Targeted bone regeneration in fractures via parathyroid hormone receptor stimulation

Abstract

Disclosed herein is a drug delivery system for bone fracture and / or bone healing, comprising at least one peptide and a targeting ligand. Some embodiments include a peptide delivery system comprising at least one acidic peptide, basic peptide, hydrophilic peptide, hydrophobic peptide, or neutral peptide linked to an acidic peptide or non-peptide polyanion, and the peptide delivery system is used to target the conjugated peptide to the fracture surface. In some embodiments, the conjugated peptide performs an anabolic function acting through PTH receptor 1, and the targeting ligand in various formats guides the drug to raw hydroxyapatite. This system offsets some of the side effects caused by free anabolic drugs, such as high blood calcium levels.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 428,492, filed November 30, 2016, and U.S. Provisional Patent Application No. 62 / 553,313, filed September 1, 2017, the disclosures of both of which are expressly incorporated herein by reference.

[0002] FIELD OF THEINVENTION Various aspects and embodiments disclosed herein generally relate to modeling, treating, reducing resistance to treatment, preventing, and diagnosing diseases / symptoms induced by bone-related diseases.Embodiments include a method of treating a bone-related disease, comprising providing to a subject at least one therapeutically effective dose of a compound disclosed herein. [Background technology]

[0003] background This section introduces aspects that may help facilitate a better understanding of the present disclosure. These statements are to be read in this light and are not to be understood as admissions about what is prior art or what is not prior art.

[0004] Healthy bone is a mix of 50-70% mineral, 20-40% organic matrix, 5-10% water, and 1-5% lipids that are constantly recycled into new bone to help maintain its rigidity and flexibility. At the beginning of this recycling process, monocytes receive several signals that cause them to differentiate into osteoclasts. Osteoblasts then express receptor activator of nuclear factor κB ligand (RANKL) for RANK (receptor activator of nuclear factor κB), a surface receptor on monocytes, inducing the TRAF6 cascade and committing monocytes to osteoclast formation. Mature osteoclasts then initiate healthy catabolic bone resorption. The anabolic process begins when mesenchymal stem cells (MSCs) are stimulated by the BMP-2 / Runx2 and Wnt / β-catenin pathways to become osteoblasts. Mature osteoblasts then deposit osteoid, a component of bone matrix composed primarily of type I collagen that mineralizes into new bone.

[0005] The annual incidence of fractures in the United States is approximately 6.3 million. This alone represents a significant burden to our health care system. In most of the developed world, this problem is compounded by the maturing baby boom generation and the general aging population. As the baby boom generation increases the incidence of osteoporosis, the incidence of compound and fatal fractures will increase dramatically. Hip fractures alone are projected to increase 160% to 500,000 per year by 2040. Even today in the 21st century, one-quarter of people over 65 with hip fractures die within one year of injury due to complications related to recumbency. This poses a major public health concern.

[0006] Current clinical treatment of fractures generally does not include the use of site-specific anabolic drugs. In fact, the only drugs approved for clinical use in such fractures are BMP-2 (approved for use in tibia trauma only) and BMP-7 (approval suspended), which are applied locally and used in the treatment of open fractures of long bones and spinal fusion. Given that 85% of anabolic drug use is unapproved, the need for more widespread application of anabolic drugs to treat bone diseases such as osteoporotic fractures is clear (Ong, KL et al., Spine 35 (2010) 1794-1800). The FDA continues to be cautious and limit approval of the use of locally administered drugs to fractures that are already open and at risk of infection.

[0007] Given this limitation, a clinically meaningful approach for the treatment of these types of fractures is needed. It would therefore be desirable to have a fracture treatment drug that is administered systemically, yet is targeted to the fracture site. Summary of the Invention [Means for solving the problem]

[0008] Summary of the Invention A first embodiment comprises at least one compound of formula XYZ, where X is at least one agent that modulates at least one activity 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; or a pharma- ceutically acceptable salt or metabolite thereof. In some embodiments, X is at least one agent that enhances at least one activity of parathyroid hormone receptors. Consistent with these embodiments is that Z is at least one negatively charged oligopeptide or equivalent thereof that binds to hydroxyapatite and / or raw bone.

[0009] A second embodiment is a peptide sequence 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 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 full-length parathyroid hormone related peptide (SEQ ID NO: 13), at least one polypeptide having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100% sequence identity and / or 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% identity to full-length abaloparatide or an analog thereof; Y is 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 and / or 41-84, and / or 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 at least one cathepsin K sensitive polypeptide; Includes compounds according to a first embodiment, wherein Z is at least one polypeptide comprising about 4 or more, about 4 to about 100, about 4 to about 50, 4 to about 20, about 4 to about 15, about 4 to about 10 acidic amino acid residues, a polyphosphate, 2-aminohexanedioic acid (2-aminoadipic acid) or a derivative thereof, and / or alendronate or a derivative thereof. In some embodiments, Z is at least one polypeptide, polyphosphate, 2-aminohexanedioic acid or a derivative thereof, and / or alendronate or a derivative thereof, 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. In other embodiments, Z is at least one negatively charged oligopeptide or equivalent that binds to hydroxyapatite and / or living bone.

[0010] In some embodiments, X has 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 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 full-length parathyroid hormone related peptide (SEQ ID NO: 13). 6%, 97%, 98%, 99%, and / or 100% sequence identity and / or 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% identity to full-length abaloparatide or an analog thereof; Y is a polypeptide having amino acid residues 35-40 of full-length parathyroid hormone related peptide or parathyroid hormone; , 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, and / or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, %, 95%, 96%, 97%, 98%, 99%, and / or 100% sequence identity to the sequence of the polypeptide of the present invention; and Z is at least one polypeptide including a polypeptide comprising about 4 or more, about 4 to about 100, about 4 to about 50, 4 to about 20, about 4 to about 15, about 4 to about 10 acidic amino acid residues, a polyphosphate, aminohexanedioic acid or a derivative thereof, and / or alendronate or a derivative thereof.In some embodiments, Z is at least one polypeptide comprising 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, a polyphosphate, an aminohexanedioic acid or a derivative thereof, and / or an alendronate or a derivative thereof.

[0011] A third embodiment 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; and Y has 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 full-length parathyroid hormone related peptide. and Z is at least one polypeptide comprising about 4 or more acidic amino acid residues, a polyphosphate, and / or an alendronate or derivative thereof. In some embodiments, Z is at least one polypeptide comprising about 4 to about 100, about 4 to about 50, about 4 to about 20, about 4 to about 15, or about 4 to about 10 acidic amino acid residues, a polyphosphate, an aminohexanedioic acid or derivative thereof, and / or an alendronate or derivative 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, a polyphosphate, an aminohexanedioic acid or a derivative thereof, and / or an alendronate or a derivative thereof.

[0012] A fourth embodiment is 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 full length abaloparatide or an analog thereof; and Y is 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% identity to amino acid residues 35-46 of full length parathyroid hormone related peptide. and Z is at least one polypeptide comprising about 4 or more, about 4 to about 100, about 4 to about 50, about 4 to about 20, about 4 to about 15, about 4 to about 10 acidic amino acid residues, a polyphosphate, 2-aminohexanedioic acid or a derivative thereof, and / or an alendronate or a derivative thereof. In some 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, a polyphosphate, 2-aminohexanedioic acid or a derivative thereof, and / or alendronate or a derivative thereof.

[0013] A fifth embodiment includes compounds according to any one of the first to fourth embodiments, wherein Z is at least one polypeptide comprising about 6, 7, 8, 9, and / or 10 acidic amino acid residues.

[0014] A sixth embodiment includes compounds according to any one of the first to fifth embodiments, wherein the acidic amino acid residue comprises L-aspartic acid or D-aspartic acid, L-glutamic acid or D-glutamic acid, or a combination thereof.

[0015] A seventh embodiment includes compounds according to any one of the first to sixth embodiments, wherein the acidic amino acid residues further include branched amino acids and / or branched chains of amino acids.

[0016] An eighth embodiment includes compounds according to any one of the first to seventh embodiments, wherein Y is at least one polypeptide comprising the formula Gly-Gly-Pro-Nle, where Nle comprises norleucine, leucine, isoleucine, and / or equivalents thereof.

[0017] A ninth embodiment includes compounds according to any one of the first to eighth embodiments, wherein the compound of formula XYZ 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. In some embodiments, the compound of formula XYZ is at least one polypeptide comprising 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.

[0018] A tenth embodiment includes compounds according to any one of the first to ninth embodiments, wherein the compound of formula XYZ is at least one polypeptide having SEQ ID NO: 10 and / or SEQ ID NO: 11. In some embodiments, the compound of formula XYZ is at least one polypeptide comprising a polypeptide having SEQ ID NO: 10 and / or SEQ ID NO: 11.

[0019] An eleventh embodiment includes compounds according to any one of the first to tenth embodiments, wherein X is at least one agonist of the parathyroid hormone receptor 1.

[0020] A twelfth embodiment is a peptide sequence in which X has 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 the first 13 amino acid residues of 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 the first 13 amino acid residues of full-length parathyroid hormone related peptide (SEQ ID NO: 13), 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, and / or 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% homology to the first 13 amino acid residues of SEQ ID NO:3.

[0021] In some embodiments, X represents 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 2, 3, 4, 6, 7, 9, 12, and / or 13 of 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 amino acid residues 2, 3, 4, 6, 7, 9, 12, and / or 13 of full-length parathyroid hormone (SEQ ID NO: 13). 4%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100% sequence identity and / or 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% homology to amino acid residues 2, 3, 4, 6, 7, 9, 12, and / or 13 of SEQ ID NO:3.

[0022] A thirteenth embodiment includes a method of treating a bone-related disease comprising providing to a subject at least one therapeutically effective dose of a compound according to any one of the first through twelfth embodiments, or a pharma- ceutically acceptable salt or metabolite thereof.

[0023] A fourteenth embodiment includes the method according to the thirteenth embodiment, wherein the subject includes a human, an animal, a cell, and / or a tissue.

[0024] A fifteenth embodiment includes methods according to the thirteenth and / or fourteenth embodiments, wherein the bone-related disease includes osteopenia, osteoporosis, rheumatoid arthritis, hematologic, autoimmunity, graft rejection, osteomyelitis, and / or bone fracture.

[0025] A sixteenth embodiment is a compound according to any one of the first to twelfth embodiments, wherein the effective dose of the compound is from about 0.0001 nanomoles per kg per day to about 1000 nanomoles per kg per day, from about 0.001 nanomoles per kg per day to about 1000 nanomoles per kg per day, from about 0.01 nanomoles per kg per day to about 1000 nanomoles per kg per day, from about 0.1 nanomoles per kg per day to about 1000 nanomoles per kg per day, from about 0.0001 nanomoles per kg per day to about 500 nanomoles per kg per day, from about 0.001 nanomoles per kg per day to about 500 nanomoles per kg per day, from about 0.01 nanomoles per kg per day to about 1000 nanomoles per kg per day. about 500 nanomoles per kg per day, about 1 nanomoles per kg per day to about 500 nanomoles per kg per day, about 0.0001 nanomoles per kg per day to about 250 nanomoles per kg per day, about 0.001 nanomoles per kg per day to about 250 nanomoles per kg per day, about 0.01 nanomoles per kg per day to about 250 nanomoles per kg per day, about 1 nanomoles per kg per day to about 250 nanomoles per kg per day, about 0.0001 nanomoles per kg per day to about 100 nanomoles per kg per day, about 0.001 nanomoles per kg per day to about 100 nanomoles per kg per day, about 0.01 nanomoles per kg per day to about 100 nanomoles per kg per day, about 1 nanomoles per kg per day to about 75 nanomoles per kg per day, about 1 nanomoles per kg per day to about 100 nanomoles per kg per day, about 1 nanomoles per kg per day to about 250 nanomoles per kg per day, about 1 nanomoles per kg per day to about 500 nanomoles per kg per day, about 10 nanomoles per kg per day to about 75 nanomoles per kg per day, about 10 nanomoles per kg per day to about 100 nanomoles per kg per day, about 10 nanomoles per kg per day to about 250 nanomoles per kg per day, about 10 nanomoles per kg per day to about 500 nanomoles per kg per day, about 20 nanomoles per kg per day to about 50 nanomoles per kg per day, about 20 nanomoles per kg per day to about 75 nanomoles per kg per day, about 20 nanomoles per kg per day to about 100 nanomoles per kg per day, about 20 nanomoles per kg per day to about 250 nanomoles per kg per day, about 20 nanomoles per kg per day to about 500 nanomoles per kg per day, and / or about 0.074 nanomoles per kg per day.

