with D-GlulO (i.e., a liner polymer of 10 D-glutamic acids) as a percent of the total counts that was found in each of the individual organs. The counts are standardized per gram of tissue weight.
• D-GlulO i.e., a liner polymer of 10 D-glutamic acids
• FIG. 22A Bar graph illustrating the relative distribution of the radiolabeled 125 I peptides (e.g., P4C, Ck2.3C, F109C, and PACAPC) conjugated with L-Glu20 (i.e., a liner polymer of 20 L-glutamic acids) as a percent of the total counts that was found in each of the individual organs. The counts are standardized per gram of tissue weight.
• L-Glu20 i.e., a liner polymer of 20 L-glutamic acids
• FIG. 22B Bar graph illustrating the relative distribution of the radiolabeled 125 I peptides (e.g., ODPC, P4C, Ck2.3C, and F109C) conjugated with D-Glu20 (i.e., a liner polymer of 20 D-glutamic acids) as a percent of the total counts that was found in each of the individual organs. The counts are standardized per gram of tissue weight.
• D-Glu20 i.e., a liner polymer of 20 D-glutamic acids
• FIG. 23A Bar graph illustrating the relative distribution of the radiolabeled 125 I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, F109C, and PACAPC) conjugated with branched L- AsplO (i.e., a branched polymer of 10 L-aspartic acids) as a percent of the total counts that was found in each of the individual organs. The counts are standardized per gram of tissue weight.
• FIG. 23B Bar graph illustrating the relative distribution of the radiolabeled 125 I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, F109C, and PACAPC) conjugated with branched L- AsplO (i.e., a branched polymer of 10 L-aspartic acids) as a percent of the total counts that was found in each of the individual organs. The counts are standardized per gram of tissue weight.
• FIG. 23B Bar graph illustrating
• Bar graph illustrating the relative distribution of the radiolabeled I peptides (e.g., ODPC, P4C, Ck2.3C, and CTCC) conjugated with branched D-AsplO (i.e., a branched polymer of 10 D-aspartic acids) as a percent of the total counts that was found in each of the individual organs.
• the counts are standardized per gram of tissue weight.
• FIG. 24A Bar graph illustrating the relative distribution of the radiolabeled 125 I peptides (e.g., P4C, Ck2.3C, F109C, and PACAPC) conjugated with L-AADIO (i.e., 10 L-amino adipic acid liner polymer) as a percent of the total counts that was found in each of the individual organs. The counts are standardized per gram of tissue weight.
• L-AADIO i.e. 10 L-amino adipic acid liner polymer
• FIG. 24B Bar graph illustrating the relative distribution of the radiolabeled 125 I peptides (e.g., ODPC, CTCC, F109C, and PACAPC) conjugated with L-SDSDD (i.e., a linear polymer having L-Ser-Asp-Ser-Asp-Asp; SEQ ID NO: 21) as a percent of the total counts that was found in each of the individual organs. The counts are standardized per gram of tissue weight.
• L-SDSDD i.e., a linear polymer having L-Ser-Asp-Ser-Asp-Asp; SEQ ID NO: 21
• FIG. 24C Bar graph illustrating the relative distribution of the radiolabeled 125 I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, F109C, and PACAPC) conjugated with (DSS) 6 (i.e., DSSDSSDSSDSSDSSDSS; SEQ ID NO: 22) as a percent of the total counts that was found in each of the individual organs. The counts are standardized per gram of tissue weight.
• DSS radiolabeled 125 I peptides
• FIG. 25A Bar graph illustrating the relative distribution of the radiolabeled 125 I PTHrPl-39C conjugated with a mono-bisphosphonate, a tri-bisphosphonate, or a polyphosphate, radiolabeled 125 I PTH1-34 conjugated with E10, and radiolabeled 125 I PTHrPl-39 conjugated with E20.
• PTHrPl-39C is PTHrPl-39 with a cysteine (C) at the 40 position, to which the different targeting ligands were conjugated.
• the counts are standardized per gram of tissue weight.
• FIG. 25B Bar graph illustrating the relative distribution of the radiolabeled 125 I
• Tyrosine conjugated with a mono-bisphosphonate a branched L-Asp4 (i.e., YPegK[DDDD] 2 ; see also SEQ ID NO:68), or a branched L-Asp8 (i.e., YPegK[DDDDDDDD] 2 ; see also SEQ ID NO:68), or a branched L-Asp8 (i.e., YPegK[DDDDDD] 2 ; see also SEQ ID NO:
• Mono-bisphosphonate YC is a peptide having tyrosine and cysteine that is conjugated with a mono-bisphosphonate. The counts are standardized per gram of tissue weight.
• FIG. 26 Graph illustrating the effects of PTH1-34E10 and saline on bone volume after treatment.
• FIG. 27 Graph illustrating the effect of PTHrPDIO on bone volume after treatment.
