Targeting Anabolic Drugs For Accelerated Fracture Repair

Abstract

Aspects of the disclosure include material and methods for the targeted delivery of growth factors, vasoactive peptides and other representative anabolic peptide drugs from different signaling cascades to bone fracture for accelerated healing is disclosed herein. Aspects of the disclosure can be used to treat bone fractures and bone defects.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/678,016, filed on May 30, 2018. This application is incorporated herein by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] Aspects of the present disclosure relate to the materials and methods for treating bone fractures and bone defects.

BACKGROUND

[0003] Src tyrosine kinase plays a crucial role in bone metabolism: despite its ubiquitous expression profile, the only apparent phenotypical abnormality in a Sarcoma-knockout (Src-KO) mouse strain was osteopetrosis. Although Src inhibitors inhibit both the formation and activity of osteoblasts (OBs) in vitro, the number of osteoclasts (OCs) derived from Src-KO mice were actually elevated in Src-KO mice, measuring more than twice that in wild-type (WT) mice. Also, a marked increase in both osteoblast number and activity was observed in vivo in Src-KO mice. These results confirm that the osteopetrosis phenotype of Src-KO mice was not a result of reduced osteoclast formation, but rather of boosted osteoblast activity as well as reduced osteoclast function. Moreover, osteoblasts derived from Src-KO mice demonstrated unremarkable morphological features compared to those harvested from WT mice, and were able to fully regulate normal osteoclast differentiation via the receptor activator of nuclear factor kappa-B ligand/receptor activator of nuclear factor kappa-B/osteoprotegerin (RANKL/RANK/OPG) pathway. Thus, this bone-resorption defect should be easily alleviated by restoring normal Src functionality in osteoclasts, reducing potential risks on the musculoskeletal system involved in long-term use of Src inhibitors for fracture healing.

[0004] Broadly, peptide anabolic drugs include different categories of protein or the fragments thereof. They are represented by bone morphogenetic protein pathway signaling peptides including P4, bone forming peptide (BFP) and peptide from bone morphogenetic protein 9 (pBMP9); insulin-like growth factor (IGF) derived peptides including mechano growth factor (MGF) and Preptin; bone stimulatory neuropeptides including Substance P and vasoactive intestinal protein (VIP); and peptides enhancing vascular functions, including C-type Natriuretic peptide (CNP), targeted prothrombin peptide (TP508) and VIP. Each of these peptides may have its own unique mechanism working to regulate bone growth, as will be outlined in the detailed description.

[0005] 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 on such fractures are bone morphogenic protein (BMP)-2 (approved for use only in tibial trauma) and BMP-7 (discontinued), which are applied locally and generally used in the treatment of open long bone fractures and spinal fusions. The need for broader application of anabolic drugs to treat bone maladies such as osteoporotic fractures with efficacy is evident.

[0006] Therefore, it is desirable to have a fracture treatment drug that is administered systemically yet targets the fracture site with evident efficacy.

SUMMARY

[0007] A first aspect of the present disclosure includes at least one compound of the formula X-Y-Z, or a pharmaceutically acceptable salt thereof, or a metabolite thereof, wherein X is at least one agent that improves bone density, mechanical strength, bone deposition, or quality; Z is at least one bone-targeting molecule; and Y is a linker that joins and/or links X and Z. In some aspects, X is at least one agent that enhances the activity or one agent that improves bone density, mechanical strength, bone deposition or other agent that promotes bone healing and/or growth. Consistent with some of these aspects, Z is at least one negatively charged oligopeptide or an equivalent thereof that binds to hydroxyapatite and/or raw bone.

[0008] The second aspect includes the compound according to the first aspect, wherein when X is a polypeptide, any 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% identity to X can be used to practice the invention.

[0009] In some aspects, Y is at least one polypeptide comprising at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% sequence identity to amino acid residues 35-40, 35-41, 35-42, 35-43, 35-44, 35-45, 35-46, 35-47, 35-48, 35-49, 35-50, 35-51, 35-52, 35-55, 35-84, 41-44, 41-45, 41-46, 41-47, 41-48, 41-49, 41-50, and/or 41-84 of a full length parathyroid hormone related peptide or parathyroid hormone, and/or at least one Cathepsin K sensitive polypeptide.

[0010] In some aspects, Z is at least one polypeptide comprising about 4 or more, from about 4 to about 100, from about 4 to about 50, from 4 to about 20, from about 4 to about 15, from about 4 to about 10 acidic amino acid residues, polyphosphate, 2-aminohexanedioic (aminoadipic) acid or derivatives thereof, and/or alendronate or derivatives thereof. In some aspects, Z is at least one polypeptide comprising about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and/or 30 acidic amino acid residues, polyphosphate, 2-aminohexanedioic acid or derivatives thereof, and/or alendronate or derivatives thereof. In other aspects, Z is at least one negatively charged oligopeptide or an equivalent thereof that binds to hydroxyapatite and/or raw bone.

[0011] The targeted delivery strategy recited in some aspects of the present disclosure enable the delivery of Src inhibitors specifically to bone fracture surfaces thereby facilitating fracture healing. This in vivo efficacy is shown by the acceleration of fracture healing observed using the Src inhibitors Dasatinib and E738.

[0012] In addition to Src inhibitors, a group of peptides targeted specifically to the fracture surfaces also demonstrates an enhanced ability to facilitate fracture healing. These peptides include osteopontin derived fragments such as osteopontin-derived peptide (ODP), collagen binding motif (CBM); BMP fragments such as P4, BFP, pBMP7; IGF fragments such as MGF and Preptin; neuropeptides such as Substance P and VIP; vasoconstrictive fragments such as CNP, TP508 and VIP; and other anabolic drugs such as osteogenic growth peptide (OGP). The in vivo efficacy of these peptides for accelerated fracture healing are demonstrated herein. All peptide conjugates are produced by solid phase synthesis.