[0026] In some embodiments, an effective dose of a compound according to any one of the first through 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 nanomoles per kg per day. In other embodiments, an effective dose of a compound according to any one of the first through 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 picomoles per kg per day.

[0027] A seventeenth embodiment includes methods according to the thirteenth to fifteenth embodiments, wherein a compound according to any one of the first to twelfth embodiments is administered orally, parenterally, rectally, transdermally, sublingually, and / or intranasally.

[0028] An eighteenth embodiment includes a compound comprising the formula XZ, where X is at least one peptide; and Z is at least one bone-targeting molecule; or a pharma- ceutically acceptable salt or metabolite thereof.

[0029] A nineteenth embodiment includes compounds according to the eighteenth embodiment, wherein Z is at least one molecule that preferentially and / or selectively targets fractured bone.

[0030] A twentieth embodiment includes compounds according to any one of the 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, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, and / or 10 amino acid residues.

[0031] A twenty-first embodiment includes compounds according to any one of the 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.

[0032] A twenty-second embodiment includes compounds according to any one of the eighteenth to twenty-first embodiments, wherein X is at least one peptide including a hydrophilic peptide, a hydrophobic peptide, a neutral peptide, a cationic peptide, and / or an anionic peptide, and / or any combination thereof.

[0033] A twenty-third embodiment includes compounds according to any one of the 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 four or more acidic amino acid residues, a polyphosphate, an aminohexanedioic acid or derivative thereof, an alendronate or derivative thereof, and / or a bisphosphonate or derivative thereof.

[0034] A twenty-fourth embodiment includes compounds according to any one of the 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 four or more acidic amino acid residues, a polyphosphate, and / or a bisphosphonate or derivative thereof.

[0035] A twenty-fifth embodiment includes compounds according to any one of the 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.

[0036] A twenty-sixth embodiment includes compounds according to any one of the 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.

[0037] A twenty-seventh embodiment includes compounds according to any one of the eighteenth to twenty-sixth embodiments, wherein Z is a polypeptide comprising at least one negatively charged amino acid residue.

[0038] A twenty-eighth embodiment includes compounds according to any one of the 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.

[0039] A twenty-ninth embodiment includes compounds according to any one of the eighteenth to twenty-eighth embodiments, wherein Z is at least one monobisphosphonate, bibisphosphonate, tribisphosphonate, tetrabisphosphonate, pentabisphosphonate, hexabisphosphonate, and / or multiple-bisphosphonate.

[0040] A 30th embodiment includes compounds according to any one of the 18th to 29th embodiments, wherein Z is at least one acidic amino acid residue, including L- or D-aspartic acid, L- or D-glutamic acid, or a combination thereof.

[0041] A thirty-first embodiment includes compounds according to any one of the eighteenth to thirtieth embodiments, wherein Z is at least one acidic amino acid residue, including branched amino acids and / or branched chains of amino acids, or combinations thereof.

[0042] A thirty-second embodiment includes compounds according to any one of the eighteenth to thirty-first embodiments, wherein Z is at least one acidic amino acid residue, including branched amino acids and / or branched chains of amino acids, or combinations thereof.

[0043] A thirty-third embodiment includes compounds according to any one of the eighteenth to thirty-second embodiments, wherein the compound of formula XZ 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.

[0044] A thirty-fourth embodiment includes compounds according to any one of the eighteenth to thirty-third embodiments, wherein the compound of formula XZ comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and / or 99% sequence identity to any one of SEQ ID NOs: 23-81, or analogs or metabolites thereof.

[0045] A thirty-fifth embodiment includes compounds according to any one of the eighteenth to thirty-fourth embodiments, wherein the compound of formula XZ comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 23-81, or analogs or metabolites thereof.

[0046] A thirty-sixth embodiment includes compounds according to any one of the eighteenth to thirty-fifth embodiments, further comprising a linking moiety, Y, where Y is a linker connecting and / or linking both X and Z.

[0047] The present disclosure includes compounds for targeted treatment bone fractures, the compounds comprising the formula XYZ, where X is an active anabolic peptide or 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 equivalent thereof that binds to hydroxyapatite and / or living bone. Conceivably, many variants of PTH and PTHrP may also have agonist activity of PTHR1 and may be used in place of PTH or PTHrP, so long as their affinity for PTHR1 is within the therapeutic range.

[0048] The present disclosure also provides a peptide of SEQ ID NO:3 with 2-methylalanyl at residue 29 and aminated at residue 34 for treating bone fracture.

[0049] In one embodiment, said active anabolic peptide or effective fragment thereof is selected from the group consisting of parathyroid hormone related peptide (PTHrP), parathyroid hormone (PTH), abaloparatide, and agonists thereof.

[0050] In some embodiments, the active anabolic peptide or effective fragment thereof has SEQ ID NO:1-9 with SEQ ID NO:3 having 2-methylalanyl at residue 29 and aminated at residue 34.

[0051] In one embodiment, the compound has the sequence of SEQ ID NO:10.

[0052] In some embodiments, the active anabolic peptide or effective fragment thereof is a variant of SEQ ID NO:1-3, where at least residues 2, 3, 4, 6, 7, 9, 12, 13 of SEQ ID NO:1-3 are conserved.

[0053] In some embodiments, the active anabolic peptide or effective fragment thereof is a variant of SEQ ID NOs: 1-3, where 1, 5, 10, 11, or 14-34 may be substituted with a conservative amino acid.

[0054] In some embodiments, the aforementioned active anabolic peptide or effective fragment thereof, which is an agonist of PTHR1, has an IC of about 0.46 nM against PTHR1. 50 IC of approximately 135 nM 50 The therapeutic affinity ranges between .

[0055] In some embodiments, the linker Y is at least some extension of any active anabolic peptide or effective fragment thereof. For example, the linker Y can be at least one non-releasable fragment selected from residues 35-173 of native PTHrP, of various lengths, or combinations thereof.

[0056] Alternatively, the linker, Y, can be at least one non-releasable fragment selected from residues 35-84 of native PTH, of various lengths, or combinations thereof.

[0057] In some embodiments, the linker Y is selected from the group consisting of various lengths of residues 35-173 of native PTHrP, or combinations thereof, and various lengths of residues 35-84 of native PTH, or combinations thereof.

[0058] In some embodiments, the linker Y is a hydrolyzable substrate susceptible to at least one abundant moiety produced in osteoclasts during bone remodeling. For example, the abundant moiety can be cathepsin K.

[0059] In some embodiments, the linker Y is a hydrolyzable substrate comprising the sequence Gly-Gly-Pro-Nle, where Nle is norleucine, leucine, isoleucine, or any equivalent hydrophobic modification thereof.

[0060] In some embodiments, the hydrolyzable substrate comprises a disulfide bond that is sensitive to glutathione.

[0061] In some embodiments, the linker, Y, is a releasable ester.

[0062] In some embodiments, the aforementioned compounds further comprise at least one spacer comprising polyethylene glycol (PEG).

[0063] In some embodiments, the negatively charged oligopeptide comprises at least 4 acidic amino acid residues and no more than 20 acidic amino acid residues.

[0064] In some embodiments, the negatively charged oligopeptide comprises an acidic amino acid residue selected from the group consisting of aspartic acid, glutamic acid, D-aspartic acid, D-glutamic acid, and combinations thereof.

[0065] In some embodiments, the negatively charged oligopeptide is a linear acidic amino acid chain.

[0066] In some embodiments, the negatively charged oligopeptide comprises at least two branched acidic amino acid chains, where the branched acidic amino acid chains are linked by at least one lysine.

[0067] In some embodiments, Z is at least one bisphosphonate. In other embodiments, Z is a polyphosphate.

[0068] In some embodiments, Z is a collagen mimetic peptide that is inserted between incomplete collagen fibrils at the fracture site. For example, the collagen mimetic peptide can have the structure [Gly-Pro-Hyp]9-OH.

[0069] In some embodiments, Z is aminohexanedioic acid (alpha-aminoadipic acid) or a derivative thereof with more than one carbon between the backbone and the acid. For example, Z can be 2-aminomalonic acid.

[0070] The present disclosure further provides a method of treating a bone fracture by administering a therapeutic amount of any of the aforementioned compounds to a patient suffering from a bone fracture.

[0071] These and other inventive features, aspects, and advantages will become better understood with reference to the following drawings, associated descriptions, and claims. [Brief description of the drawings]