• SEQ ID NO: 3 Abaloparatide 1-34 with 2-methylalanyl at residue 29 and aminated at residue 34 (AVSEHQLLHDKGKSIQDLRRRELLEKLLAKLHTA)
• SEQ ID NO: 14 Heparin-binding domain of FGF2 (“F109C") (YKRSRYTC)
• SEQ ID NO: 18 Osteopontin-derived peptide (ODPC) (D VD VPDGRGD SL AYGC)
• P4-BMP2 (KIPKASSVPTELSAISTLYLC)
• SEQ ID NO: 20 PreptinDIO (DVSTSQAVLPDDFPRYDDDDDDDD)
• SEQ ID NO: 26 F109C conjugated with D10 (YKRSRYTCMalDDDDDDDD)
• SEQ ID NO: 28. F109C conjugated with E10 (YKRSRYTCMalEEEEEEEEEEEE).
• SEQ ID NO: 30 F109C conjugated with AAD10 (YKRSRYTCMalXXXXXXXXXX, wherein X is adipic acid)
• SEQ ID NO: 31 F109C conjugated with SDSDD (YKRSRYTCMal SDSDD).
• SEQ ID NO: 37 PACAPC conjugated with A AD 10
• SEQ ID NO: 38 PACAPC conjugated with SDSDD
• SEQ ID NO: 39 PACAPC conjugated with (DSS) 6
• SEQ ID NO: 46 CTCC conjugated with (DSS) 6
• SEQ ID NO: 67 P4C conjugated with (DSS) 6
• SEQ ID NO: 71 PACAPC conjugated with branched D10
• SEQ ID NO: 72 CTCC conjugated with branched D10
• treating includes administering to a human or an animal patient at least one dose of a compound, treating includes preventing or lessening the likelihood and/or severity of at least one disease as well as limiting the length of an illness or the severity of an illness, treating may or may not result in a cure of the disease.
• the terms 'therapeutically effective dose,' 'therapeutically effective amounts,' and the like refer to a portion of a compound that has a net positive effect on health and well being of a human or other animal.
• Therapeutic effects may include an improvement in longevity, quality of life and the like these effects also may also include a reduced susceptibility to developing disease or deteriorating health or well being.
• the effects may be immediate realized after a single dose and/or treatment or they may be cumulative realized after a series of doses and/or treatments.
• a "therapeutically effective amount” in general means the amount that, when administered to a subject or animal for treating a disease, is sufficient to affect the desired degree of treatment for the disease.
• inhibitors each encompass whole or partial reduction of activity or effect of an enzyme or all and/or part of a pathway that includes an enzyme that is effected either directly or indirectly by the inhibitor or a pathway that is effected either directly or indirectly by the activity of the enzyme which is effected either directly or indirectly by the inhibitor.
• the term "pharmaceutically acceptable salt” is defined as a salt wherein the desired biological activity of the inhibitor is maintained and which exhibits a minimum of undesired toxicological effects.
• Non-limiting examples of such a salt are (a) acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids (such as e.g.
• Pharmaceutically acceptable salts include salts of compounds of the invention that are safe and effective for use in mammals and that possess a desired therapeutic activity.
• Pharmaceutically acceptable salts include salts of acidic or basic groups present in compounds of the invention.
• Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., l,l'-methylene-bis-
• Certain compounds of the invention may form pharmaceutically acceptable salts with various amino acids.
• Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.
• Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.
• compositions for administration may be formulated as pharmaceutical compositions for administration.
• Such pharmaceutical compositions and processes for making the same are known in the art for both humans and non- human mammals. See, e.g., remington: The Science and practice of pharmacy, (A. Gennaro, et al, eds., 19 th ed., Mack Publishing Co., 1995).
• Formulations can be administered through various means, including oral administration, parenteral administration such as injection (intramuscular, subcutaneous, intravenous, intraperitoneal) or the like; transdermal administration such as dipping, spray, bathing, washing, pouring-on and spotting-on, and dusting, or the like. Additional active ingredients may be included in the formulation containing a compound of the invention or a salt thereof.
• the pharmaceutical formulations of the present invention include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular and intravenous) and rectal administration.
• the formulations may be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active ingredient, i.e., the compound or salt of the present invention, with the carrier.
• the formulations are prepared by uniformly and intimately bringing into association the active ingredient with a liquid carrier or, a finely divided solid carrier or both, and then, if necessary, forming the associated mixture into the desired formulation.
• the pharmaceutical formulations of the present invention suitable for oral administration may be presented as discrete units, such as a capsule, cachet, tablet, or lozenge, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or non-aqueous liquid such as a syrup, elixir or a draught, or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
• the formulation may also be a bolus, electuary or paste.
• the pharmaceutical formulations of the present invention suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, and may also include an antioxidant, buffer, a bacteriostat and a solution which renders the composition isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may contain, for example, a suspending agent and a thickening agent.