[0013] Some aspects of this disclosure include compounds comprising: a compound of the formula X-Y-Z, wherein X is at least one agent that modulates bone growth such as activity of Src tyrosine kinase; Z is at least one bone-targeting molecule; and Y is a linker that joins and/or links X and Z; or a pharmaceutically acceptable salt thereof, or a metabolite thereof. In some aspects, Z is at least one polypeptide comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and/or 20 acidic amino acid residues. In some aspects, X is selected from the group consisting of Dasatinib and E738. In some aspects, Y is a releasable linker selected from disulfide; ester; or protease specific amide bond. In some aspects, Y is a nonreleasable bond selected from carbon-carbon bond; or amide bond.

[0014] In some aspects, this disclosure includes a compound of the formula X-Y-Z, wherein X is at least one peptide or a fragment thereof that modulates activity of bone and cartilage formation; Z is at least one bone-targeting molecule; and Y is a linker that joins and/or links X and Z; or a pharmaceutically acceptable salt thereof, or a metabolite thereof. In some aspects, Y is a releasable linker selected from disulfide; ester; or protease specific amide bond. In some aspects, Y is a nonreleasable bond selected from carbon-carbon bond; or amide bond. In some aspects, Y is a peptide belonging to the natural sequence of Z. In some aspects, Y is a (polyethylene glycol) PEG linker. In some aspects Y is a PEG linker comprised of 2-8 oxyethylene units. In some aspects, Z comprises at least 10 aspartic or glutamic acids conjugated to X. In some aspects, Z comprises at least 20 aspartic or glutamic acids conjugated to X. In some aspects, the compound may be produced by solid phase synthesis.

[0015] In some aspects, X is a bone anabolic peptide derived from BMP. In some aspects, X is a bone anabolic peptide derived from IGF. In some aspects, X is a bone anabolic peptide derived from a neuropeptide. In some aspects, X is a bone anabolic peptide that improves vascular function and/or vascularization. In some aspects, X is OGP. In some aspects, the peptide is BFP, P4, or pBMP9. In some aspects, the peptide is MGF or preptin. In some aspects, the peptide is Substance P or VIP. In some aspects, the peptide is TP508, VIP, or CNP. Unless indicated otherwise, the invention may be practiced by combining any X with any Z and optionally any suitable linking group Y. [0016] 1. A compound comprising: [0017] a compound of the formula X-Y-Z, wherein [0018] X is at least one agent that modulates activity selected from the group consisting of: Src Inhibitors, Sphingosine1-phosphate (SIP), and Neuropeptides: [0019] Z is at least one bone-targeting molecule; and [0020] Y is an optional linker that joins and/or links X and Z; [0021] or a pharmaceutically acceptable salt thereof, or a metabolite thereof. [0022] 2. The compound according to claim 1, wherein [0023] Z is at least one polypeptide comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and/or 20 acidic amino acid residues. [0024] 3. The compound according to claims, 1-2 wherein Z includes multiple aspartates and/or multiple glutamates. [0025] 4. The compound according to claim 3, wherein Z is comprised of at least one polypeptide selected from the group consisting of: at least 5 aspartic acids, at least 5 glutamic acids, at least 10 aspartic acids, at least 10 glutamic acids, at least 20 aspartic acids, at least 20 glutamic acids. [0026] 5. The compounds according to claims 1-4, wherein Z is selected from the group consisting of: a polypeptide comprising 10 aspartic acid residues (SEQ ID NO. 23) and a polypeptide comprising 10 glutamic acid residues (SEQ ID NO. 24). [0027] 6. The compound according to claims 1-4, wherein Z is at least one polypeptide comprising 4 or more acidic amino acid residues, polyphosphate, aminohexanedioic acid or derivatives thereof, and/or alendronate or derivatives thereof. [0028] 7. The compound according to claims 1-6, wherein Y is selected from the group consisting of: releasable linkers and non-releasable linkers. [0029] 8. The compound according to claim 7, wherein the releasable linker includes at least of the following groups: a disulphide, an ester, or a Protease specific amide bond. [0030] 9. The compound according to claim 7, the non-releasable linker includes at least one of the following groups; a carbon-carbon bond, or an amide. [0031] 10. The compound according to claims 1-6, wherein Y is polyethylene glycol (PEG). [0032] 11. The compound according to claim 10, wherein the PEG linker is comprised of 2-8 oxyethylene units. [0033] 12. The compound according to claims 1-6, wherein Y is a peptide belonging to the natural sequence of Z. [0034] 13. The compound according to claims, 1-9, wherein the Src1 inhibitor is at least one compound selected from the group consisting of: Dasatinib, and E739. [0035] 14. The compound according to claims, 1-9, wherein the SIP is least one compound selected from the group consisting of: Ozanimod (SIP1R agonist), and 4-deoxypyridoxine (DOP) (SIP lyase inhibitor). [0036] 15. The compound according to claims, 1-9, wherein the Neuropeptide, is at least one compound selected from the group consisting of: Substance P, vasoactive intestinal peptide (VIP), Pituitary adenylate cyclase-activating polypeptide (PACAP), Amylin (1-8) and Calcitronin gene like related peptide (CGRP). [0037] 16. The compound according to claims, 1-9, wherein the Osteogenic peptide is histone h4. [0038] 17. Use of a compound according to any of claims 1-16, for the manufacture of a medicament for therapeutic application.

[0039] A method of treating a patient, comprising the step of administering at least one dose of a compound according to claims 1-16. These and other features, aspects and advantages of the present disclosure will become better understood with reference to the following figures, associated descriptions and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1 depicts the bone volume divided by total volume of the 100 thickest micro computed tomography (CT) slices of the fracture callus bone density ("BV/TV") using a Dasatinib and targeted Dasatinib conjugate (both 10 .mu.mol/kg). Both were subcutaneously dosed daily to fracture-bearing Notre Dame breed (ND4) of Swiss Webster mice. Bone density of the fracture callus from the targeted Dasatinib group is twice as dense as the saline group, and 50% denser than the free Dasatinib group.

[0041] FIG. 2 depicts BV/TV and Trabecular Thickness using targeted E738 conjugate (1 mol/kg), subcutaneously dosed every-other-day to fracture-bearing Charles River's breed (CFW) of Swiss Webster mice. Targeted E738 conjugate significantly improved the bone density and trabecular thickness at the fracture callus.

[0042] FIG. 3 depicts structures for Dasatinib and E738.