[0072] [Figure 1] Figure 1 illustrates bone density data collected by microCT. 100 CT frames (slices) selected from the ossification of each fracture were analyzed. The stack of frames was selected from the fracture area with the greatest ossification. [Diagram 2] FIG. 1 is a graph illustrating trabecular spacing data collected by microCT. A decrease in trabecular spacing is associated with increased bone density and may indicate healing progression. 100 CT frames (slices) selected from each fracture ossification were analyzed. [Diagram 3] 1 is a graph illustrating bone mineral density data collected by microCT. 100 CT frames (slices) selected from the ossification of each fracture were analyzed. The stack of frames was selected from the fracture area with the greatest ossification. [Figure 4]1 is a graph illustrating the expression ratio of alkaline phosphatase (ALP) for both targeted and non-targeted PTHrP in MC3T3E1 cells.ALP ratio is calculated by (ALP expression in treated / ALP expression in vehicle control).Expression is normalized against GAPDH. [Diagram 5] 1 is a graph illustrating the expression ratio of osteopontin (OPN) for both targeted and non-targeted PTHrP in MC3T3E1 cells. OPN ratio is calculated by (OPN expression with treatment / OPN expression with vehicle control). [Figure 6] 1 is a graph illustrating the gene expression ratio of several key bone markers for both targeted and non-targeted PTHrP in MC3T3E1 cells.Activity markers include alkaline phosphatase (ALP), type I collagen alpha (Col1 alpha), osteocalcin (OC), osteoprotegerin (OPG), osteopontin (OPN), and osterix (OSX).Gene expression ratios are calculated by (gene expression with treatment / gene expression with vehicle control). [Figure 7] 1 is a graph illustrating the effect of PTHrPD10 (targeted) or PTHrP (free) on bone volume after treatment. [Figure 8] 1 is a graph illustrating the effect of abaloparatide D10 (targeted), abaloparatide (non-targeted), and saline on bone volume after treatment. [Figure 9] FIG. 1 is a graph illustrating the effect of polyphosphate-targeted PTHrP or saline on bone volume following treatment. [Figure 10] FIG. 1 is a graph illustrating the effect of single alendronate, branched trialendronate targeted PTHrP, or free PTHrP on bone volume following treatment. [Figure 11] 1 is a chemical formula illustrating linear polymers of acidic amino acids of varying carbon chain length. [Figure 12] 1 is a chemical formula illustrating branched polymers of acidic amino acids of varying carbon chain length. [Figure 13] 1 is a chemical formula illustrating non-amino acid based bone targeting ligands. The top diagram represents a bisphosphonate. The bottom diagram represents a polyphosphate targeting ligand. [Figure 14] Figure 14A: A bar graph illustrating the fold difference in relative counts between fractured and healthy femurs using various targeting ligands radiolabeled with 125I. Figure 14B: A bar graph illustrating the counts per minute of fractured femurs compared to healthy femurs using various targeting ligands radiolabeled with 125I. [Figure 15] 1 is a near-infrared image illustrating targeting by LS288 conjugated to a linear polymer of 10L-aspartic acid 10 days after osteotomy in the right femur. Some off-target signal is seen at the back due to the injection site. [Figure 16] Near-infrared images illustrating targeting by LS288 conjugated to a linear polymer of 10L-aspartic acid 3 (bottom row), 6 (middle row), and 10 (top row) days after osteotomy in the right femur. In each pair of femurs, the left femur represents the fractured femur, while the right femur represents the healthy femur. [Figure 17] Figure 17A: A bar graph illustrating the relative distribution of radiolabeled conjugated peptide, preptin D10, as a percentage of the total counts found in each individual organ. Counts are normalized per gram of tissue weight. Figure 17B: A bar graph illustrating the fold difference in relative counts between fractured and healthy femurs using radiolabeled 125I-preptin D10. [Figure 18]Figure 18A: A bar graph illustrating the fold difference in relative counts between fractured and healthy (non-fractured) femurs using various radiolabeled 125I peptides conjugated with L-Asp10, D-Asp10, L-Glu10, or D-Glu10. Figure 18B: A bar graph illustrating the fold difference in relative counts between fractured and healthy (non-fractured) femurs using various radiolabeled 125I peptides conjugated with L-Asp20, L-Glu20, or D-Glu20. Figure 18C: A bar graph illustrating the fold difference in relative counts between fractured and healthy (non-fractured) femurs using various radiolabeled 125I peptides conjugated with branched L-Asp10, branched D-Asp10, branched L-Asp4, or branched L-Asp8. [Figure 19] Figure 19A: A bar graph illustrating the fold difference in relative counts between fractured and healthy (non-fractured) femurs using various conjugated peptides radiolabeled with 125I. Figure 19B: A bar graph illustrating the fold difference in relative counts between fractured and healthy (non-fractured) femurs using various conjugated peptides radiolabeled with 125I. [Figure 20]Figure 20A: A bar graph illustrating the relative distribution of radiolabeled 125I peptides (e.g., Ck2.3C and PACAPC) conjugated with L-Asp10 (i.e., a linear polymer of 10 L-aspartic acid) as a percentage of the total counts found in each of the individual organs. Figure 20B: A bar graph illustrating the relative distribution of radiolabeled 125I peptides (e.g., ODPC, P4C, Ck2.3C, and PACAPC) conjugated with D-Asp10 (i.e., a linear polymer of 10 D-aspartic acid) as a percentage of the total counts found in each of the individual organs. Figure 20C: A bar graph illustrating the relative distribution of radiolabeled 125I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, and F109C) conjugated with L-Asp20 (i.e., a linear polymer of 20 L-aspartic acid) as a percentage of the total counts found in each individual organ. Counts are normalized per gram of tissue weight. [Figure 21] Figure 21A: A bar graph illustrating the relative distribution of radiolabeled 125I peptides (e.g., P4C, Ck2.3C, and CTCC) conjugated with L-Glu10 (i.e., a linear polymer of 10 L-glutamic acid) as a percentage of the total counts found in each of the individual organs. Counts are normalized per gram of tissue weight. Figure 21B: A bar graph illustrating the relative distribution of radiolabeled 125I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, and F109C) conjugated with D-Glu10 (i.e., a linear polymer of 10 D-glutamic acid) as a percentage of the total counts found in each of the individual organs. Counts are normalized per gram of tissue weight. [Figure 22]Figure 22A: A bar graph illustrating the relative distribution of radiolabeled 125I peptides (e.g., P4C, Ck2.3C, F109C, and PACAPC) conjugated with L-Glu20 (i.e., a linear polymer of 20 L-glutamic acid) as a percentage of the total counts found in each of the individual organs. Counts are normalized per gram of tissue weight. Figure 22B: A bar graph illustrating the relative distribution of radiolabeled 125I peptides (e.g., ODPC, P4C, Ck2.3C, and F109C) conjugated with D-Glu20 (i.e., a linear polymer of 20 D-glutamic acid) as a percentage of the total counts found in each of the individual organs. Counts are normalized per gram of tissue weight. [Figure 23] Figure 23A: A bar graph illustrating the relative distribution of radiolabeled 125I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, F109C, and PACAPC) conjugated with branched L-Asp10 (i.e., branched polymers of 10 L-aspartic acid) as a percentage of the total counts found in each of the individual organs. Counts are normalized per gram of tissue weight. Figure 23B: A bar graph illustrating the relative distribution of radiolabeled 125I peptides (e.g., ODPC, P4C, Ck2.3C, and CTCC) conjugated with branched D-Asp10 (i.e., branched polymers of 10 D-aspartic acid) as a percentage of the total counts found in each of the individual organs. Counts are normalized per gram of tissue weight. [Figure 24]Figure 24A: A bar graph illustrating the relative distribution of radiolabeled 125I peptides (e.g., P4C, Ck2.3C, F109C, and PACAPC) conjugated with L-AAD10 (i.e., linear polymer with 10 L-aminoadipic acid) as a percentage of the total counts found in each of the individual organs. Counts are normalized per gram of tissue weight. Figure 24B: A bar graph illustrating the relative distribution of radiolabeled 125I peptides (e.g., ODPC, CTCC, F109C, and PACAPC) conjugated with L-SDSDD (i.e., linear polymer with L-Ser-Asp-Ser-Asp-Asp; SEQ ID NO: 21) as a percentage of the total counts found in each of the individual organs. Counts are normalized per gram of tissue weight. Figure 24C: A bar graph illustrating the relative distribution of radiolabeled 125I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, F109C, and PACAPC) conjugated with (DSS)6 (i.e., DSSDSSDSSDSSDSSDSS; SEQ ID NO: 22) as a percentage of the total counts found in each individual organ. Counts are normalized per gram of tissue weight. [Diagram 25]Figure 25A: A bar graph illustrating the relative distribution of radiolabeled 125I PTHrP1-39C conjugated with monobisphosphonate, tribisphosphonate, or polyphosphate, radiolabeled 125I PTHrP1-34 conjugated with E10, and radiolabeled 125I PTHrP1-39 conjugated with E20. PTHrP1-39C is PTHrP1-39 with a cysteine ​​(C) at position 40 conjugated with different targeting ligands. Counts are normalized per gram of tissue weight. Figure 25B: A bar graph illustrating the relative distribution of radiolabeled 125I tyrosine conjugated with monobisphosphonate, branched L-Asp4 (i.e., YPegK[DDDD]2; see also SEQ ID NO: 68), or branched L-Asp8 (i.e., YPegK[DDDDDDDD]2; see also SEQ ID NO: 69). "Monobisphosphonate YC" is a peptide with tyrosine and cysteine ​​conjugated with monobisphosphonate. Counts are normalized per gram of tissue weight. [Figure 26] 1 is a graph illustrating the effect of PTH1-34E10 and saline on bone volume after treatment. [Figure 27] 1 is a graph illustrating the effect of PTHrPD10 on bone volume after treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] Brief Description of the Sequence Listing SEQ ID NO:1. PTHrP 1-34 (AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA) SEQ ID NO:2. PTH 1-34 (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF) SEQ ID NO:3: Abaloparatide 1-34 (AVSEHQLLHDKGKSIQDLRRRELLEKLLAKLHTA), with 2-methylalanyl at residue 29 and aminated at residue 34 SEQ ID NO: 4. PTHrP 1-35 (AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAG) SEQ ID NO:5. PTHrP 1-36 (AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAGI) SEQ ID NO:6. PTHrP 1-37 (AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAGIR) SEQ ID NO:7. PTHrP 1-38 (AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAGIRA) SEQ ID NO:8. PTHrP 1-39 (AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAGIRAT) SEQ ID NO: 9. PTHrP 1-40 (AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAGIRATS) SEQ ID NO: 10. PTHrP1-46D10 (VSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAGIRATSEVSPNSDDDD DDDDDD) SEQ ID NO: 11. PTH 1-46D10 (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAP LAPRDADDDDDDDDDD) SEQ ID NO: 12. PTHrP (AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEIRATSEVSPNSKPSPNTKNHPVRFGSDDEGRYLTQETNKVETYKEQPLKTPGKKKKGKPGKRKEQEKKKRRTRSAWLDSGVTGSGLEGDHLSDTSTTSLELDSRRH) SEQ ID NO: 13. PTH (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ) SEQ ID NO: 14: Heparin-binding domain of FGF2 ("F109C") (YKRSRYTC) SEQ ID NO:15: Pituitary adenylate cyclase activating polypeptide ("PACAPC") (HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNKC) SEQ ID NO: 16: Chemotactic cryptic peptide derived from the CTX domain of type III collagen ("CTCC") (YIAGVGGEKSGGFYC) SEQ ID NO: 17: Casein kinase 2 beta chain ("Ck2.3C") (RQIKIWFQNRRMKWKKIPVGESLKDLIDQC) SEQ ID NO: 18: Osteopontin derived peptide ("ODPC") (DVDVPDGRGDSLAYGC) SEQ ID NO:19: P4-BMP2 ("P4C") (KIPKASSVPTELSAISTLYLC) SEQ ID NO: 20: Preptin D10 (DVSTSQAVLPDDFPRYDDDDDDDDDD) SEQ ID NO: 21. SDSDD SEQ ID NO: 22. DSSDSSDSSDSSDSSDSS SEQ ID NO: 23. PTH1-34E10 (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFEEEEEEEEEE) SEQ ID NO: 24. PTHrP1-36E10 (AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAGIEEEEEEEEEE) SEQ ID NO: 25: PTHrP1-39E20 (AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAGIRATCMalEEEEEEEEEEEEEEEEEE) [wherein Mal is maleimide in all sequences presented herein] SEQ ID NO:26: F109C conjugated with D10 (YKRSRYTCMaIDDDDDDDDDD) SEQ ID NO:27: F109C conjugated with D20 (YKRSRYTCMaIDDDDDDDDDDDDDDDDDDDD) SEQ ID NO: 28: F109C conjugated with E10 (YKRSRYTCMaleEEEEEEEEEE) SEQ ID NO: 29: F109C conjugated to E20 (YKRSRYTCMaIEEEEEEEEEEEEEEEEEE) SEQ ID NO:30: F109C conjugated with AAD10 (YKRSRYTCMalXXXXXXXXXX, where X is adipic acid) SEQ ID NO: 31: F109C conjugated with SDSDD (YKRSRYTCMaISDSDD) SEQ ID NO:32: F109C conjugated with (DSS)6 (YKRSRYTCMaIDSSDSSDSSDSSDSSDSS) SEQ ID NO: 33: PACAPC conjugated with D10 (HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNKCMalDDDDDDDDDD) SEQ ID NO:34: PACAPC conjugated with D20 (HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNKCMalDDDDDDDDDDDDDDDDDDDD) SEQ ID NO: 35: PACAPC conjugated with E10 (HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNKCMalEEEEEEEEEE) SEQ ID NO: 36: PACAPC conjugated with E20 (HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNKCMalEEEEEEEEEEEEEEEEEE) SEQ ID NO: 37: PACAPC conjugated with AAD10 (HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNKCMalXXXXXXXXXX [wherein X is adipic acid]) SEQ ID NO: 38: PACAPC conjugated with SDSDD (HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNKCMalSDSDD) SEQ ID NO: 39: PACAPC conjugated with (DSS)6 (HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNKCMalDSSDSSDSSDSSDSSDSS) SEQ ID NO: 40: CTCC conjugated with D10 (YIAGVGGEKSGGFYCMalDDDDDDDDDD) SEQ ID NO: 41: CTCC conjugated with D20 (YIAGVGGEKSGGFYCMalDDDDDDDDDDDDDDDDDDDD) SEQ ID NO: 42: CTCC conjugated with E10 (YIAGVGGEKSGGFYCMalEEEEEEEEEE) SEQ ID NO: 43: CTCC conjugated with E20 (YIAGVGGEKSGGFYCMalEEEEEEEEEEEEEEEEEEEE) SEQ ID NO: 44: CTCC conjugated with AAD10 (YIAGVGGEKSGGFYCMalXXXXXXXXXX [wherein X is adipic acid]) SEQ ID NO: 45: CTCC conjugated with SDSDD (YIAGVGGEKSGGFYCMalSDSDD) SEQ ID NO: 46: CTCC conjugated with (DSS)6 (YIAGVGGEKSGGFYCMalDSSDSSDSSDSSDSSDSS) SEQ ID NO: 47: Ck2.3C conjugated with D10 (RQIKIWFQNRRMKWKKIPVGESLKDLIDQCMalDDDDDDDDDD) SEQ ID NO: 48: Ck2.3C conjugated with D20 (RQIKIWFQNRRMKWKKIPVGESLKDLIDQCMalDDDDDDDDDDDDDDDDDDDD) SEQ ID NO: 49: Ck2.3C conjugated with E10 (RQIKIWFQNRRMKWKKIPVGESLKDLIDQCMalEEEEEEEEEE) SEQ ID NO:50: Ck2.3C conjugated with E20 (RQIKIWFQNRRMKWKKIPVGESLKDLIDQCMalEEEEEEEEEEEEEEEEEE) SEQ ID NO:51: Ck2.3C conjugated with AAD10 (RQIKIWFQNRRMKWKKIPVGESLKDLIDQCMalXXXXXXXXXX [wherein X is adipic acid]) SEQ ID NO: 52: Ck2.3C conjugated with SDSDD (RQIKIWFQNRRMKWKKIPVGESLKDLIDQCMalSDSDD) SEQ ID NO:53: Ck2.3C conjugated with (DSS)6 (RQIKIWFQNRRMKWKKIPVGESLKDLIDQCMalDSSDSSDSSDSSDSSDSS) SEQ ID NO:54: ODPC conjugated with D10 (DVDVPDGRGDSLAYGCMalDDDDDDDDDD) SEQ ID NO:55: ODPC conjugated with D20 (DVDVPDGRGDSLAYGCMalDDDDDDDDDDDDDDDDDDDD) SEQ ID NO:56: ODPC conjugated with E10 (DVDVPDGRGDSLAYGCMalEEEEEEEEEE) SEQ ID NO:57: ODPC conjugated with E20 (DVDVPDGRGDSLAYGCMalEEEEEEEEEEEEEEEEEEEE) SEQ ID NO:58: ODPC conjugated with AAD10 (DVDVPDGRGDSLAYGCMalXXXXXXXXXX [wherein X is adipic acid]) SEQ ID NO: 59: ODPC conjugated with SDSDD (DVDVPDGRGDSLAYGCMalSDSDD) SEQ ID NO: 60: ODPC conjugated with (DSS)6 (DVDVPDGRGDSLAYGCMalDSSDSSDSSDSSDSSDSS) SEQ ID NO: 61: P4C conjugated with D10 (KIPKASSVPTELSAISTLYLCMalDDDDDDDDDD) SEQ ID NO: 62: P4C conjugated with D20 (KIPKASSVPTELSAISTLYLCMalDDDDDDDDDDDDDDDDDDDD) SEQ ID NO: 63: P4C conjugated with E10 (KIPKASSVPTELSAISTLYLCMalEEEEEEEEEE) SEQ ID NO: 64: P4C conjugated with E20 (KIPKASSVPTELSAISTLYLCMaIEEEEEEEEEEEEEEEEEE) SEQ ID NO: 65: P4C conjugated with AAD10 (KIPKASSVPTELSAISTLYLCMalXXXXXXXXX [wherein X is adipic acid]) SEQ ID NO: 66: P4C conjugated with SDSDD (KIPKASSVPTELSAISTLYLCMalSDSDD) SEQ ID NO: 67: P4C conjugated with (DSS)6 (KIPKASSVPTELSAISTLYLCMaIDSSDSSDSSDSSDSSDSS) SEQ ID NO: 68: Branched D4 Y (YPegKDDDDDDDD [wherein Peg is polyethylene glycol]) SEQ ID NO: 69: Branched D8 Y (YPegKDDDDDDDDDDDDDDDD [wherein Peg is polyethylene glycol]) SEQ ID NO: 70: F109C conjugated with branched D10 (YKRSRYTCMaIK[DDDDDDDDDD]2) SEQ ID NO: 71: PACAPC conjugated with branched D10 (HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNKCMalK[DDDDDDDDDD]2) SEQ ID NO: 72: CTCC conjugated with branched D10 (YIAGVGGEKSGGFYCMalK[DDDDDDDDDD]2) SEQ ID NO: 73: Ck2.3C conjugated with branched D10 (RQIKIWFQNRRMKWKKIPVGESLKDLIDQCMalK[DDDDDDDDDD]2) SEQ ID NO: 74: ODPC conjugated with branched D10 (DVDVPDGRGDSLAYGCMalK[DDDDDDDDDD]2) SEQ ID NO: 75: P4C conjugated with branched D10 (KIPKASSVPTELSAISTLYLCMaIK[DDDDDDDDDD]2) SEQ ID NO: 76: F109C conjugated with branched E10 (YKRSRYTCMaIK[EEEEEEEEEE]2) SEQ ID NO: 77: PACAPC conjugated with branched E10 (HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNKCMalK[EEEEEEEEEE]2) SEQ ID NO: 78: CTCC conjugated with branched E10 (YIAGVGGEKSGGFYCMalK[EEEEEEEEEE]2) SEQ ID NO: 79: Ck2.3C conjugated with branched E10 (RQIKIWFQNRRMKWKKIPVGESLKDLIDQCMalK[EEEEEEEEEE]2) SEQ ID NO: 80: ODPC conjugated with branched E10 (DVDVPDGRGDSLAYGCMalK[EEEEEEEEEE]2) SEQ ID NO: 81: P4C conjugated with branched E10 (KIPKASSVPTELSAISTLYLCMaIK[EEEEEEEEEE]2)