• the formulations may be presented in a single unit-dose or multi-dose containers, and may be stored in a lyophilized condition requiring the addition of a sterile liquid carrier prior to use.
• Pharmaceutically acceptable carrier Pharmaceutically acceptable carrier, unless stated or implied otherwise, is used herein to describe any ingredient other than the active component(s) that maybe included in a formulation.
• the choice of carrier will to a large extent depend on factors such as the particular mode of administration, the effect of the carrier on solubility and stability, and the nature of the dosage form.
• a tablet may be made by compressing or moulding the active ingredient with the pharmaceutically acceptable carrier.
• Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form, such as a powder or granules, in admixture with, for example, a binding agent, an inert diluent, a lubricating agent, a disintegrating and/or a surface active agent.
• Moulded tablets may be prepared by moulding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
• the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient.
• bone related diseases include, but are not limited to, osteopenia, osteoporosis, rheumatoid arthritis, hematologic, autoimmunity, transplant rejection, bone cancer, and/or bone fracture.
• a "therapeutic affinity index” is an affinity range of a therapeutic ligand, usually expressed by IC50 of the therapeutic ligand or agonists thereof to engage a receptor mediated signaling cascade (event) to achieve the desired therapeutic effect. For example, if a ligand has a IC50 of 4nM-15nM to trigger a G-protein coupled receptor mediated response that achieve the ligand' s therapeutic effect, then the ligand has a therapeutic affinity index of IC50 about 4nM to about 15nM. Therapeutic affinity index may be complicated when the therapeutic ligand has different modes of actions to achieve its different therapeutic effects. For example, if the receptor for the therapeutic ligand has two different conformations, each conformation may have a distinct therapeutic affinity index.
• PTHrPl-46D10 PTHrP D10
• targeted PTHrP can be used interchangeably to define the same compound.
• PTHrP 1-34 PTHrP
• PTHrP free PTHrP
• non-targeted PTHrP non-targeted PTHrP
• Osteoporosis is defined as low bone mineral density and/or poor bone microarchitecture associated with increased risk of fractures. This chronic disease mainly affects postmenopausal women, but it may also older men. This disease is increasingly being considered an age-related morbidity. The skeletal alterations observed in patient with in osteoporosis are a consequence of a relative deficit of bone formation relative to bone resorption. Osteoporosis therapies have mostly relied on antiresorptive drugs.
• One current, an alternative therapeutic approach for treating osteoporosis is based on the intermittent administration of parathyroid hormone (PTH). PTH is secreted by the chiefly by cells of the parathyroid glands as a prohormone polypeptide that include 84 amino acids.
• PTH parathyroid hormone
• PTH acts to increase the concentration of ionic calcium (Ca 2+ ) in the blood.
• PTH essentially acts to increase the concentration of calcium in the blood by acting upon the parathyroid hormone 1 receptor, which is present at high levels in bone and kidney.
• the protein hPTH-(l-34) crystallizes as a slightly bent, long helical dimer. Analysis reveals that the extended helical conformation of hPTH-(l-34) is the likely bioactive conformation.
• the N-terminal fragment (1-34 of parathyroid hormone (PTH) has been crystallized and the structure has been refined to 0.9 A resolution.
• PTH to increase osteoblastogenesis and osteoblast survival.
• PTHrP peptides might be considered a promising bone anabolic therapy.
• Parathyroid hormone/parathyroid hormone-related peptide receptor also known as parathyroid hormone receptor 1 (PTHRl) is a protein that in humans is encoded by the PTHRl gene. PTHRl functions as a receptor for parathyroid hormone (PTH) and for parathyroid hormone-related protein (PTHrP).
• PTH parathyroid hormone
• PTHrP parathyroid hormone-related protein
• PTHRl belongs to family B G-protein-coupled receptor (GPCR) that is expressed primarily in bone, kidney and cartilage but also in other tissues including the vasculature and certain developing organs.
• GPCR G-protein-coupled receptor
• N-terminal fragments consisting of the first 34 residues of PTH and PTHrP are generally thought to contain the key functional determinants of receptor interaction present in the corresponding full-length, mature polypeptide chains, which also includes 84 and 141 amino acid residues, respectively.
• PTH and PTHrP are distinct among the family B peptide ligands in that they include extended C-terminal segments. The biological roles of these segments remains obscure, although some functional responses have been identified, such as a capacity of fragments corresponding to the C-terminal portion of PTH to induce pro-apoptotic effects in osteocytesl7 and of fragments encompassing the mid-region of PTHrP.
• Membrane binding assays developed to evaluate the affinity of ligands for PTHRl in conformations formed upon coupling to a heterotrimeric G protein (R conformation) or when PTHRl is not coupled to a G protein (R° conformation) provided the initial clues that structurally distinct PTH and PTHrP analogues can bind with altered affinities to the different receptor conformational states.