[0043] FIG. 4 depicts structures for targeted conjugates of Dasatinib and E738.

[0044] FIG. 5 depicts peak load of Fractured Femurs after 2 weeks.

[0045] FIG. 6 depicts BV/TV two weeks after fractured femur received various concentration of Preptin D10 treatment.

[0046] FIG. 7 depicts TbTh (the trabecula thickness of the 100 thickest micro CT slices of the fracture callus) two weeks after fractured femur received various concentration of Preptin D10 treatment.

[0047] FIG. 8 depicts BV (the overall bone volume of the 100 thickest micro CT slices of the fracture callus) two weeks after fractured femur received various concentration of Preptin D10 treatment.

[0048] FIG. 9 depicts BV/TV two weeks after fractured femur received various concentration of OGPD10.

[0049] FIG. 10 depicts TbTh two weeks after fractured femur received various concentration of OGP D10.

[0050] FIG. 11 depicts TbSp (the spacing between the trabecula in the 100 thickest micro CT slices of the fracture callus) two weeks after fractured femur received various concentration of OGP D10.

[0051] FIG. 12 depicts BV/TV two weeks after fractured femur received various concentration of BFPD10.

[0052] FIG. 13 depicts TbSp two weeks after fractured femur received various concentration of BFPD10.

[0053] FIG. 14A depicts BV/TV four weeks after a fractured femur received various concentration of substance P4 mini peg D10; FIG. 14B depicts the max load of substance P4 D10 four weeks after a fractured femur received the max load of substance P4 D10.

[0054] FIG. 15 depicts BV/TV four weeks after fractured femur received various concentration of Ghrelin D10.

[0055] FIG. 16 depicts BV four weeks after fractured femur received various concentration of pBMP9 D10.

[0056] FIG. 17 depicts BV/TV four weeks after fractured femur received various concentration of pBMP9 D10.

[0057] FIG. 18 depicts BV/TV four weeks after fractured femur received various concentration of CNP D10.

[0058] FIG. 19 depicts BV/TV four weeks after fractured femur received 1 nmol/day of ODP D10.

[0059] FIG. 20 depicts BV/TV three weeks after fractured femur received various concentrations of CBM D10 as compared to a fractured femur that received parathyroid hormone 1-34 (PTH).

[0060] FIG. 21 depicts BV/TV four weeks after fractured femur received various concentrations of P4 D10.

[0061] FIG. 22 depicts BV four weeks after fractured femur received 1 nmol/day of P4 D10.

[0062] FIG. 23 depicts BV/TV four weeks after fractured femur received various concentrations of MGF D10.

[0063] FIG. 24 depicts BV/TV four weeks after fractured femur received various concentrations of TP 508_D10.

[0064] FIG. 25a depicts BV/TV four weeks after fractured femur received 1 nmol/day of VIP_D10.

[0065] FIG. 25b depicts TbTh four weeks after fractured femur received 1 nmol/day of VIP_D10.

[0066] FIG. 26a depicts BV/TV three weeks after fractured femur received 0.05 nmol/day, 0.1 nmol/day, and 0.5 nmol/day of VIP_D10.

[0067] FIG. 26b depicts Max Load three weeks after fractured femur received 0.05 nmol/day, 0.1 nmol/day, and 0.5 nmol/day of VIP_D10.

[0068] FIG. 26c depicts Work to Fracture three weeks after fractured femur received 0.05 nmol/day, 0.1 nmol/day, and 0.5 nmol/day of VIP_D10.

[0069] FIG. 27 depicts the structure for BMP9 (SEQ ID NO: 14).

[0070] FIG. 28 depicts the structure for Ghrelin D10 (SEQ ID NO: 15).

[0071] FIG. 29 depicts the structure for Preptin D10 (SEQ ID NO: 3).

[0072] FIG. 30 depicts the structure for CNP-D10 (SEQ ID NO: 16).

[0073] FIG. 31 depicts the structure for VIP D10 (SEQ ID NO: 17).

[0074] FIG. 32 depicts the structure for Substance P with 4 mini PEG conjugated to D10 (SEQ ID NO: 18).

[0075] FIG. 33 depicts the structure for CBM D10 (SEQ ID NO: 9).

[0076] FIG. 34 depicts the structure for ODP D10 (SEQ ID NO: 19).

[0077] FIG. 35 depicts the structure for Ozanimod.

[0078] FIG. 36 depicts the structure for DOP.

[0079] FIG. 37 depicts the structure for D.sub.10-Ozanimod.

[0080] FIG. 38 depicts the structure for D.sub.10-DOP.

[0081] FIG. 39 depicts BV/TV 3 weeks after fractured femur received various concentrations of D.sub.10-DOP.

[0082] FIG. 40 depicts alkaline phosphatase (ALP) activity of MC3T30E1 cells following exposure to ozanimod of various concentrations.

[0083] FIG. 41 depicts BV/TV 3 weeks after fractured femur received various concentrations of D.sub.10-Ozanimod.

[0084] FIG. 42 depicts BV/TV 3 weeks after fractured femur received various concentrations of PACAP (D)E.sub.10.

[0085] FIG. 43 depicts Max Load 3 weeks after fractured femur received various concentrations of PACAP (D)E.sub.10.

[0086] FIG. 44 depicts Stiffness 3 weeks after fractured femur received various concentrations of PACAP (D)E.sub.10.

[0087] FIG. 45 depicts the sequence for Amylin(1-8) D.sub.10 (SEQ ID NO: 20).

[0088] FIG. 46 depicts BV/TV 3 weeks after fractured femur received various concentrations of Amylin(1-8)

[0089] FIG. 47 depicts Max Load 3 weeks after fractured femur received various concentrations of Amylin(1-8) D.sub.10.

[0090] FIG. 48 depicts Work to Fracture 3 weeks after fractured femur received various concentrations of Amylin(1-8) D.sub.10.

[0091] FIG. 49 depicts the sequence for CGRP E.sub.10 (SEQ ID NO: 21).

[0092] FIG. 50 depicts BV/TV 3 weeks after fractured femur received various concentrations of CGRP E.sub.10.