[0074] Detailed Description While the concepts of the present disclosure have been illustrated and described in detail in the drawings and description herein, it is understood that the results in the drawings and their description are to be considered as illustrative in nature and not restrictive, and that only exemplary embodiments have been shown and described, and that all changes and modifications that come within the spirit of the present disclosure are desired to be protected.

[0075] Unless otherwise defined, scientific and technical terms have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0076] Unless expressly stated or clearly implied otherwise, the term "about" as used herein refers to a range of a value plus or minus 10 percent, e.g., about 1.0 encompasses values ​​from 0.9 to 1.1.

[0077] Unless otherwise stated or implied, the term "treating" as used herein includes administering to a human or animal patient at least one dose of a compound, where treating includes preventing or reducing the likelihood and / or severity of at least one disease, as well as limiting the duration or severity of the disease, which may or may not result in a cure of the disease.

[0078] Unless expressly stated or clearly implied otherwise, the terms "therapeutically effective dose", "therapeutically effective amount", and the like, as used herein, refer to the portion of a compound that has a net positive effect on the health and well-being of a human or other animal. Therapeutic effects can include improvements in lifespan, quality of life, and the like, and these effects can also include reduced susceptibility to disease development or deterioration of health or well-being. Effects can be achieved rapidly after a single administration and / or treatment, or cumulatively after a series of administrations and / or treatments. A "therapeutically effective amount" generally means an amount sufficient to affect the desired degree of treatment for a disease when administered to a subject or animal for treating the disease.

[0079] As used herein, "inhibition" or "inhibitory activity" each encompasses a total or partial reduction in the activity or effect of an enzyme, or a pathway involving the enzyme caused directly or indirectly by the inhibitor, or a pathway caused directly or indirectly by the activity of an enzyme caused directly or indirectly by the inhibitor.

[0080] The term "pharmaceutically acceptable salts," as used herein, is defined as salts that retain the desired biological activity of the inhibitors and exhibit minimal undesired toxicological effects. Non-limiting examples of such salts are: (a) acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like) and salts formed with organic acids (e.g., acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like); (b) base addition salts formed with metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or cations formed from ammonia, N,N-dibenzylethylenediamine, D-glucosamine, tetraethylammonium, or ethylenediamine; or (c) combinations of (a) and (b); for example, zinc tannate, and the like.

[0081] Pharmaceutically acceptable salts include salts of the compounds of the invention that are safe and effective for use in mammals and that possess the desired therapeutic activity. Pharmaceutically acceptable salts include salts of acidic or basic groups present in the 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, gentisate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). Certain compounds of the present invention may form pharma- ceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum salts, calcium salts, lithium salts, magnesium salts, potassium salts, sodium salts, zinc salts, and diethanolamine salts.For further information on some pharma- ceutically acceptable salts that can be used to practice the present invention, see Berge, et al., 66 J. PHARM. SCI. 1-19 (1977); Haynes, et al., J. Pharma. Sci., Vol. 94, No. 10, Oct. 2005, pgs. 2111-2120, and see, for example, P. Stahl, et al., Handbook of Pharmaceutical Salts: Properties, Selection and Use, (VCHA / Wiley-VCH, 2002); SM Berge, et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977.

[0082] Pharmaceutical formulations: The compounds of the present invention and their salts can be formulated as pharmaceutical compositions for administration. Such pharmaceutical compositions and processes for making them are known in the art for both human and non-human mammals. See, for example, Remington: The Science and practice of pharmacy (A. Gennaro, et al., eds., 1999). th ed., Mack Publishing Co., 1995). The formulations can be administered via a variety of means, including oral administration, parenteral administration such as injection (intramuscular, subcutaneous, intravenous, intraperitoneal); transdermal administration such as dipping, spraying, basting, washing, pour-on and spotting-on, and dusting. Additional active ingredients may also be included in the formulation containing the compound of the present invention or its salt.

[0083] The pharmaceutical preparation of the present invention includes pharmaceutical preparations suitable for oral administration, parenteral administration (including subcutaneous, intradermal, intramuscular, and intravenous) and rectal administration.The preparation can be presented in unit dosage form and can be prepared by any of the methods well known in the pharmaceutical arts.All methods include the step of associating the active ingredient, i.e., the compound or salt of the present invention, with a carrier.In general, the preparation is prepared by uniformly and intimately associating the active ingredient with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, forming the associated mixture into a desired preparation.

[0084] Pharmaceutical formulations of the invention suitable for oral administration can be presented as discrete units, such as capsules, sachets, tablets, or lozenges, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid, such as a syrup, elixir, or draught; or as a liquid oil-in-water emulsion or a liquid water-in-oil emulsion. The formulation can also be a bolus, electuary, or paste.

[0085] Pharmaceutical formulations of the present invention suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, but also aqueous and non-aqueous sterile suspensions which may contain antioxidants, buffers, bacteriostats, and solutions which render the composition isotonic with the blood of the recipient, as well as suspending agents and thickening agents, for example. The formulations can be presented in single unit-dose or multi-dose containers, but can also be stored in a freeze-dried condition requiring the addition of a sterile liquid carrier prior to use.

[0086] Pharmaceutically acceptable carrier: As used herein, unless otherwise stated or implied, pharma- ceutically acceptable carrier is used to describe any ingredient, other than the active component, that may be included in a formulation. The choice of carrier will depend in large part 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.

[0087] Tablets can be made by compressing or molding the active ingredient with a pharma- ceutically acceptable carrier. Compressed tablets can be prepared by compressing the active ingredient in a free-flowing form, such as powder or granules, in a suitable machine, for example, with a binder, an inert diluent, a lubricant, a disintegrant, and / or a surfactant. Molded tablets can be prepared by molding a mixture of the powdered active ingredient moistened with an inert liquid diluent in a suitable machine. Tablets can be optionally coated or scored, and can be formulated to provide slow or controlled release of the active ingredient.

[0088] "Bone-related disorders" as used herein include, but are not limited to, osteopenia, osteoporosis, rheumatoid arthritis, hematological disorders, autoimmunity, transplant rejection, bone cancer, and / or bone fractures.

[0089] The "therapeutic affinity index" described herein is the affinity range of a therapeutic ligand, usually represented by the IC50 of the therapeutic ligand or its agonist, to participate in a receptor-mediated signaling cascade (event) to achieve a desired therapeutic effect. For example, if a ligand has an IC50 of 4nM to 15nM to induce a G protein-coupled receptor-mediated response that achieves the therapeutic effect of the ligand, the ligand has a therapeutic affinity index of IC50 of 4nM to 15nM. If a therapeutic ligand has different modes of action to achieve its different therapeutic effects, the therapeutic affinity index may be complicated. For example, if the receptor of a therapeutic ligand has two different conformations, each conformation may have a significantly different therapeutic affinity index.

[0090] As used herein, unless expressly stated or clearly implied otherwise, the terms "PTHrP1-46D10," "PTHrP D10," and "targeted PTHrP" may be used interchangeably to define the same compound.

[0091] Unless expressly stated or clearly implied otherwise, the terms "PTHrP1-34," "PTHrP," ​​"free PTHrP," ​​and "non-targeted PTHrP" as used herein may be used interchangeably to define the same compound.

[0092] Osteoporosis is defined as a decrease in bone mineral density and / or deterioration of bone microarchitecture associated with an increased risk of fracture. This chronic disease mainly affects postmenopausal women, but can also affect elderly men. The disease is increasingly considered an age-related morbidity. The skeletal degeneration observed in patients with osteoporosis is the result of a relative deficiency of bone formation compared to bone resorption. Osteoporosis treatment is largely based on antiresorptive drugs. One recent alternative therapy for treating osteoporosis is based on the intermittent administration of parathyroid hormone (PTH). PTH is mainly secreted by cells of the parathyroid gland as a prohormone polypeptide containing 84 amino acids. Effective hormone-receptor interaction requires only the N-terminal 34 amino acids. PTH is a circulating calcium ion (Ca 2+ ) concentration. PTH essentially acts to increase blood calcium concentration by acting on the parathyroid hormone 1 receptor, which is present at high levels in bone and kidney. The protein hPTH(1-34) crystallizes as a slightly bent, long-chain helical dimer. Analysis reveals that the extended helical conformation of hPTH(1-34) is likely to be the bioactive conformation. The N-terminal fragment (parathyroid hormone (PTH) 1-34) has been crystallized and the structure refined to 0.9 Å resolution.

[0093] The bone anabolism caused by PTH therapy is mainly explained by the ability of PTH to increase osteoblast formation and osteoblast survival. PTH and PTH-related protein (PTHrP), a local factor abundant in bone, interact with the common PTH type 1 receptor in osteoblasts with similar affinity. Studies mainly in osteoporotic rodent models and limited data in postmenopausal women suggest that N-terminal PTHrP peptides could be considered as a promising bone anabolic therapy.