• Direct comparative studies of PTH(l-34) and PTHrP(l-36) demonstrated that although these two peptides maintain similar affinity for the R state, they do not have the same affinity for the R° state, with PTH(l-34) displaying a much higher affinity for R° than PTHrP(l-36).
• Abaloparatide, PTHrP, and PTH share high homology in the first 13 amino acids of each protein.
• each active anabolic drug comprises a peptide that triggers signaling by the parathyroid hormone and includes or contains a minimum homology at residues of 2, 3, 4, 6, 7, 9, 12 and 13, anabolic peptide variants can be combined with various linkers, optionally spacers and targeting ligands described in this disclosure to effect targeted delivery of bone fracture healing agent.
• linkers, optionally spacers and targeting ligands described in this disclosure to effect targeted delivery of bone fracture healing agent.
• the functional consequences of this altered selectivity were typically revealed in cAMP assays.
• PTH(l-34) and PTHrP(l-36) had similar potencies in conventional cAMP dose- response assays (in accordance with their similar affinities for the R state and a GaS-mediated mechanism of intracellular cAMP production).
• the duration of the responses induced by the two ligands was different, with PTH(l-34) showing a more prolonged response than PTHrP(l-36).
• the duration of the cAMP responses observed in the cell-based studies correlate with the different affinities that PTH(l-34)
• PTH, PTHrP or their agonists are tested for either local or systematic administration to provide anabolic agent to heal any bone fracture.
• these tests have revealed that there are side effects to the use of those proteins.
• local application of PTH or PTHrP requires exposing the bone and resulted in with increased healing time, pain and discomfort, and even the possibility of infection.
• systematic application of PTH and PTHrP tend to have off target effects, including an increase in blood calcium levels. Therefore, the development of a bone anabolic agent delivery system that and may mitigate the above referenced side effects is desirous.
• a drug delivery system comprising a drug, a linker and a targeting ligand is disclosed herein.
• Some aspects of the present disclosure provide compounds for targeting and healing of bone fractures. These compounds may comprise at least three distinct structural/functional regions: an effective anabolic peptide or any agonist that engages parathyroid hormone receptor 1 (PTHR1) and subsequent signaling cascade that leads to the healing; a linker with or without a spacer to provide a flexible arm length for the anabolic peptide to reach the bone fracture site; and a targeting ligand which typically comprises a negatively charged oligopeptide or its equivalent to guide the compound to the fracture site and specifically bind to hydroxyapatite and/or raw bone.
• PTHR1 parathyroid hormone receptor 1
• the drug may be the first 34 amino acids in parathyroid hormone related peptide (PTHrP).
• the linker can include amino acid 35-46 of PTHrP, which spaces the drug from the targeting ligand and also gives leeway to the length of the drug, as some believe that amino acid 35-40 may increase receptor binding.
• the targeting ligand can include aspartic acid decapeptide, although other polymers functionalized by carboxylic acid are likely to bind in a similar manner (e.g. D or L glutamic acid, D or Laspartic acid and Aminohexanedioic acid in various combinations and arrangements).
• a peptide comprising SEQ ID NO:3 (Abaloparatide 1-34) with residue 29 methyl
• Ala, and residue 34 aminated can serve as a potent active anabolic agent without further linker or targeting ligand to treat bone fracture.
• PTH and PTHrP may have variants possessing agonistic activity of PTHR1 and be used in place of PTH or PTHrP to engage and achieve a therapeutic effect of bone fracture healing.
• each active anabolic drug comprises a peptide that triggers signaling by the parathyroid hormone and includes or contains a minimum homology at residues of 2, 3, 4, 6, 7, 9, 12 and 13, the proposed anabolic peptide variants may be combined with various linkers, optionally spacers and targeting ligands described in this disclosure to effect targeted delivery of bone fracture healing agent.
• conservative substitutions or modifications at residues 1, 5, 10, 11, or 14-34 for PTH or PTHrP along with any combinations of herein described linker sequence and targeting ligand are contemplated for the protection in this disclosure.
• the targeting ligand comprises an acidic oligopeptide comprising a plurality of aspartic acid residues.
• the number of D or L aspartic acid residues may be from about 4 to about 10, or from about 10 to about 20 residues.
• the oligopeptide may be linear or it may be branched. In one illustrative embodiment, a lysine residue is used as the branch point.
• the aspartic acid may be either L-aspartic acid, D-aspartic acid or a mixture of either enantiomer.
• the D-aspartic acid in the oligopeptide is that it may be less susceptible to proteolytic degradation as compared to an oligopeptide comprising only the naturally-occurring L-aspartic acid.
• the acidic oligopeptide may be no more than 20 L or D-glutamic acid.
• the acidic oligopeptide may be the combination of no more than 20 L or D-aspartic acid, or L or D- glutamic acid.