[0093] FIG. 51 depicts BV 3 weeks after fractured femur received various concentrations of CGRP

[0094] FIG. 52 depicts Max Load 3 weeks after fractured femur received various concentrations of CGRP E.sub.10.

[0095] FIG. 53 depicts Work to Force 3 weeks after fractured femur received various concentrations of CGRP E.sub.10

[0096] FIG. 54 depicts the sequence for PACAP (D)E.sub.10 (SEQ ID NO: 22).

BRIEF DESCRIPTION OF THE SEQUENCES

[0097] SEQ ID NO: 1: bone forming peptide conjugated with 10 aspartate acids (BFP D10).

[0098] SEQ ID NO: 2: osteogenic growth peptide conjugated with 10 aspartate acids (OGP D10).

[0099] SEQ ID NO: 3: Preptin conjugated with 10 aspartate acids (Preptin D10).

[0100] SEQ ID NO: 4: substance P with 4 mini PEG linker and conjugated with 10 aspartate acids (substance P 4 mini PEG D10).

[0101] SEQ ID NO: 5: Ghrelin D10 with Serine (Ser)-3 replaced with diaminopropinoic acid.

[0102] SEQ ID NO: 6: BMP9 D10.

[0103] SEQ ID NO: 7: C-type Natriuretic peptide (CNP) conjugated with 10 aspartate acids (CNP 10).

[0104] SEQ ID NO: 8: Vasoactive intestinal peptide conjugated with D10.

[0105] SEQ ID NO: 9: collagen binding motif conjugated with 10 aspartate acids (CBM D10).

[0106] SEQ ID NO: 10: P4 conjugated with 10 aspartate acids (P4 D10).

[0107] SEQ ID NO: 11: Mechano-growth factor conjugated with 10 aspartate acids (MGF D10).

[0108] SEQ ID NO: 12: Thrombin fragment TP508 conjugated with 10 aspartate acids (TP 508 D10).

[0109] SEQ ID NO: 13: Osteopontin-derived peptide conjugated with 10 aspartate acids (ODP D10).

[0110] SEQ ID NO: 14: BMP9 (BMP9).

[0111] SEQ ID NO: 15: Ghrelin D10 (Ghrelin D10).

[0112] SEQ ID NO: 16: CNP-D10.

[0113] SEQ ID NO: 17: VIP D10.

[0114] SEQ ID NO: 18:4 mini PEG D10.

[0115] SEQ ID NO:19: ODPD10.

[0116] SEQ ID NO: 20: Pituitary Adenylyl Cyclase Activating Peptide (PACAP)_conjugated with 10 aspartate acids (PACAP-(D)E.sub.10).

[0117] SEQ ID NO: 21: Pituitary Adenylate Cyclase-Activating Peptide conjugated with 10 aspartate acids (Amylin 1-8).

[0118] SEQ ID NO: 22: Calcitonin Gene-Rated Peptide (CGRP) conjugated with 10 glutamic acids (CGRP-E.sub.10).

[0119] SEQ ID NO: 23: Targeting group consisting of a polypeptide, DDDDDDDDDD.

[0120] SEQ ID NO: 24: Targeting group consisting of a polypeptide, EEEEEEEEEE

DETAILED DESCRIPTION

[0121] While the concepts of the present disclosure are illustrated and described in detail in the figures and the description herein, results in the figures and their description are to be considered as examples and not restrictive in character; it being understood that only the illustrative aspects are shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

[0122] Unless defined otherwise, the scientific and technology nomenclatures have the same meaning as commonly understood by a person in the ordinary skill in the art pertaining to this disclosure.

[0123] Aspects of the fracture targeted technology disclosed herein can help both civilians and military personnel. Bone fractures occur at an annual rate of 2.4 per 100 people and cost the US healthcare system approximately $28 billion per year. Of the 6.3 million bone fractures that occur annually in the US, 300,000 result in delayed union or non-union healing. Approximately 887,679 hospitalizations result each year from fractures. Over half (57%) of fractures resulting in hospitalizations occur in persons aged 65 and over. Estimated health care costs are indicated in Table 1, below.

TABLE-US-00001 TABLE 1 Cost without Cost with Fracture Healing time surgery surgery Leg 10-12 weeks) $2,500 $16,000 Hip 12+ weeks $11,500 $66,500 Vertebral (8+ weeks) $5,000-15,000 $50,000-150,000 Arm 6-10 weeks $2,500 $16,000

[0124] Currently, a substantial fraction of national defense outlays is devoted to combat-related medical expenditures, with a significant proportion of these costs devoted to treatment of orthopedic injuries. Indeed, .about.65% of all wounds associated with military conflicts since WWI have included orthopedic injuries, and 26% of all injuries to an extremity have involved one or more broken bones. Treatment of bone fractures not only removes a soldier from service for an extended period of time, but also requires the attention of multiple additional personnel to treat, monitor and rehabilitate the injured soldier. Unfortunately, some orthopedic injuries are so severe that resolution of the damage never occurs, and the armed services are then obligated to care for the damaged combatant in perpetuity.

[0125] Fractured bones are not only an adverse consequence of combat, they also constitute a prominent repercussion of military training exercises. During the course of a soldier's schooling, a female recruit will have a 3.4-21% chance of suffering a stress fracture, while a male recruit will have a 1-7.9% probability of experiencing the same injury. While such maladies may at first seem trivial, statistics reveal that they cost the military .about.$34,000 per soldier which totals up to .about.$100 million in aggregate per year. Not surprisingly, many affected recruits eventually leave the military as a consequence of their stress fracture, which results in further expenses arising from wasted recruiting and training efforts. Therapies for fractured bones both within and outside of the military rely almost exclusively on mechanical stabilization of the damaged bone (i.e. use of a cast, pin, rod, or plate, etc.). In fact, the only FDA-approved drug for enhancing fracture repair is a bone anabolic agent that must be applied topically to the fracture surface during surgery. Needless to say, such a therapy is inappropriate when the surgery is not otherwise indicated, can only be administered once (i.e. during the brief period when the fracture surface is exposed), cannot be easily adapted for treatment of multiple fractures, and is never used for therapy of stress fractures. What is critically needed is obviously a systemically administered bone anabolic agent (i.e. as drug that can stimulate rapid bone fracture healing) that will concentrate selectively on the bone fracture surface and induce accelerated bone formation only at the damaged site. Surprisingly, nothing of this sort has ever been described in the literature.