[0094] The parathyroid hormone / parathyroid hormone-related peptide receptor, also known as parathyroid hormone receptor 1 (PTHR1), is a protein that, in humans, is encoded by the PTHR1 gene. PTHR1 functions as a receptor for parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP).

[0095] PTHR1 belongs to the B-type family of G protein-coupled receptors (GPCRs) that are expressed primarily in bone, kidney, and cartilage, but also in other tissues, including the vasculature and certain developing organs.

[0096] The N-terminal fragments consisting of the first 34 residues of PTH and PTHrP are generally believed to contain the key functional determinants of receptor interaction present in the corresponding full-length mature polypeptide chains, which also contain 84 and 141 amino acid residues, respectively. PTH and PTHrP differ notably among the B-type family peptide ligands in that they contain extended C-terminal segments. The biological roles of these segments remain unclear, although some functional responses have been identified, such as the ability of a fragment corresponding to the C-terminal portion of PTH to induce a proapoptotic effect in bone cells17, and of a fragment encompassing the central region of PTHrP.

[0097] The conformation that is formed when a ligand is coupled to a heterotrimeric G protein (R G conformation) or the conformation in which PTHR1 is not coupled to a G protein (R 0 Membrane binding assays developed to assess the affinity of PTH and PTHrP analogs for PTHR1 in the 1-3 conformation provided the first clues that structurally distinct PTH and PTHrP analogs may bind with altered affinity to different conformational states of the receptor. Direct comparative studies with PTH(1-34) and PTHrP(1-36) demonstrated that these two peptides bind to the R GFor states, we maintain the same affinity, but for R 0 PTH(1-34) does not have the same affinity for the R 0 These results demonstrated that PTHrP(1-36) exhibits a much greater affinity for PTHrP than PTHrP(1-36).

[0098] Abaloparatide, PTHrP, and PTH share high homology in the first 13 amino acids of each protein. To the extent that each active anabolic agent induces signaling by parathyroid hormone and includes peptides that include or contain minimal homology at residues 2, 3, 4, 6, 7, 9, 12, and 13, the anabolic peptide variants can be combined with the various linkers, optionally spacers, and targeting ligands described in this disclosure to provide targeted delivery of the fracture healing agent.

[0099] The functional consequences of this altered selectivity were typically demonstrated in cAMP assays. PTH(1-34) and PTHrP(1-36) had similar potencies in conventional cAMP dose-response assays (R G In accordance with their similar affinity for the IL-1 signaling pathway and the GαS-mediated mechanism of intracellular cAMP production, the duration of the responses induced by the two ligands (assessed using a time-course washout assay) differs, with PTH(1-34) showing a more sustained response than PTHrP(1-36). In general, the duration of the cAMP response observed in cell-based studies is significantly longer than that assessed in the membrane assay, R G PTH(1-34) and PTHrP(1-36), rather than their affinity for the condition, 0 R 0 The R state is uncoupled from G proteins and is therefore inactive with respect to cAMP signaling. 0 The complex is quite stable over time, but may be capable of isomerizing into a functional G protein-coupled state.

[0100] R differs between PTH ligands and PTHrP ligands G R, as opposed to affinity in 0 One key structural determinant for affinity for PTHrP(1-36) can be traced to the identity of the residue at position 5, so that by replacing His5 in PTHrP(1-36) with the corresponding isoleucine in PTH, R 0 The affinity of the antibody to cAMP is significantly enhanced, and the duration of the cAMP signaling response induced in the target cells is extended.

[0101] Currently, PTH, PTHrP, or their agonists are being investigated for local or systemic administration in an attempt to provide an anabolic agent to heal any fracture. However, these studies have revealed that side effects are observed for the use of these proteins. For example, local application of PTH or PTHrP requires exposure of the bone, which results in increased healing time, pain, discomfort, and even the possibility of infection. Alternatively, systemic application of PTH and PTHrP tends to have off-target effects, including increased blood calcium levels. Therefore, it is desirable to develop a bone anabolic agent delivery system that can reduce the side effects mentioned above.

[0102] Disclosed herein is a drug delivery system that includes a drug, a linker, and a targeting ligand. Some embodiments of the present disclosure provide compounds for bone fracture targeting and healing. These compounds may include at least three distinct structural / functional regions: an effective anabolic peptide or any agonist that is involved in the parathyroid hormone receptor 1 (PTHR1) and the subsequent signaling cascade that leads to healing; a linker with or without a spacer that provides variable arm length for the anabolic peptide to reach the fracture site; and a targeting ligand that typically includes a negatively charged oligopeptide or its equivalent that directs the compound to the fracture site and specifically binds to hydroxyapatite and / or living bone. In one particular embodiment, the drug may be the first 34 amino acids in parathyroid hormone-related peptide (PTHrP). The linker may include amino acids 35-46 of PTHrP, which separates the drug from the targeting ligand and also provides some leeway in drug length, since amino acids 35-40 are believed, in part, to enhance receptor binding. Finally, the targeting ligand may include an aspartic acid decapeptide, although other polymers functionalized with carboxylic acids would likely be attached in a similar fashion (e.g., D- or L-glutamic acid, D- or L-aspartic acid, and aminohexanedioic acid, in various combinations and configurations).

[0103] A peptide comprising SEQ ID NO:3 with methyl Ala at residue 29 and aminated at residue 34 (abaloparatide 1-34) can be used to treat bone fractures as a potent active anabolic agent without additional linkers or targeting ligands.

[0104] It is envisioned that PTH and PTHrP can have variants that retain agonist activity for PTHR1 and can be used in place of PTH or PTHrP to participate in fracture healing and achieve a therapeutic effect in fracture healing. As long as each active anabolic agent induces signaling by parathyroid hormone and includes or contains a peptide with minimal homology at residues 2, 3, 4, 6, 7, 9, 12, and 13, the proposed anabolic peptide variants can be combined with various linkers, optionally spacers, and targeting ligands described in the present disclosure to provide targeted delivery of fracture healing agents. For example, conservative substitutions or modifications at residues 1, 5, 10, 11, or 14-34 of PTH or PTHrP, along with any combination of linker sequences and targeting ligands described herein, are also envisioned for protection in the present disclosure.

[0105] In one aspect of the present disclosure, the targeting ligand comprises an acidic oligopeptide comprising a plurality of aspartic acid residues. The number of D-aspartic acid residues or L-aspartic acid residues can be from about 4 to about 10 or from about 10 to about 20 residues. The oligopeptide can be linear or branched. In an exemplary embodiment, a lysine residue is used as a branch point. In another aspect of the present invention, the aspartic acid can be L-aspartic acid, D-aspartic acid, or a mixture of both optical isomers. The advantage of including D-aspartic acid in an oligopeptide is that it can be less susceptible to proteolysis compared to an oligopeptide that contains only L-aspartic acid in nature.

[0106] In other aspects of the disclosure, the acidic oligopeptide can be no more than 20 L-glutamic acids or D-glutamic acids. In yet other aspects of the disclosure, the acidic oligopeptide can be no more than 20 L- or D-aspartic acids, or a combination of L-glutamic acids or D-glutamic acids.

[0107] In some aspects of the present disclosure, the targeting ligand can be a polyphosphate or at least one bisphosphate. In yet other aspects of the present disclosure, the targeting ligand can be a collagen mimetic peptide. Such collagen mimetic peptides are inserted between incomplete collagen fibrils at the fracture site. In one aspect of the present disclosure, the collagen mimetic peptide can have the structure [Gly-Pro-Hyp]9-OH.

[0108] In some embodiments of the present disclosure, the targeting ligand can be aminohexanedioic acid (alpha-aminoadipic acid) or a derivative thereof with more than one carbon between the backbone and the acid. For example, the targeting ligand can be 2-aminomalonic acid.

[0109] In some aspects of the present disclosure, the targeted delivery compound further comprises at least one spacer comprising PEG (polyethylene glycol).

[0110] There can be linker sequences of variable length between the active anabolic compounds, such as PTH or PTHrP, or their respective agonists and targeting ligands. In some embodiments of the present disclosure, the linker can be any portion of the extension of the active fragment of PTH or PTHrP, i.e., residues 35-84 of PTH or residues 35-173 of PTHrP. Such active fragment extensions are typically not releasable, and the linker sequence can be any portion of the extension or a combination of different portions of the extension.

[0111] In some other aspects of the present disclosure, the linker can be a hydrolyzable substrate that is sensitive to at least one abundant moiety produced in osteoclasts during bone remodeling. For example, cathepsin K is a moiety produced in osteoclasts during bone remodeling. A linker sequence that includes Gly-Gly-Pro-Nle (wherein Nle is norleucine, leucine, isoleucine, or any other equivalent with hydrophobic modification) can be used as a substrate for cathepsin K. When the targeting compound is delivered to the osteoclast site, cathepsin K can hydrolyze the linker and release the active anabolic compound to affect bone healing.

[0112] Yet another hydrolyzable linker can contain a disulfide bond, which can be released by glutathione in osteoclasts.

[0113] Yet another hydrolyzable linker can be a releasable ester.

[0114] These features of the present disclosure are further supported by the following examples. EXAMPLES

[0115] material and method synthesis Peptides were synthesized by solid phase peptide synthesis or recombinant expression.

[0116] Solid Phase Peptide Synthesis Briefly, 2-chlorotrityl resin (1.11 mmol per g) was loaded overnight in a solid-phase peptide synthesis vial capable of being bubbled with nitrogen with the first amino acid at 0.4 mmol per g in DCM and DIPEA. The resin was then capped with four 5 mL washes with DCM / MeOH / DIPEA (17:2:1), followed by three washes with DCM and DMF, respectively. After each amino acid coupling reaction, the Fmoc group was removed by incubation with 20% (v / v) piperidine in DMF for three 10 min each. The resin was then washed three times with DMF before the next amino acid was added. Each amino acid was added in a 5-fold excess with HBTU / DIPEA. Upon completion of the synthesis, the peptide was cleaved using 95:2.5:2.5 trifluoroacetic acid:water:triisopropylsilane. Cysteine-containing peptides were cleaved using 95:2.5:2.5 trifluoroacetic acid:triisopropylsilane:water and a 10-fold excess of TCEP (tris(2-carboxyethyl)phosphine).

[0117] Recombinant protein expression An ampicillin resistance plasmid was created containing the T7 promoter, thioredoxin coding sequence, HisTag sequence, tryptophan residues, and peptide coding sequence. Competent cells were transformed with the plasmid and plated on agar plates containing ampicillin. Single colonies were selected and expanded overnight in ampicillin (100ug / ml) LB medium at 37°C. Competent cells were then further expanded in 1 liter of ampicillin (100ug / ml) LB medium for 15 hours. After 15 hours, IPTG was added to reach a final concentration of 1 mM, and the medium was stirred at 37°C, 180 rpm for 5 hours. Cells were then pelleted and lysed by sonication in 20 mM Tris-HCL, pH 8, containing 6 M guanidine HCl. The fusion protein was then isolated by elution from the HisTag using imidazole. The fusion protein-containing fraction was dialyzed and lyophilized. Proteolytic cleavage was performed using the iodosobenzoic acid method. The final peptide was purified using an anion exchange column, dialyzed, and lyophilized for further use.

[0118] Characterization The molecular weight of the peptide was confirmed using HPLC / MS.

[0119] Induction of fractures in mice All animal studies were performed in accordance with protocols established by Purdue's animal care and use committee and as described in the literature. CD4 Swiss mice (30-35 g) obtained from the Harlan Laboratory were used for these experiments. Fixed femoral fractures were performed under aseptic conditions with isoflurane anesthesia. The skin around the knee was shaved and cleaned first with an alcohol pad and then with Betadine solution. A skin incision was made via a medial parapatellar approach. The patella was then removed and an incision was made below the patella. The intramedullary canal was reamed using a 25-gauge needle. A 22-gauge locking nail (both ends were flattened to provide rotational stability) was then inserted. The wound was ligated and then fractured using a three-point bending device with built-in stops to prevent overinjury. Subcutaneous buprenorphine (0.05-0.1 mg / kg) was administered at the time of surgery and then every 12 hours for 3-7 days after operation.

[0120] Dosage: Mice were dosed subcutaneously daily with 31 nmoles of peptide / kg or saline control, the first dose administered 6 hours after fracture, continued throughout the study, and the final dose administered the day before euthanasia.

[0121] Bone density analysis CT images and bone data were collected using a Scanco μCT 40. Bones were scanned while submerged in PBS to prevent dehydration. Images were analyzed for bone mineral density, total volume (TV), relative bone volume (BV / TV), trabecular width (Tb.Th), and trabecular center distance (Tb.Sp) using ImageJ software. Volumes of interest included the fracture ossification and both cortical and trabecular bone between points on the cortical bone at the fracture site.