• the targeting ligand may be polyphosphate or at least one bisphosphate.
• the targeting ligand may be a collagen mimetic peptide.
• Such collagen mimetic peptide intercalates imperfect collagen fibrils at bone fracture site.
• the collagen mimetic peptide may have the structure of [Gly-Pro-HypJg-OH.
• the targeting ligand may be aminohexanedioic acid (alpha-aminodadipic acid) or its derivatives with more than one carbon between the backbone and the acid.
• the targeting ligand may be 2-aminomalonic acid.
• the targeted delivery compound further comprises at least one spacer comprising PEG (polyethylene glycol).
• the linker may be any portion of the extension of PTH or PTHrP' s active fragment, namely from residues 35-84 of PTH or 35-173 of PTHrP.
• Such extension of the active fragment is usually non-releasable and the linker sequence can be any portion of the extension or the combinations of different portions of the extension.
• the linker can be a hydrolysable substrate sensitive to at least one abundant moiety produced in an osteoclast during bone remodeling.
• Cathespin K is a moiety that is produced in an osteoclast during bone remodeling.
• a linker sequence comprising Gly-Gly-Pro-Nle (where Nle is norleucine, Leucine, isoleucine or any other equivalent with hydrophobic modification may serve as the substrate of Cathespin K.
• Cathespin K may hydrolyze the linker and release the active anabolic compound to work on the bone healing.
• Yet another hydrolysable linker may comprise disulfide bonds, and it may be released by glutathione at the osteoclast.
• hydrolysable linker may be a releasable ester.
• Peptides were synthesized by either solid-phase peptide synthesis or by recombinant expression.
• peptide were cleaved using 95:2.5:2.5 trifluoroacetic acid:water:triisopropylsilane.
• Cysteine containing peptides were cleaved using 95:2.5:2.5 and 10 fold excess TCEP trifluoroacetic acid: triisopropylsilane: water: TCEP (tris(2-carboxyethyl)phosphine).
• Ampicillian resistant plasmids were generated containing a T7 promoter, thioredoxin coding sequence, HisTag sequence, tryptophan residue, and peptide coding sequence. Competent cells were transformed with the plasmids and plated on ampicillin containing auger plates. Single colonies were selected and expanded overnight in ampicillin (lOOug/ml) LB media at 37C. The Competent cells were then expanded further in 11 of ampicillin (lOOug/ml) LB media for 15 hours. At 15 hour IPTG was added to reach a final concentration of lmM and the media was agitated at 180 rpm at 37C for 5 hours.
• Subcutaneous Buprenorphine (0.05 - 0.1 mg/kg) was administered at the time of surgery, followed by a dose every 12 h for 3-7 days post operation.
• mice were dosed subcutaneously, daily with 31nmol/kg peptide or saline control. The first dose was administered 6 hours following fracture and continued on throughout the study, the last dose being administered the day before euthanasia.
• Scanco ⁇ 40 was used to collect CT images and data of bone. The bones were scanned while immersed in PBS to prevent dehydration. ImageJ software was used to analyze the images for bone density, total volume (TV), relative bone volume (BV/TV), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp). Volumes of interest included the fracture callus, and both cortical and trabecular bone between the points on the cortical bone at the fracture site.
• TV total volume
• BV/TV relative bone volume
• Tb.Th trabecular thickness
• Tb.Sp trabecular spacing
• EXAMPLE 1 A PTHrP delivery system for targeted bone fracture healing [00250]
• a fracture targeted pharmaceutical comprising a drug, a linker and a targeting ligand were synthesized.
• SEQ ID NO: 10 The sequence of the pharmaceutical is listed in SEQ ID NO: 10, which comprises amino acid residues 1-46 of PTHrP followed by 10-Aspartic acids. As described above, residues 1-34 are the active portion of PTHrP, residues 35-46 are the linker portion of the proposed pharmaceutical for healing bone fracture, and the 10-Aspartic acids are the targeting ligand.
• mice were dosed daily for 4 weeks with either targeted PTHrP (31nmol/kg/day). PTHrP l-34(31nmol/kg/day) or saline. At the end of the study, mice were euthanized by C02, femurs were harvested, and bone densities were determined by MicroCT.
• the targeted version of PTHrP increased bone densities around fractures significantly higher than that of free PTHrP and Saline. This indicates that the strategy of using an effective anabolic agent linked to a targeting ligand sequence may work for bone fracture healing.
• FIG. 2 Another way of indicating the progress of bone healing is to measure trabecular spacing data collected from microCT.
• a selection of 100 CT frames (slices) from each fracture callus were analyzed.
• the stack of frames were chosen from the area of the fracture where the callus was the largest.
• From left to right the targeted PTHrP (PTHrP 1- 46 followed by 10 aspartic acids) and saline control.
• Targeted PTHrP has statistically tighter spacing than does the saline control.