[0126] Recognizing the enormous need for a systemically administered bone fracture-targeted healing agent, peptides and other molecules with structures that home specifically to bone fracture surfaces following intravenous or subcutaneous administration were identified. A second group of bone anabolic agents (for example, both bone growth stimulating hormones and cytokines as well as various low molecular weight bone growth-inducing drugs, etc.), that when linked to one of our bone fracture-homing peptides, would promote accelerated fracture repair, were also identified. Fortunately, several fracture-targeted bone anabolic drugs met all initial requirements for advancement into large animal studies. That is, the targeted conjugates were found to: i) reduce the time for fractured femur repair in mice by roughly half, ii) induce no detectable systemic toxicity at its effective dose, iii) cause no ectopic bone formation at either the injection site or elsewhere), iv) lead to regeneration of bone at the fracture site that was biomechanically stronger than the contralateral (unbroken) femur, and v) result in eventual remodeling of the fractured region into normal cortical bone.

[0127] All in vivo data included herein are from swiss Webster mice. All mice received an osteotomy on their right femur and received subcutaneous drug administration daily for either 2, 3 or 4 weeks, as indicated, for each compound. 1.times. concentration represents 1 nmol/day, 10.times. represents 10 nmol/day, 100.times. represents 100 nmol/day most studies have an n of 5.

[0128] Aspects of the disclosure include conjugates sometimes written in the form of X-Y-Z, wherein each conjugate includes at least one moiety (X) that has the ability to effect bone growth, development and/or healing, for example, anabolic agents, and a targeting moiety (Z) which has an affinity for bone and helps to direct the conjugate to bone. In some of these conjugates, the X and Z portions are joined together by a linker region (Y).

[0129] Targeting moieties (Z), many of which are explicit or implicit disclosed herein, have the potential to target bone anabolic agents to bone fractures, ostectomies, and osteotomy sites. The compounds described here are composed of molecules with high affinity towards hydroxyapatite and a bone anabolic agent. Although targeting has been exemplified primarily with acidic oligopeptides, all molecules with affinity towards hydroxyapatite could be attached to a bone anabolic agent to improve fracture repair. These molecules include but are not limited to ranelate, bisphosphonates, tetracyclines, polyphosphates, molecules with multiple carboxylic acids, calcium chelating molecules, metal chelators, acidic amino acid chains of either d or L chirality. Each of the previously listed targeting molecules can be single units, polymers, dendrimers or multiple units. Other molecules can also be substituted for the targeting agent. These include peptides, proteins and manmade molecules that intercalate, bind to, adsorb to, or hybridize with: collagen, the extracellular matrix, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, elastin, fibronectin, laminin, proteoglycans, basement membrane, extracellular polymeric substances, integrins, blood clotting factors, fibrinogen, thrombin, fibrin, and other extracellular macromolecules, It is also possible to target using a combination of the listed targeting molecules.

Definitions

[0130] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0131] The term "BV/TV" means the bone volume divided by total volume of the 100 thickest micro CT slices of the fracture callus.

[0132] The term "TbTh" means the trabecular thickness of the 100 thickest micro CT slices of the fracture callus.

[0133] The term "By" means the overall bone volume of the 100 thickest micro CT slices of the fracture callus.

[0134] The term "TbSp" means the spacing between the trabecula in the 100 thickest micro CT slices of the fracture callus.

[0135] The term "Peak Load" means a postmortem 4 point bend of the healed femur. Peak load represents the maximum force the healed femur withstood before it refractured.

[0136] The term "D10" at the end of any name represents that the peptide is targeted to bone by a chain of 10 aspartic acids. D10 can be at the N-terminus or C-terminus of the specified peptide.

[0137] The term "E10" of "(D)E10" at the end of any name represents that the peptide is targeted to bone by a chain of 10 (D) glutamic acids.

[0138] The term "mp4" in the middle of any name represents that the therapeutic is linked to the targeting peptide via a polymer of 4 minipegs aka 8-Amino-3,6-Dioxaoctanoic Acid.

[0139] The term "P4" means a fragment that represents the knuckle epitope in hBMP-2.

[0140] The term "P-4" corresponds to residues 73-92 of BMP-2 in which Cys-78, Cys-79, and Met-89 are changed to Ser, Ser, and threonine (Thr). BMPs are well known regulators of bone and cartilage formation. BMPs bind as dimers to type I and type II Ser/Thr receptor kinases, forming an oligomeric complex that activates intracellular small mothers against decapentaplegic (Smad) proteins leading to their translocation into the nucleus where they serve as transcription factors to activate different OB differentiation markers (such as Runx2), leading to osteoblastogenesis. BMPs have also been shown to stimulate mesenchymal stem cells (MSC) differentiation to OBs by promoting recruitment of osteoprogenitor cells.

[0141] Compounds which effect bone growth and may be used to practice aspects of the present disclosure include but are not limited to the following: growth factors, fragments of neuropeptides or neurotransmitters, synthetic peptides or small molecules that mimic the action of neuropeptides or neurotransmitters, a sarcoma (SRC) kinase inhibitor peptides or small molecules that inhibits the activity of SRC kinase, or a sphingosine-1-phosphate receptors (S1P-R) modulator kinase inhibitor peptides or small molecules that modulates the activity of S1P-R. Examples of which may include but are not limited to: PACAP, VIP, Calcitonin, Calcitonin gene like peptide, amylin, Neuropeptide Y, Serotonin, Dopamine, SSRIs, Purines and pyrimidines, Glutamate, norepinephrine, epinephrine, Substance P, Nerve growth factor, Dasatinib Imatinib, Saracatinib (AZD0530), Bosutinib (SKI-606), KK2-391, NVP-BHG712, PP2, PP121, PP1, MNS (3,4-Methylenedioxy-beta-nitrostyrene), UM-164, Repotrectinib, WH-4-023, CCT196969, MLR-1023, SU6656, AD80, eCF506, AZM 475271, Herbimycin A, KB SRC 4, Lavendustin A (RG 14355), SU 6656, S1P, FTY720, AAL(R), KRP-203, Ceralifimod, ponesimod, siponimod, or CYM-5442, RP-001.