[0122] Statistical analysis was performed using Prism GraphPad software. Data are presented in Results as mean ± standard error of the mean (SEM). Unpaired Student's t-test was used to determine statistical significance, and P values ​​less than 0.05 were considered statistically significant.

[0123] Example 1 PTHrP Delivery System for Targeted Fracture Healing In this example, a bone fracture targeting drug was synthesized, comprising a drug, a linker, and a targeting ligand.

[0124] A pharmaceutical sequence comprising amino acid residues 1-46 of PTHrP followed by 10 aspartic acids is listed in SEQ ID NO: 10. As described above, residues 1-34 are the active portion of PTHrP, residues 35-46 are the linker portion of the pharmaceutical proposed for healing fractures, and the 10 aspartic acids are the targeting ligand.

[0125] Closed femoral fractures were performed in three groups of mice. Mice were administered targeted PTHrP (31 nmoles per kg per day), PTHrP 1-34 (31 nmoles per kg per day), or saline daily for four weeks. At the end of the study, mice were euthanized by CO2, femurs were harvested, and bone mineral density was determined by MicroCT.

[0126] As shown in Figure 1, the targeted form of PTHrP increased peri-fracture bone density significantly more than free PTHrP and saline, indicating that a strategy using a potent anabolic agent linked to a targeting ligand sequence may work for fracture healing.

[0127] Another way to indicate bone healing progress is to measure trabecular center distance data collected by microCT. As shown in Figure 2, 100 CT frames (slices) selected from the ossification of each fracture were analyzed. The stack of frames was selected from the fracture area with the greatest ossification. From left to right, targeted PTHrP (PTHrP1-46 followed by 10 aspartates) and saline control. Targeted PTHrP shows a statistically significant reduction in center distance over saline control.

[0128] Compared to traditional single anabolic agent administration, which typically results in high calcium levels in the blood, the present application provides an alternative that is superior to the need for localized application of fracture healing agents, which reduces the risk of bone exposure associated with high blood calcium levels or infection.

[0129] It is envisioned that various conservative substitutions into the first 34 amino acids will result in the same or better restoration of bone density in mice with fractures.

[0130] It is also contemplated that other portions of the PTHrP extension sequence beyond 1-34 may be used to provide equal or greater attachment to the 10 aspartic acid targeting ligand.

[0131] It is further envisioned to use any linker and targeting ligand described in this disclosure. For example, the linker can be a natural peptide variant of the PTH sequence, or any cathepsin K-sensitive linker, such as Gly-Gly-Pro-Nle, where Nle is norleucine or another hydrophobic amino acid, such as leucine or isoleucine. The linker can be a disulfide linker that can be released in a reducing environment. Glutathione is usually released in certain types of injury and can reduce disulfide bonds. It is envisioned that the disulfide linker can increase the efficacy of anabolic agents. The linker can also be an ester that is hydrolyzed and released to increase healing efficiency. The targeting ligand is usually an acidic oligopeptide chain that contains four or more acidic amino acid residues and binds to hydroxyapatite and / or living bone. These acidic amino acid residues can be either aspartic acid or glutamic acid or a combination thereof. In some cases, the acidic oligopeptide can be a branched acidic oligopeptide with at least one lysine to increase accumulation of the drug at the fracture site. The branch can be multiple, such as two, three, or four branches.

[0132] Another option for the targeting ligand is one or more bisphosphonates, i.e. polybisphosphonates. The collagen mimetic peptide has the sequence [Gly-Pro-Hyp]9-OH.

[0133] Yet another option for targeting ligands is aminohexanedioic acid (alpha-aminoadipic acid) or its derivatives with more than one carbon between the backbone and the acid. For example, 2-aminomalonic acid can be used as a targeting ligand for PTHrP or its variants.

[0134] Spacers such as PEG (polyethylene glycol) can be added to the synthesized targeted drug delivery systems to reduce the possibility that the targeting ligand will interfere with anabolic efficiency.

[0135] The different combinations of PTHrP variant-linker-targeting ligand options can be tested for bone density recovery in the same manner as described in this embodiment.Specifically, the synthesized drug-linker-targeting ligand is compared with PTHrP variant itself and saline solution for their effect on bone density around fracture.It is expected that the targeting form of PTHrP variant will increase bone density around fracture more significantly than free PTHrP variant and saline solution.

[0136] Example 2 A variety of different combinations of anabolic drugs with linker options and targeting ligands to provide targeted delivery of drugs to bone fractures for healing. In this example, the first 34 amino acids of PTH are synthesized with a suitable linker as described herein, and a suitable targeting ligand. As in Example 1, the linker can be any segment of the active PTH extension, including residues 35-84, or other linkers described in Example 1. The targeting ligand can be any of the targeting ligands described in Example 1. As shown in FIG. 3, the linker is 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) can be compared to the PTH variant itself and saline for their effect on bone density around fractures. The targeted form of the PTH variant is predicted to increase bone density around fractures significantly more than the free PTH variant and saline. FIG. 3 showed that targeted PTH 1-46D10 produced a statistically significant increase in bone mineral density compared to saline controls.

[0137] It is believed that some other effective anabolic drug could be used to replace PTH(1-34) in this example to achieve fracture healing.

[0138] Example 3 Abaloparatide 1-34 as a free-standing fracture healing agent In this example, abaloparatide 1-34 (SEQ ID NO: 3) was examined, with modified residues at 29 as methyl Ala and at 34 as aminated Ala. Abaloparatide has been examined in treating osteoporosis to prevent fractures. Its ability to heal actual fractures, a different process, is also being examined. Of course, linking abaloparatide 1-34 to the linkers and targeting ligands described in the previous examples would likely increase bone density at the fracture site for targeted delivery.

[0139] Now, referring to FIG. 1, 100 CT frames (slices) selected from the ossification of each fracture were analyzed. The CT frames were taken after 4 weeks. The stack of frames was selected from the fracture area with the greatest ossification. From left to right: targeted PTHrP (PTHrP1-46 followed by 10 aspartates), free unconjugated PTHrP (PTHrP1-34), and saline control. The targeted form of PTHrP increased bone density around the fracture significantly more than did free PTHrP and saline.

[0140] Now referring to FIG. 2, a decrease in central distance is associated with an increase in bone density and may indicate progress in healing. CT frames were taken after 4 weeks. 100 CT frames (slices) selected from the ossification of each fracture were analyzed. A stack of frames was selected from the fracture area with the greatest ossification. From left to right, targeted PTHrP (PTHrP1-46 followed by 10 aspartates) and saline control. Targeted PTHrP shows a statistically significant reduction in central distance compared to saline control.

[0141] Now referring to FIG. 3, 100 CT frames (slices) selected from the ossification of each fracture were analyzed. The CT frames were taken after 2 weeks. The stack of frames was selected from the fracture area where ossification was maximal. From left to right, targeted PTH (PTH 1-46 followed by 10 aspartates) and saline control. Targeted PTH results in a statistically significant increase in bone mineral density compared to the saline control.

[0142] Now, referring to Figure 4, the expression ratio of alkaline phosphatase (ALP) for both targeted and non-targeted PTHrP was analyzed in MC3T3E1 cells. The ALP ratio is calculated by (ALP expression with treatment / ALP expression with vehicle control). The increased expression level is associated with increased osteoblast activity and is a key protein involved in bone mineralization. The addition of targeting ligand does not reduce the efficacy of the drug.

[0143] Now, referring to Figure 5, the expression ratio of osteopontin (OPN) for both targeted and non-targeted PTHrP was analyzed in MC3T3E1 cells. OPN ratio is calculated by (OPN expression with treatment / OPN expression with vehicle control). Increased expression level is associated with increased osteoblast activity and is a key protein involved in bone mineralization. Addition of targeting ligand does not reduce the efficacy of the drug.

[0144] Now, referring to FIG. 6, the gene expression ratios of several key bone markers for both targeted and non-targeted PTHrP were analyzed in MC3T3E1 cells. The activity markers include alkaline phosphatase (ALP), type I collagen alpha (Col1 alpha), osteocalcin (OC), osteoprotegerin (OPG), osteopontin (OPN), and osterix (OSX). The gene expression ratios are calculated by (gene expression with treatment / gene expression with vehicle control). The activity of targeted PTHrP is as low as 1 pM.

[0145] [Table 1]

[0146] Now, with reference to Table 1, mice were treated with subcutaneous injections of PTHrPD10 (the "targeted drug") or saline at 31 nmoles per kg per day. Swiss ND4 mice were treated for 28 days. Mice were sacrificed at the end of the study and livers and kidneys were excised. Organs were fixed in formalin and paraffin sections were prepared from each. A veterinary pathologist performed a randomized blinded analysis of the organs. No detectable toxicity was observed. Lesions recorded were of minimal importance and not associated with obvious tissue damage (necrosis). It is believed that this type of lesion is unlikely to cause clinical signs or disease. These lesions are likely within normal limits for these animals.

[0147] Now, referring to FIG. 7, mice were treated with subcutaneous injections of PTHrPD10 (targeted) or PTHrP (free) at 31 nmoles per kg per day. Swiss ND4 mice were treated for 14, 28, or 56 days. Mice were sacrificed at the end of the treatment period for each study, and femurs were excised. Fracture bone density was measured using a Scanco microCT. 100 slices of each fracture with the largest bone diameter were selected for measurement. "Targeted 2" represents mice administered targeted PTHrP for 2 weeks (14 days). "Free 2" represents mice administered unmodified PTHrP for 2 weeks (14 days). "Targeted 4" represents mice administered targeted PTHrP for 4 weeks (28 days). "Free 4" represents mice administered with unmodified PTHrP for 4 weeks (28 days). "Targeted 8" represents mice administered with targeted PTHrP for 8 weeks (56 days). "Free 8" represents mice administered with unmodified PTHrP for 8 weeks (56 days). Targeted PTHrP can be observed at any time with a higher density than free PTHrp. The maximum difference between targeted PTHrP and free PTHrP is at 2 weeks. These results indicate that targeted drugs not only improve fracture healing, but also accelerate fracture healing.

[0148] Now, referring to FIG. 8, mice were treated with subcutaneous injections of abaloparatide D10 (targeted), abaloparatide (non-targeted) and saline at 31 nmoles per kg per day. Swiss ND4 mice were treated for 28 days. Mice were sacrificed at the end of the study and femurs were excised. Fracture bone density was measured using a Scanco microCT. 100 slices of each fracture with the largest bone diameter were selected for measurement. Abaloparatide has already been used to treat osteoporosis. These results indicate that abaloparatide can also be used to treat fractures. These results indicate that targeted abaloparatide is more effective than free abaloparatide.

[0149] Now, referring to FIG. 9, mice were treated with subcutaneous injections of polyphosphate-targeted PTHrP or saline at 31 nmoles per kg per day. Swiss ND4 mice were treated for 28 days. Mice were sacrificed at the end of the study and femurs were excised. Fracture bone density was measured using a Scanco microCT. 100 slices with the largest bone diameter for each fracture were selected for measurement. Polyphosphate-targeted PTHrP enhanced fracture healing compared to saline.

[0150] Now, referring to FIG. 10, mice were treated with subcutaneous injections of single alendronate, branched trialendronate targeted PTHrP, or free PTHrP at 31 nmoles per kg per day. Swiss ND4 mice were treated for 28 days. Mice were sacrificed at the end of the study and femurs were excised. Fracture bone density was measured using a Scanco microCT. 100 slices with the largest bone diameter for each fracture were selected for measurement. Single alendronate or branched trialendronate targeted PTHrP enhanced fracture healing compared to free PTHrP.

[0151] Example 4 The targeting ligand can include, but is not limited to, oligo acidic amino acids. Exemplary oligo acidic amino acids include linear polymers 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, including L and / or D amino acids; linear polymers 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, including L and / or D amino acids; linear polymers of 10 acidic amino acids, including L and / or D acidic amino acids; 4 on each branch, including L and / or D amino acids; branched polymers of glutamic acid 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; and / or branched polymers of acidic amino 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-acidic amino acids, but are not limited to these. 11-12, exemplary linear and branched polymers of acidic amino acids are shown, and FIG 13 presents exemplary targeting ligands that are not derived from amino acids.