• PTHrP extension sequences beyond 1-34 may provide similar or better connection to the targeting ligand of 10 Aspartic acids.
• the linker can be variations of the native peptide of PTH sequence, or any Cathepsin K sensitive linker such as Gly-Gly-Pro-Nle where Nle is a norleucine or another hydrophobic amino acid such as leucine or isoleucine.
• the linker may be a disulfide linker that can be released in a reductive environment. Glutathione is usually released in certain types of injury and may reduce disulfide bonds. It is contemplated a disulfide linker may increase the potency of the anabolic agent.
• the linker may also be an ester that is hydrolyzed and released to increase the healing efficiency.
• the targeting ligands are usually acidic oligopeptide chains containing 4 or more acidic amino acid residues and they bind to hydroxyapatite and/or raw bone. These acidic amino acid residues can be any of aspartic or glutamic acid or the combination thereof.
• acidic oligopeptide may be branched with at least one Lysine to increase the drug accumulation in the fracture site.
• the branched chains can be multiple branches, such as 2, 3, or 4 etc.
• Another choice of targeting ligand is one or more bisphosphate, i.e. poly bisphosphate.
• targeting ligand is aminohexanedioic acid (alpha-aminodadipic acid) or its derivatives with more than one carbon between the backbone and the acid.
• 2-aminomalonic acid may be used as the targeting ligand for PTHxP or its variants.
• a spacer such as PEG polyethylene glycol
• PEG polyethylene glycol
• the testing of bone density recovery can be performed similarly like described in this Example. Specifically, the synthesized drug-linker-targeting ligand is compared to the PTHrP variant itself and saline for their effect on bone densities around fractures.
• the targeted version of PTHrP variants is expected to increase bone densities around fracture significantly higher than that of free PTHrP variant and Saline.
• EXAMPLE 2 Various different combinations of anabolic drug with linker choices and targeting ligands to make targeted delivery of the drug to bone fracture for healing
• the first 34 amino acid of PTH is synthesized with a suitable linker described herein and a suitable targeting ligand.
• the linker may be any segment of the extension of the active PTH, including residues 35-84, or other linkers described in Example 1.
• the targeting ligand may be any of those described in Example 1.
• the linker is the amino acid residues 36-46 and the targeting ligand is 10 aspartic acid.
• the synthesized drug-linker-targeting ligand (PTH 1-46D10, SEQ ID NO: 11) may be compared to the PTH variant itself and saline for their effect on bone densities around fractures.
• the targeted version of PTH variants is expected to increase bone densities around fracture significantly higher than that of free PTH variant and Saline.
• Figure 3 has shown targeted PTH 1-46D10 is statistically denser than saline controls.
• CT frames were taken at 4 weeks. The stack of frames was chosen from the area of the fracture where the callus was the largest. From left to right the targeted PTHrP (PTHrP 1-46 followed by 10 aspartic acids), free unconjugated PTHrP (PTHrP 1-34), and saline control. The targeted version of PTHrP increased bone densities around fractures significantly higher than that of free PTHrP and Saline.
• CT frames were taken at 4 weeks. A selection of 100 CT frames (slices) from each fracture callus was analyzed. The stack of frames was chosen from the area of the fracture where the callus was the largest. From left to right the targeted PTHrP (PTHrP 1-46 followed by 10 aspartic acids) and saline control. Targeted PTHrP has statistically tighter spacing than does the saline control.
• CT frames were taken at 2 weeks. The stack of frames was chosen from the area of the fracture where the callus was the largest. From left to right the targeted PTH (PTH 1-46 followed by 10 aspartic acids) and saline control. Targeted PTH is statistically denser than saline controls.
• ALP alkaline phosphatase
• FIG. 4 alkaline phosphatase (ALP) expression ratio for both targeted and non-targeted PTHrP were analyzed in MC3T3E1 cells. The ALP ratio is calculated by (Treated ALP expression/Vehicle control ALP expression). Higher expression levels are associated with greater osteoblast activity and is a key protein involved in bone mineralization. The addition of targeting ligand does not reduce the efficacy of the drug.
• osteopontin (OPN) expression ratio for both targeted and non-targeted PTHrP were analyzed in MC3T3E1 cells.
• the OPN ratio is calculated by (Treated OPN expression/Vehicle control OPN expression). Higher expression levels are associated with greater osteoblast activity and is a key protein involved in bone mineralization. The addition of targeting ligand does not reduce the efficacy of the drug.
• gene expression ratio of several key bone markers for both targeted and non-targeted PTHrP were analyzed in MC3T3E1 cells.
• Activity markers include alkaline phosphatase (ALP), Collagen I-alpha (Coll -alpha), osteocalcin(OC), osteoprotegerin (OPG), osteopontin (OPN, and Osterix (OSX).