Bone Growth Modifiers and Delivery Peptides

TABLE-US-00002 [0142] BFP1D10 (SEQ ID NO: 1) DDDDDDDDDDGQGFSYPYKAVFSTQ

[0143] BFP (bone forming Peptide) a fragment of immature BMP7 is a 15-amino acid peptide corresponding to residues 100-115 of the immature form of BMP-7 which like BMP2 is involved in osteogenic differentiation, proliferation, and formation of new bone. This short peptide also induces osteogenesis calcium content in MSCs.

BMP-9

[0144] BMP-9 is also a potent regulator of osteogenesis and chondrogenesis and is a potent inducer of differentiation of osteoblasts. pBMP9 is a 23-residue peptide derived from residues 68-87 of the knuckle epitope of human BMP-9. The mechanism of action of this peptide is likely to involve the Smad pathway. The structure of BMP9 is depicted in FIG. 27.

Ghrelin D10

[0145] Ghrelin is a 28-residue peptide hormone synthesized primarily by the gastric fundus in response to fasting, and acts as a ligand of the growth hormone secretagogue (GHS) receptor (GHSR) to promote growth hormone release from the pituitary. Ghrelin stimulation at the GHSR leads to the proliferation of osteoblasts and prevents the apoptosis of osteoblasts through mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) and phosphoinositide 3-kinase/protein kinase B (PKB) (PI3K/AKT) pathways. Ghrelin also stimulates osteoprotegerin (OPG) gene expression, which inhibits the coupling between the osteoclasts and Osteoblasts, leading to reduced osteoblast-related osteoclast differentiation. Increased OPG also and decreases osteoclast activity. Ghrelin is only active when the Ser-3 is acylated with octanoic acid. Our construct contains a stabilized version of this where Ser-3 was replaced with diaminopropionic acid. The structure of Ghrelin D10 is depicted in FIG. 28.

Preptin D10

[0146] Preptin is a 34-residue peptide hormone that is secreted by the .beta.-cells of the pancreatic islets. This peptide corresponds to Asp-69 to Leu-102 of the E-peptide of proinsulin-like growth factor-II (pro-IGF-II). Preptin's anabolic effects on bone are exerted through its ability to stimulating osteoblasts s proliferation, differentiation, and promoting their survival. Preptin's proliferative effect is predicted to be facilitated through a G-protein-coupled receptor triggering phosphorylation of p42/44 MAP kinases. Some of Preptin's anabolic effects are believed to be due to it stimulating an increase in a known bone anabolic connective tissue growth factor. While the native peptide effects glucose metabolism the first 16 amino acids are important for its anabolic effects and have no effects on glucose metabolism. The structure of Preptin D10 is depicted in FIG. 29.

CNP-D10 is a C-Type Natriuretic Peptide Targeted with D10

[0147] C-Type Natriuretic Peptide (CNP) contains 22 residues stabilized by an intramolecular disulfide linkage between Cys-6 and Cys-22 it functions as a local regulator of vascular tone, possibly due to its strong vasorelaxant properties. CNP also acts on the differentiation and proliferation of OBs, OCs, and chondrocytes via an autocrine/paracrine process through binding to the natriuretic peptide receptor B (NPR-B). CNP activates bone turnover and remodeling. Endochondral ossification is another mechanism of bone formation affecting chondrocytes. It involves the conversion of an initial cartilage template into bone such as long bones and vertebrae. CNP has been shown to be an important anabolic regulator of endochondral ossification. The structure of CNP-D10 is depicted in FIG. 30.

VIP D10 is Vasoactive Intestinal Peptide Targeted with D10

[0148] Vasoactive intestinal peptide (VIP), a neuropeptide that consists of 28 amino acids and originally isolated from porcine intestine. VIP has several effects however its receptors are present on the nerves that rapidly innervate the fracture callus. It has been shown to be an important regulator of bone formation. VIP exerts its biological effects through the G-protein-coupled receptors (VPAC1, VPAC2, and PAC1). Signaling through these receptors also enhanced cell osteoblast differentiation and proliferation. It also increases expressions of collagen type I, osterix, and ALP through signaling at the VPAC2 receptor by triggering an increase in intracellular calcium. VIP also increases the expressions of BMPs and the nuclear presence of Smad1 transcription factor, which can activate various bone-specific genes. VIP also enhances osteoblast proliferation and mineralization through increased gap junction intercellular communication (GJIC) between osteoblasts. VIP also affects the differentiation of osteoclasts thus leading to an increase in bone resorption. The structure of VIP D10 is depicted in FIG. 31.

Substance P with 4 Mini PEG Conjugated to D10

[0149] Substance P- is an 11-amino acid long pro-inflammatory neuropeptide belonging to the tachykinin family. Substance P improves mineralization of osteoblasts and the expression of osteogenic markers at late-stage bone formation, by activating neurokinin-1 receptor, a G-protein coupled receptor found in the central and peripheral nervous systems. Also, substance P reduces osteoclastogenesis and bone resorption. Substance P upregulates the expressions of collagen type 1, ALP, Runx2 and osteocalcin in osteoblasts this effect involves the activation of Wnt/.beta.-catenin signaling pathway. Substance P promotes differentiation and migration capability of rat bone marrow MSCs and activates BMP-2 expression in osteoblasts. Some of substance P's anabolic effects are attributed to in human to increases in osteoblast proliferation and mineralization through increased gap junction intercellular communication between osteoblasts. Gap junction intercellular communication has important roles in conveying the anabolic effects of hormones and growth factors and regulating transcription of osteogenic markers. The structure of Substance P with 4 mini PEG conjugated to D10 is depicted in FIG. 32.