[0152] The targeting ligand is coupled with a maleimide-containing linker. The targeting ligand with the maleimide-containing linker is further conjugated with a variety of peptides with significantly different chemical properties through a cysteine ​​maleimide coupling process. These peptides can include, but are not limited to, a sequence representing the heparin-binding domain of FGF2 (SEQ ID NO: 14), a sequence representing the pituitary adenylate cyclase activating polypeptide (SEQ ID NO: 15), a sequence representing the chemotactic cryptic peptide derived from the CTX region of collagen III (SEQ ID NO: 16), a sequence representing casein kinase 2 beta chain (SEQ ID NO: 17), a sequence representing an osteopontin-derived peptide (SEQ ID NO: 18), and / or a sequence representing P4-BMP2 (SEQ ID NO: 19). 125 The conjugated peptides are iodized via iodination reagents from Pierce, in which I is covalently attached to histidine, tyrosine, and / or tryptophan residues of the conjugated peptide.

[0153] F109C conjugated to branched D10 has the formula: YKRSRYTCMalK[DDDDDDDDDD]2 (see also SEQ ID NO:70).

[0154] PACAPC conjugated with branched D10 has the formula: HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNKCMalK[DDDDDDDDDD]2 (see also SEQ ID NO:71).

[0155] CTCC conjugated with branched D10 has the formula: YIAGVGGEKSGGFYCMalK[DDDDDDDDDD]2 (see also SEQ ID NO:72).

[0156] Ck2.3C conjugated with branched D10 has the formula: RQIKIWFQNRRMKWKKIPVGESLKDLIDQCMalK[DDDDDDDDDD]2 (see also SEQ ID NO:3).

[0157] An ODPC conjugated with branched D10 has the formula: DVDVPDGRGDSLAYGCMalK[DDDDDDDDDD]2 (see also SEQ ID NO:74).

[0158] P4C conjugated with branched D10 has the formula: KIPKASSVPTELSAISTLYLCMalK[DDDDDDDDDD]2 (see also SEQ ID NO:75).

[0159] F109C conjugated to branched E10 has the formula: YKRSRYTCMalK[EEEEEEEEEE]2 (see also SEQ ID NO:76).

[0160] PACAPC conjugated to branched E10 has the formula: HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNKCMalK[EEEEEEEEEE]2 (see also SEQ ID NO:77).

[0161] CTCC conjugated to branched E10 has the formula: YIAGVGGEKSGGFYCMalK[EEEEEEEEEE]2 (see also SEQ ID NO:78).

[0162] Ck2.3C conjugated to branched E10 has the formula: RQIKIWFQNRRMKWKKIPVGESLKDLIDQCMalK[EEEEEEEEEE]2 (see also SEQ ID NO:79).

[0163] An ODPC conjugated to branched E10 has the formula: DVDVPDGRGDSLAYGCMalK[EEEEEEEEEE]2 (see also SEQ ID NO:80).

[0164] P4C conjugated to branched E10 has the formula: KIPKASSVPTELSAISTLYLCMalK[EEEEEEEEEE]2 (see also SEQ ID NO:81).

[0165] Now, referring to Figures 14A and 14B, adult Swiss Weber mice were administered 1 mCi of 125 I-labeled conjugates were injected. Cysteine ​​tyrosine dipeptides are coupled to maleimide linkers. Cysteine ​​tyrosine dipeptides with maleimide linkers are conjugated to each of the indicated targeting ligands. PTHrP_ASP10 represents a conjugated peptide having 1-46 of PTHrP with a linear polymer of 10 aspartic acids. Preptin_Asp10 represents a conjugated peptide having 1-16 of preptin with a linear polymer of 10 aspartic acids. 24 hours after injection, autopsies were performed and tissues were excised. Tissues were then counted via a gamma counter. Here, the relative counts of fractured femurs to healthy femurs are displayed as ratios.

[0166] Now, referring to FIG. 15, adult female Swiss Weber mice were subcutaneously injected with LS288 conjugated to a linear polymer of 10L-aspartic acid 10 days after osteotomy in the right femur. The mice were imaged via an excitation beam of 780 nm for 1 second. Emission fluorescence was collected at 810 nm for 1 second. Since the injection site was near the back of the mouse, the mouse exhibits high fluorescence on the back.

[0167] Now, referring to FIG. 16, adult female Swiss Weber mice were subcutaneously injected with LS288 conjugated to a linear polymer of 10L-aspartic acid 3, 6, and 10 days after osteotomy in the right femur. Mice were imaged via an excitation beam of 780 nm for 1 second. Emission fluorescence was collected at 810 nm for 1 second. The top row represents 10 days after fracture, the middle row represents 6 days after fracture, and the bottom row represents 3 days after fracture. The left femur in every group is the fractured femur compared to the right healthy femur. This confirms that targeting the fracture improves the healing process.

[0168] Now referring to FIG. 17, adult Swiss Weber mice were administered 0.25 mCi of radiolabeled IFN-γ-α-glucose (100 mM NaCl) at 10 days after osteotomy. 125 The mice were injected with the I-preptin-D10 conjugate and sacrificed at different time points. Each tissue was harvested and then quantified in a gamma counter (FIG. 17A). Radiolabeling 125 I-Preptin D10 was used to calculate the fold difference in relative counts between fractured and healthy femurs (FIG. 17B).

[0169] Now, with reference to Figures 18 and 19, adult Swiss Weber mice (12 weeks old) were administered 0.22 mCi of radiolabeled erythropoietin 10 days after osteotomy. 125 Each mouse was injected with 100% β-lactam 1 (100% β-lactam 1) or 100% β-lactam 1 (100% β-lactam 1) conjugated peptide. 14 hours after injection, each mouse was sacrificed and each of the listed organs was collected and quantified. Counts were normalized according to sample weight. The fold difference in relative counts between fractured and healthy (non-fractured) femurs for each conjugated peptide was calculated.

[0170] Now, referring to FIG. 20, adult Swiss Weber mice (12 weeks old) were administered 0.22 mCi of radiolabeled IL-10 conjugated with L-Asp10 (FIG. 20A), D-Asp10 (FIG. 20B), or L-Asp20 (FIG. 20C) 10 days after osteotomy. 125 I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, F109C, and PACAPC) were injected. 14 hours after injection, each mouse was sacrificed and each of the listed organs was collected and quantified. Counts were normalized according to sample weight. All of the conjugated peptides tested exhibited preferential and / or selective targeting to fractured bone over other organs.

[0171] Now, referring to FIG. 21, adult Swiss Weber mice (12 weeks old) were treated with 0.22 mCi of radiolabeled IL-10 conjugated to L-Glu10 (FIG. 21A) or D-Glu10 (FIG. 21B) 10 days after osteotomy. 125 I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, and F109C) were injected. 14 hours after injection, each mouse was sacrificed and each of the listed organs was collected and quantified. Counts were normalized according to sample weight. All of the tested conjugated peptides exhibited preferential and / or selective targeting to the fractured bone over other organs. The high signal observed in the kidney may be due to the high abundance of glutamate transporters in the kidney. These transporters contribute to the reabsorption of glutamate into the circulation, rather than the actual uptake into cells. This means that the signal is likely to decay over time, contrary to the uptake observed in the fractured femur, where negatively charged molecules are strongly attached to bone (see, e.g., FIG. 17). For further details regarding glutamate absorption, see Hediger, MA Glutamate transporters in kidney and brain. Am. J. Physiol. - Ren. Physiol. 277, F487-F492 (1999); Kanai, Y. & Hediger, MA Primary structure and functional characterization of a high-affinity glutamate transporter. Nature 360, 467-471 (1992); and Kanai, Y. & Hediger, MA The glutamate and neutral amino acid transporter family: physiological and pharmacological implications. Eur. J. Pharmacol. 479, 237-247 (2003).

[0172] Now, referring to FIG. 22, adult Swiss Weber mice (12 weeks old) were treated with 0.22 mCi of radiolabeled Glu20 conjugated to L-Glu20 (FIG. 22A) or D-Glu20 (FIG. 22B) 10 days after osteotomy. 125 I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, F109C, and PACAPC) were injected. 14 hours after injection, each mouse was sacrificed and each of the listed organs was collected and quantified. Counts were normalized according to sample weight. Most of the conjugated peptides tested exhibited preferential and / or selective targeting to fractured bone or kidney over other organs. Extending the glutamic acid polymer to 20 appeared to slightly improve selectivity to fractured bone, but kidney uptake was still maintained.

[0173] Now, referring to FIG. 23, adult Swiss Weber mice (12 weeks old) were administered 0.22 mCi of radiolabeled IgG10 conjugated with branched L-Asp10 (FIG. 23A) or branched D-Asp10 (FIG. 23B) 10 days after osteotomy. 125 I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, F109C, and PACAPC) were injected. 14 hours after injection, each mouse was sacrificed and each of the listed organs was collected and quantified. Counts were normalized according to sample weight. All of the conjugated peptides tested exhibited preferential and / or selective targeting to fractured bone over other organs. Some appeared to show high uptake in the kidney.

[0174] Now, referring to FIG. 24, adult Swiss Weber mice (12 weeks old) were administered 0.22 mCi of radiolabeled IFN-γ-γ-γ conjugated to L-AAD10 (FIG. 24A), L-SDSDD (FIG. 24B), or (DSS)6 (FIG. 24C) 10 days after osteotomy. 125I peptides (e.g., ODPC, P4C, Ck2.3C, CTCC, F109C, and PACAPC) were injected. 14 hours after injection, each mouse was sacrificed, and each of the listed organs was collected and quantified. Counts were normalized according to sample weight. Peptides conjugated with L-AAD10 are believed to have some targeting ability to fractured bone. Peptides conjugated with L-SDSDD exhibited moderate targeting ability to fractured bone, and increased uptake in the kidney was also observed. Peptides conjugated with (DSS)6 are believed to have no targeting ability to fractured bone.

[0175] Now referring to FIG. 25A, adult Swiss Weber mice (12 weeks old) were administered 0.22 mCi of radiolabeled bisphosphonate conjugated to monobisphosphonate, tribisphosphonate, polyphosphate, E10, or E20 10 days after osteotomy. 125 25B, adult Swiss Weber mice (12 weeks old) were injected with 0.22 mCi of radiolabeled bisphosphonate YC, branched L-Asp4, or branched L-Asp8 conjugated to monobisphosphonate ("monobisphosphonate YC"), branched L-Asp4, or branched L-Asp8 ("branched (L)D8 Y"). 125 I was injected with tyrosine.

[0176] Now referring to Figure 26, the effect of PTH1-34E10 and saline on bone volume after treatment is shown. Mice were treated with PTH1-34E10 daily for 2 weeks. After 2 weeks, the mice were sacrificed and femurs were excised for microCT analysis.

[0177] Now referring to Figure 27, the effect of PTHrPD10 and saline on normalized bone volume after treatment with the indicated doses is shown. Mice were treated with PTHrPD10 daily for 4 weeks. After 4 weeks, mice were sacrificed and femurs were excised for microCT analysis.

[0178] The glutamate-containing targeting ligands showed greater uptake in the kidney than the other targeting ligands tested, with uptake ranging from 20-50% of the measured dose per gram. This uptake is likely due to glutamate reuptake receptors expressed in the kidney. The glutamate-containing targeting ligands showed greater selectivity for fractured bone over other bones, as indicated by the ratio of delivery to fractured bone to non-fractured bone ranging from 7-12 for the glutamate-containing targeting ligands. Extending the glutamate polymer to 20 resulted in a slight improvement in delivery, from 20-50% of the measured dose per gram for the 10-mer to 40-70% of the measured dose per gram for the 20-mer. This improvement is likely due to the increased affinity that the additional glutamate provides to the exposed hydroxyapatite at the fracture site. The extended 20-mer glutamate targeting ligands also suffered from the same kidney uptake problems as the shorter 10-mers. Changing from the L to the D enantiomer does not appear to have a consistent effect on glutamate targeting ligands.

[0179] Aspartic acid targeting ligands appear to provide the greatest delivery of the targeting ligands, with delivered accumulations ranging from 40-70% of the measured dose per gram. However, aspartic acid targeting ligands appear to be somewhat less selective for fractured versus non-fractured bone, with a ratio of accumulation in fractured versus non-fractured bone of 4-6 for L-amino acid aspartic acid targeting and 6-9 for D-amino acid aspartic acid targeting ligands. However, aspartic acid targeting ligands do not suffer as high an accumulation in the kidney as glutamic acid targeting ligands, typically maintaining accumulations below 15% of the measured dose per gram. Branched forms appear not to outperform linear forms in their ability to deliver more to the fracture site.

[0180] Extending the length from 10 to 20, conjugated with L-Glu20 (denoted "(L)D20"), improved consistency of targeting ligands across different peptides, with all peptides delivered at 50-70% of the measured dose per gram. The change from the L enantiomer to the D enantiomer appears to improve the stability of the aspartic acid targeting ligand, increasing targeting as evidenced by higher delivery rates and better selectivity ratios.