• ALP alkaline phosphatase
• Collagen I-alpha Coll -alpha
• osteoprotegerin OPG
• osteopontin OPN
• OSX osteopontin
• the gene expression ratio is calculated by (Treated gene expression/Vehicle control gene expression).
• the targeted PTHrP has activity as low as lpM.
• mice were treated with 31nmol/kg/day subcutaneous injections of PTHrPDlO ("Targeted Drug") or saline.
• Swiss ND4 mice were treated for 28 days. Mice were sacrificed at the end of the study and liver and kidneys were excised. Organs were fixed in formalin and paraffin sections were made from each. A veterinary pathologist performed a randomized blind analysis on the organs. No detectable toxicity was observed, the lesion noted are minimal in significance and unassociated with obvious tissue damage (necrosis). It appears unlikely that the type of lesion would cause clinical signs or illness. They are more likely within the normal limits for these animals.
• mice were treated with 31nmol/kg/day subcutaneous injections of PTHrPDlO (targeted) or PTHrP (free).
• the Swiss ND4 mice were treated for 14, 28, or 56 days. Mice were sacrificed at the end of the dosing period for each study and femurs were excised. Fracture callus densities were measured using a scanco microCT. 100 slice section at the thickest diameter each fracture callus were selected for the measurement.
• 'Targeted 2' represents mice dosed by targeted PTHrP for 2 weeks (14 days).
• 'Free 2' represents mice dosed by unmodified PTHrP for 2 weeks (14 days).
• 'Targeted 4' represents mice dosed by targeted PTHrP for 4 weeks (28 days).
• 'Free 4' represents mice dosed by unmodified PTHrP for 4 weeks (28 days).
• 'Targeted 8' represents mice dosed by targeted PTHrP for 8 weeks (56 days).
• 'Free 8' represents mice dosed by unmodified PTHrP for 8 weeks (56 days).
• Greater densities can be observed in the targeted PTHrP over the free PTHrp at every time point. The greatest differences between targeted and free PTHrP is at 2 weeks. Those results indicate that the targeted drug not only improves fracture healing but that it also accelerates fracture healing.
• mice were treated with 31nmol/kg/day subcutaneous injections of abaloparatide D10 (targeted), abaloparatide (not targeted) and saline.
• Swiss ND4 mice were treated for 28 days. Mice were sacrificed at the end of the study and femurs were excised. Fracture callus densities were measured using a scanco microCT. 100 slice section at the thickest diameter each fracture callus were selected for the measurement.
• Abaloparatide has been used previously for the treatment of osteoporosis. These results indicate that Abaloparatide can also be used for treating bone fractures. These results indicate that targeted abaloparatide performs better than free abaloparatide.
• mice were treated with 31nmol/kg/day subcutaneous injections of PTHrP targeted with polyphosphate or saline.
• Swiss D4 mice were treated for 28 days. Mice were sacrificed at the end of the study and femurs were excised. Fracture callus densities were measured using a scanco microCT. 100 slice section at the thickest diameter each fracture callus were selected for the measurement. PTHrP targeted with polyphosphate increases fracture healing compared to Saline.
• mice were treated with 31nmol/kg/day subcutaneous injections of PTHrP targeted with a single alendronate, tri -branched alendronate or free PTHrP.
• Swiss D4 mice were treated for 28 days. Mice were sacrificed at the end of the study and femurs were excised. Fracture callus densities were measured using a scanco microCT. 100 slice section at the thickest diameter each fracture callus were selected for the measurement.
• PTHrP targeted either a single alendronate or tri-branched alendronate increases fracture healing compared to free PTHrP.
• Targeting ligands can include, but are not limited to, oligo acidic amino acids.
• oligo acidic amino acids include, but are not limited to, a linear polymer of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and/or 30 aspartic acids comprising L and/or D amino acids, a linear polymer of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and/or 30 glutamic acids comprising L and/or D amino acids, a linear polymer of 10 acidic amino acids comprising L and/or D acidic amino acids, a branched polymer of aspartic acids with 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and/or 20 residues on each branch comprising L and/or D amino acids, a branched polymer of glutamic acids with 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and/or 20 residues on each branch comprising L and/or D amino acids, a
• Targeting ligands are coupled with a maleimide-containing linker.
• the targeting ligands having a maleimide-containing linker are further conjugated with various peptides having distinct chemical properties via a cysteine maleimide coupling process.
• peptides can comprise, but are not limited to, a sequence representing a heparin-binding domain of FGF2 (SEQ ID NO: 14), a sequence representing a pituitary adenylate cyclase-activating polypeptide (SEQ ID NO: 15), a sequence representing a chemotactic cryptic peptide derived from the CTX region of collagen type III (SEQ ID NO: 16), a sequence representing a casein kinase 2 beta chain (SEQ ID NO: 17), a sequence representing a osteopontin-derived peptide (SEQ ID NO: 18), and/or a sequence representing a P4-BMP2 (SEQ ID NO: 19).