CBMD10-is the Collagen Binding Motif of Osteopontin Targeted by D10

[0150] CBM collagen binding motif is the highly conserved 28-residue collagen binding motif (CBM) (residues 150-177) of human osteopontin. Osteopontin, a glycosylated phosphoprotein prominently localized in the extracellular matrix (ECM) of mineralized bone tissue to form a complex with collagen in bone tissue, thereby inducing mineralization of collagen fibrils. CBM enhances osteoblast differentiation of human MSC. CBM causes osteogenic differentiation of human bone marrow MSCs and increases mineralized of bone. CBM works in human MSCs by increasing extracellular Ca' influx, which leads to the activation of CaMKII and the subsequent phosphorylation of ERK1/2, ultimately influencing OB differentiation. The structure of VIP D10 is depicted in FIG. 33.

ODPD10

[0151] Osteopontin-derived peptide (ODP), a 15-residue peptide derived from rat osteopontin. ODP like CBM is a fragment of extracellular protein involved in the mineralization of collagen. ODP enhanced the differentiation and mineralization of MSCs. ODP improves the attachment via receptor mediated attachment and migration of osteoblasts and fibroblasts to the fracture site. ODP improves the proliferation and migration of osteoblasts. Though the signaling pathways aren't completely elucidated for this molecule its believed that it works in a similar mechanism as CBM. The structure of ODPD10 is depicted in FIG. 34.

TABLE-US-00003 OGP-D10 (SEQ ID NO: 2) DDDDDDDDDDALKRQGRTLYGFGG

[0152] OGP-targeted Osteogenic growth peptide (OGP) is composed of a 14-aa residue identical to the C-terminus of histone 4 conjugated to an acidic oligopeptide at the N-terminus. Systemic administration of free OGP has been shown to improve fracture repair by improving the mineralization of cartilaginous fracture callus.

TABLE-US-00004 (SEQ ID NO: 11) MGF-DDDDDDDDDDYQPPSTNKNTKSQRRKGSTFEEHK

[0153] Targeted mechano growth factor (MGF) E peptide is a splice variant of insulin-like growth factor I (IGF-I) with a targeting acidic oligopeptide on the N terminus. MGF causes osteoblast proliferation through the MAPK-ERK signaling pathway. Local injections (57 ug/kg) in rabbit bone defects (5 mm) demonstrated accelerated healing through osteoblast proliferation.

TABLE-US-00005 (SEQ ID NO: 12) TP508-DDDDDDDDDDAGYKPDEGKRGDACEGDSGGPFV

[0154] Targeted TP-508 is a prothrombin peptide that has been modified on the N-terminus with an acidic oligopeptide. The anabolic portion of TP-508 has been used in clinical trials for repairing foot ulcers. Free tp-508 has a proliferative effect on osteoblasts. Local injections have demonstrated accelerated fracture repair in older rats.

Sphingosine-1-Phosphate (S1P)--S1P Lyase Inhibitor (DOP) and S1PR1 Agonist (Ozanimod)

[0155] Sphingosine-1-phosphate (S1P) is an important extracellular signaling molecule that mediates a variety of physiological functionalities. There are five cell-surface receptors responsible for the downstream signaling pathways related to S1P, namely S1PR1 to S1PR5. In particular, the S1P-S1PR1 and S1P-S1PR2 axes are essential in bone metabolism. It has been reported that S1PR1 knockdown would lead to reduced trabecular thickness and trabecular density in vivo, resulting in an osteoporotic phenotype.1 Meanwhile, S1PR2 signaling boosts OPG production and promotes osteoblast differentiation, which in turn facilitates bone formation. Naturally, stimulation of S1P signaling at the fracture site might promote bone growth.

[0156] Small molecule agonists for S1P receptors have been widely reported. S1P lyase is the sole enzyme responsible for the irreversible degradation of S1P. The inhibition of this enzyme would increase the availability of S1P, resulting in enhanced S1P signaling. Targeted delivery of a S1P inhibitor or a S1PR1 agonist to the fracture surface would boost S1P signaling at the fracture site, which might accelerate fracture repair.

[0157] By utilizing a targeted delivery strategy, S1P lyase inhibitors or S1PR1/S1PR2 agonists were delivered specifically to the fracture surface for enhanced ability to facilitate fracture healing. Local injections demonstrated acceleration of fracture healing from representative S1P lyase inhibitor (DOP) and S1PR1 agonist (ozanimod). The structure of Ozanimod is depicted in FIG. 35. The structure of DOP is depicted in FIG. 36 The structure of D.sub.10-ozanimod in FIG. 37. The structure of D.sub.10-DOP is depicted in FIG. 38.

TABLE-US-00006 Pituitary Adenylate Cyclase-Activating Peptide (D)E.sub.10-PACAP(D)E.sub.10 (SEQ ID NO: 14) HHSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK

[0158] Vasoactive Intestinal Peptide (VIP) and Pituitary Adenylyl Cyclase Activating Peptide (PACAP) are two neuropeptides that have demonstrated an activating and proliferative effect on osteoblasts, the cells that produce the mineral component of bone. VIP is important in bone remodeling and has shown stimulating effects on alkaline phosphatase (ALP), a marker of osteoblast mineralization. PACAP is capable of affecting osteoblasts independently of VIP, and in cell cultures has shown osteogenic effects. VIP and PACAP both act on g-protein coupled receptors VPAC-1 and VPAC-2 with equal affinity, while PACAP acts on the G-protein coupled receptor (GPCR) PAC-1 with 1000 times greater affinity. These receptors are found throughout the body, and VIP and PACAP play roles in the brain, intestines, immune system, endocrine system, and others as well as bone. By synthesizing these peptides in sequence with acidic oligopeptide targeting ligands, accelerated healing and minimized of off target effects of PACAP are demonstrated by targeting bone fracture sites. The structure of PACAP (D)E.sub.10 is depicted in FIG. 54.

Pituitary Adenylate Cyclase-Activating Peptide--Amylin (1-8)-D.sub.10 (SEQ ID NO 15)

[0159] Amylin is a natural 37 amino acid peptide formed primarily by the .beta.-cells of the pancreatic islets. It comprises an amidated C-terminus and a disulfide bond between the Cys residues at sites 2 and 7 in the primary sequence. The present compound is amino acid 1-8 of the native sequence attached to 4 peg2 spacers then attached to ten aspartic acid to home it to hydroxyapatite. Amylin is structurally homologous to calcitonin gene-related peptide (CGRP) and more distantly to calcitonin itself. Like calcitonin, amylin was found to decrease Osteoclasts (OC) development in mouse bone marrow cultures, stimulate adenosine monophosphate (AMP) formation, induce quiescence in OCs, and thus, reducing the extent of bone resorbed. In addition to its activity on OCs, amylin has also been found to affect osteoblasts (OB), possibly through an increase of cAMP and the activation of MAPK/protein kinase C signaling pathway. The structure for Amylin (1-8) is depicted in FIG. 45.