[0181] The peptide conjugated with AAD10 appears to have only a moderate targeting ability. The peptide conjugated with AAD10 still maintains a more systemic distribution. It was still able to maintain its improved ability to deliver 2-5 times the labeled compound to fractured bone versus non-fractured bone. The peptide conjugated with SDSDD also only had a moderate targeting ability, with delivery in the range of 20-40%. The peptide conjugated with SDSDD also still maintains a more systemic distribution. The peptide conjugated with SDSDD still maintains its improved ability to deliver 3-5 times the labeled compound to fractured bone versus non-fractured bone. The peptide conjugated with (DSS)6 also appears to have only a moderate targeting ability to fractured bone. (DSS)6 also still maintains a more systemic distribution. The peptide conjugated with (DSS)6 was still able to maintain its improved ability to deliver labeled compound to fractured versus non-fractured bone by 2-3 fold.

[0182] Although the novel technology has been illustrated and described in detail in the drawings and the foregoing description, these should be considered as illustrative in nature and not restrictive, with the understanding that only preferred embodiments have been shown and described, and that all changes and modifications that fall within the spirit of the novel technology are desired to be protected. Similarly, while specific examples, theoretical reasoning, explanations, and illustrations have been used to illustrate the novel technology, these illustrations along with the discussion should not be construed as limiting the technology. All patents, patent applications, and references to textbooks, scientific treatises, publications, and the like referenced in this application are incorporated herein by reference in their entirety to the extent that they do not contradict the explicit teachings of this specification. The present invention may include the following aspects. [1] Compound of formula XYZ [In the formula, X is at least one agent that modulates the activity of the parathyroid hormone receptor; Z is at least one bone targeting molecule; Y is a linker connecting and / or linking X and Z]; or a pharma- ceutically acceptable salt thereof or a metabolite thereof A compound comprising: [2] X is at least one polypeptide having at least 80% sequence identity to full length parathyroid hormone related peptide (SEQ ID NO:12), at least 80% sequence identity to full length parathyroid hormone (SEQ ID NO:13), and / or at least 80% identity to full length abaloparatide or an analog thereof; Y is at least one polypeptide comprising at least 80% sequence identity to amino acid residues 35-40, 35-41, 35-42, 35-43, 35-44, 35-45, 35-46, and / or 35-84 of full-length parathyroid hormone-related peptide or parathyroid hormone, and / or at least one cathepsin K-sensitive polypeptide; Z is at least one polypeptide containing four or more acidic amino acid residues, a polyphosphate, an aminohexanedioic acid or a derivative thereof, and / or an alendronate or a derivative thereof; The compound according to the above [1]. [3] X is at least one polypeptide having at least 80% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9; Y is at least one polypeptide comprising at least 80% sequence identity to amino acid residues 35-46 and / or 41-46 of full-length parathyroid hormone-related peptide; Z is at least one polypeptide containing four or more acidic amino acid residues, a polyphosphate, and / or an alendronate or a derivative thereof; The compound according to any one of the above [1] and [2]. [4] X is at least one polypeptide having 80% identity to full-length abaloparatide or an analog thereof; Y is at least one polypeptide comprising at least 80% sequence identity to amino acid residues 35-46 of full-length parathyroid hormone-related peptide; Z is at least one polypeptide containing four or more acidic amino acid residues, a polyphosphate, and / or an alendronate or a derivative thereof; The compound according to any one of the above [1] to [3]. [5] Z is at least one polypeptide comprising 6, 7, 8, 9, and / or 10 acidic amino acid residues; The compound according to any one of the above [1] to [4]. [6] The compound according to any one of the above [1] to [5], wherein the acidic amino acid residue comprises L-aspartic acid or D-aspartic acid, L-glutamic acid or D-glutamic acid, or a combination thereof. [7] The compound according to any one of the above [1] to [6], wherein the acidic amino acid residues further comprise branched amino acids and / or branched chains of amino acids. [8] Y is at least one polypeptide having the formula Gly-Gly-Pro-Nle, where Nle includes norleucine, leucine, isoleucine, and / or equivalents thereof; The compound according to any one of the above [1] to [7]. [9] The compound according to any one of [1] to [8] above, wherein the compound of formula XYZ is at least one polypeptide having at least 80%, 90%, and / or 95% sequence identity to SEQ ID NO: 10 and / or SEQ ID NO: 11.

[10] The compound according to any one of the above [1] to [9], wherein the compound of formula XYZ is at least one polypeptide having SEQ ID NO: 10 and / or SEQ ID NO: 11.

[11] The compound according to any one of the above [1] to

[10] , wherein X is at least one agonist of parathyroid hormone receptor 1.

[12] The compound according to any one of the above [1] to

[11] , wherein X is at least one polypeptide having at least 80% sequence identity to the first 13 amino acids of full-length parathyroid hormone related peptide (SEQ ID NO: 12), at least 80% sequence identity to the first 13 amino acids of full-length parathyroid hormone (SEQ ID NO: 13), and / or at least 80% homology to the first 13 amino acids of SEQ ID NO: 3.

[13] 1. A method for treating a bone-related disease, comprising: providing to a subject at least one therapeutically effective dose of a compound according to any one of [1] to

[12] above, or a pharma- ceutically acceptable salt or metabolite thereof; The method includes:

[14] The method according to

[13] above, wherein the subject includes a human, an animal, a cell, and / or a tissue.

[15] The method according to any one of the above

[13] and

[14] , wherein the bone-related disease comprises osteopenia, osteoporosis, rheumatoid arthritis, blood disease, autoimmunity, transplant rejection, and / or bone fracture.

[16] The method according to any one of the above

[13] to

[15] , wherein the effective dose of the compound according to any one of the above [1] to

[10] comprises 0.001 nmol per kg per day to 1000 nmol per kg per day, 0.01 nmol per kg per day to 1000 nmol per kg per day, 0.1 nmol per kg per day to 1000 nmol per kg per day, 1 nmol per kg per day to 500 nmol per kg per day, 1 nmol per kg per day to 250 nmol per kg per day, 1 nmol per kg per day to 100 nmol per kg per day, 10 nmol per kg per day to 75 nmol per kg per day, and / or 20 nmol per kg per day to 50 nmol per kg per day.

[17] The method according to any one of

[13] to

[16] above, wherein the compound according to any one of [1] to

[12] above is administered orally, parenterally, rectally, and / or transdermally.

[18] Compound of formula XZ [In the formula, X is at least one peptide; Z is at least one bone-targeting molecule]; or a pharma- ceutically acceptable salt thereof or a metabolite thereof A compound comprising:

[19] The compound according to

[18] above, wherein Z is at least one molecule that preferentially and / or selectively targets fractured bone.

[20] The compound according to any one of the above

[18] and

[19] , wherein X is at least one peptide having fewer than 60, 50, 40, 30, 20, and / or 10 amino acid residues.

[21] The compound according to any one of the above

[18] to

[20] , wherein X is at least one peptide having more than 1, 2, 3, 4, and / or 5 amino acid residues.

[22] The compound according to any one of the above

[18] to

[21] , wherein X is at least one peptide, including a hydrophilic peptide, a hydrophobic peptide, a neutral peptide, a cationic peptide, and / or an anionic peptide, and / or any combination thereof.

[23] X is at least one polypeptide comprising a sequence having at least 80% sequence identity to 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; Z is at least one polypeptide containing four or more acidic amino acid residues, a polyphosphate, an aminohexanedioic acid or a derivative thereof, an alendronate or a derivative thereof, and / or a bisphosphonate or a derivative thereof; The compound according to any one of the above

[18] to

[22] .

[24] X is at least one polypeptide comprising a sequence having at least 90% sequence identity to 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; Z is at least one polypeptide, polyphosphate, and / or bisphosphonate or derivative thereof containing four or more acidic amino acid residues; The compound according to any one of the above

[18] to

[23] .

[25] The compound according to any one of

[18] to

[24] above, wherein X is at least one polypeptide comprising a sequence having at least 95% sequence identity to 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.

[26] The compound according to any one of

[18] to

[25] above, wherein X is at least one polypeptide comprising a sequence having at least 100% sequence identity to 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.

[27] The compound according to any one of the above

[18] to

[26] , wherein Z is at least one polypeptide comprising negatively charged amino acid residues.

[28] The compound according to any one of the above

[18] to

[27] , wherein Z is at least one polypeptide comprising 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and / or 20 negatively charged amino acid residues.

[29] The compound according to any one of the above

[18] to

[28] , wherein Z is at least one monobisphosphonate, bibisphosphonate, tribisphosphonate, and / or multiple-bisphosphonate.

[30] The compound according to any one of

[18] to

[29] above, wherein Z is at least one acidic amino acid residue, including L-aspartic acid or D-aspartic acid, L-glutamic acid or D-glutamic acid, or a combination thereof.

[31] The compound according to any one of the above

[18] to

[30] , wherein Z is at least one acidic amino acid residue, including branched amino acids and / or branched chains of amino acids.

[32] The compound according to any one of the above

[18] to

[31] , wherein Z is at least one acidic amino acid residue, including branched amino acids and / or branched chains of amino acids.

[33] The compound of any one of the above

[18] to

[32] , wherein the compound of formula XZ comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 23 to 81, or analogs or metabolites thereof.

[34] The compound of any one of the above

[18] to

[33] , wherein the compound of formula XZ comprises a sequence having 95% sequence identity to any one of SEQ ID NOs: 23 to 81, or analogs or metabolites thereof.

[35] The compound of any one of the above

[18] to

[34] , wherein the compound of formula XZ comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 23 to 81, or analogs or metabolites thereof.

[36] The compound according to any one of the above

[18] to

[35] , further comprising a linking moiety Y, wherein Y is a linker connecting and / or linking both X and Z.

Claims

1. A compound having the structure X-Y-Z [In the formula, X is a polypeptide having the amino acid sequence of SEQ ID NO:3; Y is a linker, and Z is a bone-targeting molecule, said bone-targeting molecule being at least one polypeptide comprising four or more acidic amino acid residues, a polyphosphate, an aminoadipic acid, an alendronate, and / or a bisphosphonate; or a pharma- ceutically acceptable salt thereof.

2. 2. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein Y is a non-releasable linker.

3. 2. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein Y comprises a polypeptide.

4. 4. The compound of claim 3, or a pharma- ceutically acceptable salt thereof, wherein Y is a full-length parathyroid hormone related peptide or a polypeptide having at least 80% sequence identity to amino acid residues 35-84 of parathyroid hormone.

5. 5. The compound of claim 4, or a pharma- ceutically acceptable salt thereof, wherein Y is a polypeptide having at least 80% sequence identity to amino acid residues 35-46 of full-length parathyroid hormone related peptide.

6. 2. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein Z comprises 4 or more and 70 or less amino acids.

7. 7. The compound of claim 6, or a pharma- ceutically acceptable salt thereof, wherein Z comprises 4 or more and 40 or less amino acids.

8. 8. The compound of claim 7, or a pharma- ceutically acceptable salt thereof, wherein Z comprises 6 or more and 35 or less amino acids.

9. 2. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein Z is charged.

10. 7. The compound of claim 6, or a pharma- ceutically acceptable salt thereof, wherein at least one amino acid is aspartic acid or glutamic acid.

11. 2. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein Z comprises 6 or more and 35 or less glutamic acid residues.

12. 2. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein Z comprises 6 or more and 35 or less aspartic acid residues.

13. A compound having the structure X-Y-Z [In the formula, X is a polypeptide having the amino acid sequence of SEQ ID NO:3; Y is a non-releasable polypeptide linker, and Z comprises a bone-targeting molecule of 6 to 40 glutamic acid residues or 6 to 40 aspartic acid residues; or a pharma- ceutically acceptable salt thereof.

14. 14. The compound of claim 13, or a pharma- ceutically acceptable salt thereof, wherein Z comprises 6 or more and 35 or less glutamic acid residues.

15. 14. The compound of claim 13, or a pharma- ceutically acceptable salt thereof, wherein Y is a polypeptide having at least 80% sequence identity to amino acid residues 35-46 of full-length parathyroid hormone related peptide.

16. A pharmaceutical composition for treating a bone-related disease, comprising:

16. A pharmaceutical composition comprising a compound according to any one of claims 1 to 15 or a pharma- ceutically acceptable salt thereof.

17. 17. The pharmaceutical composition of claim 16, wherein the bone-related disease comprises osteopenia, osteoporosis, rheumatoid arthritis, hematological disorders, autoimmunity, transplant rejection and / or bone fracture.

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