• the conjugated peptides are iodinated via Pierce iodination reagent, where 125 I is covalently bound to the histidine, tyrosine and/or tryptophan residues of the conjugated peptides.
• PACAPC conjugated with branched D10 has the formula
• CTCC conjugated with branched D10 has the formula
• DVDVPDGRGDSLAYGCMalK[DDDDDDDD] 2 (see also SEQ ID NO: 74)
• CTCC conjugated with branched E10 has the formula
• DVDVPDGRGDSLAYGCMalK[EEEEEEEE] 2 see also SEQ ID NO: 80.
• FIG. 14A and 14B adult female Swiss Weber mice were injected with 1 mCi of 125 I labeled conjugates 10 days post osteotomy.
• a cysteine tyrosine dipeptide is coupled to a maleimide linker.
• the cysteine tyrosine dipeptide having a maleimide linker is conjugated to each of the indicated targeting ligands.
• PTHrP ASPIO represents a conjugated peptide having 1-46 of PTHrP with a linear polymer of 10 aspartic acids.
• Preptin AsplO represents a conjugated peptide having 1-16 of preptin with a linear polymer of 10 aspartic acids.
• a necropsy 24 hours post injections was performed and tissues were excised. The tissues were then counted via a gamma counter. The relative counts of the fractured femur over the healthy femur are displayed here as a ratio.
• mice were injected subcutaneously with LS288 conjugated to a linear polymer of 10 L-aspartic acids 10 days post osteotomy on the right femur.
• the mouse was imaged via a 1 sec 780nm excitation beam. Emission fluorescence was collected at 810nm for 1 second.
• the injection site was near the back of the mouse, and therefore, the mouse exhibits high fluorescence in the back.
• FIG. 17 adult Swiss Weber mice were injected with 0.25mCi of radiolabeled 125 I PreptinD IO conjugate 10 days post osteotomy and sacrificed at the different time points. Each tissue was collected and then quantified with a gamma counter (FIG. 17A). Fold difference of relative counts between fractured femur and healthy femur using radiolabeled 125 I PreptinDIO were calculated at different time points (FIG. 17B).
• FIG. 23 adult female Swiss Weber mice (12 weeks old) were injected with 0.22 mCi of radiolabeled 125 I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, F109C, and PACAPC) conjugated with branched L- Asp 10 (FIG. 23 A) or branched D- Asp 10 (FIG. 23B) 10 days post osteotomy. 14 hours post injection each mouse was sacrificed and each of the listed organs were collected and quantified. The counts were standardized to the weight of the samples. All of the conjugated peptides tested exhibited preferential and/or selective targeting towards to fractured bone over other organs. Some appeared to have higher uptake in the kidneys.
• radiolabeled 125 I peptides e.g., ODPC, P4C, Ck2.3C, CTCC, F109C, and PACAPC
• FIG. 26 the effects of PTH1-34E10 and saline on bone volume after treatment. Mice were treated daily with PTH1-34E10 for 2 weeks. At two week mice were sacrificed and femurs were excised for microCT analysis.
• the Glutamic acid containing targeting ligands have higher up take in the kidneys than the other targeting ligands tested with uptakes ranging from 20-50% of measured dose per gram. This uptake is likely due glutamic reuptake receptors that are expressed in the kidneys. They are more selective for fractured bone over other bone than other targeting ligands as is indicated by the ratio of fractured to non-fracture delivery ranging from 7-12 in glutamic acid targeting ligands. Extending the glutamic polymers to 20 generated a modest improvement of delivery from the 20-50%> of measured dose per gram for the lOmers up to 40-70%> of measured dose per gram for the 20mers.
• the aspartic acid targeting ligands appear to have the highest delivery of the targeting ligands with delivery accumulation ranging from 40-70%) of measured dose per gram.
• the aspartic acids targeting ligands appear to be slightly less selective between fractured and non- fractured bone with ratios of fractured bone to non-fractured bone accumulation around 4-6 for L amino acid aspartic acid targeting and 6-9 for D amino acid aspartic acid targeting ligands.
• Peptides conjugated with AAD10 appear to have only moderate targeting abilities. It still maintains a more systemic distribution. But it was still able to maintain an improvement in its ability to deliver 2-5 times as much of the labeled compound to the fractured bone over non- fractured bone.
• Peptides conjugated with SDSDD appear to have only moderate targeting ability's with deliveries ranging from 20-40%. It still maintains a more systemic distribution. But it was still able to maintain an improvement in its ability to deliver 3-5 times as much of the labeled compound to the fractured bone over non-fractured bone.
• Peptides conjugated with (DSS) 6 appear to have only moderate targeting abilities towards fractured bone. It still maintains a more systemic distribution. But it was still able to maintain an improvement in its ability to deliver 2-3 times as much of the labeled compound to the fractured bone over non-fractured bone.

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