Calcitonin Gene-Related Peptide (CGRP) (SEQ ID NO 16)

[0160] The targeted construct of CGRP is the full natural 37 amino acids with the 2nd and 7th amino acids cyclized then on the C terminus is 4 minipeg spacers followed by ten D glutamic acids.

[0161] Primary afferent sensory nerve fibers within the periosteum release peptides important for osteogenesis including the osteoanabolic neuropeptide, CGRP (calcitonin gene-related peptide). Thus, activation of sensory nerves release or exogenous treatment with CGRP is demonstrated to accelerate bone fracture repair by localizing it just to the bone fracture site. The structure of CGRP is depicted in FIG. 49. CGRP is also demonstrated to improve fracture healing, likely through promotion of both osteogenic differentiation and angiogenesis. Its effect can also be contributed to a potential decrease in osteoclasts.

Material and Methods

Solid Phase Peptide Synthesis

[0162] Unless noted otherwise, the conjugates of the present disclosure are synthesized using the following synthesis. In a solid phase peptide synthesis vial capable of bubbling nitrogen, Wang resin (0.39 mmol/g) was loaded at 0.39 mmol/g with the first amino acid overnight in dichloromethane (DCM) and diisopropylethylamine (DIPEA). The resin was then capped with acetic anhydride and pyridine for 30 minutes, followed by three washes of DCM and dimethylformamide (DMF), respectively. Following each amino acid coupling reaction, fluorenylmethyloxycarbonyl (Fmoc)-groups were removed by three 10 minute incubations with 20% (v/v) piperidine in DMF. The resin was then washed 3.times. with DMF prior to the next amino acid being added. Each amino acid was added in a 5-fold excess with N,N,N',N'-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU)/DIPEA. Upon completion of the synthesis, peptides were 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 10 fold tris(2-carboxyethyl)phosphine (TCEP).

Small Molecules Synthesis

[0163] For the initial loading of the solid phase peptide synthesis (SPPS) resin, 2-chlorotrityl chloride resin (0.4 g, 1.4 mmol/g) was swollen in DCM (10 mL/g resin) followed by addition of Fmoc-L-Asp(OtBu)-OH (1.15 g, 2.8 mmol) and DIPEA (1.66 mL, 9.5 mmol) dissolved in DCM (14 mL). The mixture was agitated by bubbling argon for 1 hour, after which the solution was drained before 20 mL of capping cocktail (DCM:MeOH:DIPEA=17:2:1) was added and the solution was again bubbled for 20 minutes. The resin was then subjected to standard washing procedures which consisted of washes with DMF (3 times), DCM (3 times) and isopropyl alcohol (IPA) (3 times) following each coupling reaction, and washes with DMF (3 times) following each deprotection. After the initial loading, all subsequent coupling reactions were performed with solutions of Fmoc-L-Asp(O-tert-butyl)-OH (1.15 g, 2.8 mmol) or Fmoc-S-trityl-L-cysteine (1.64 g, 2.8 mmol), PyBOP (1.42 g, 2.75 mmol), and DIPEA (1.66 mL, 9.5 mmol) in DMF (14 mL). One-hour standard coupling time was used for all aspartic acid and cysteine residues. Fmoc-deprotection was done with 20% piperidine solution in DMF for two sessions of 5 minutes and 10 minutes each. The 11-mer peptidic product was cleaved off the resin using a cleavage cocktail consisting of 90% trifluoroacetic acid (TFA), 3.3% triisopropyl silane (TIPS), 3.3% water and 3.3% ethane dithiol (EDT). Following cleavage, the crude product was concentrated under reduced pressure to remove most TFA, water, TIPS, and EDT, and then washed 3.times. with ethyl ether (Et.sub.2O), dried under reduced pressure for 24 hours to give 1 as a white powder (680 mg, 81.3% overall yield, 98.1% average coupling efficiency).

[0164] 3-maleimidopropionic acid (200 mg, 1.18 mmol) and benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (572 mg, 1.10 mmol) were dissolved in 5 mL anhydrous DMF in a 50-mL round-bottom flask degassed with argon. The flask was cooled on ice and diisopropylethylamine (1.03 mL, 5.9 mmol) was added and stirred for 5 minutes. Dasatinib (360 mg, 0.738 mmol) dissolved in 2 mL anhydrous DMF was then added dropwise to the mixture and the reaction mixture was warmed slowly to room temperature with stirring over the ensuing 4 hours. Ethyl acetate (.about.50 ml) was added to the reaction flask and the diluted mixture was washed 2.times. with deionized (DI) water followed by 4 washes with saturated aqueous sodium chloride (NaCl). The organic phase was then collected, dried over sodium sulfate and concentrated in vacuo to yield the crude product. The crude product was purified by flash column chromatography on Teledyne CombiFlash Rf+ Lumen (0-25% MeOH in DCM) to give 2 as a pale-yellow powder (320 mg, 68%).

Cyclization Method for Disulfide Bridged Cyclic Peptides

[0165] For CNP, the standard synthesis of the linear form of the cyclic peptides Fmoc Cystine with Acetamidomethyl protecting group on the sulfur was used. Then, to cyclize the peptide, the Cys(Acm) On-Resin was suspended the linear peptide resin in N,N-dimethylformamide (DMF) (approximately 1 mL/gram of resin). Then, the resin was treated with 10 equiv. of iodine (I2) in DMF/H2O 4:1 (v/v), approximately 1 mL/gram of resin). Then, argon gas was bubbled through the reaction mixture at room temperature for 40 minutes. Then, the resin was filtered and washed 3 times with DMF, 2 times with 2% ascorbic acid in DMF, 5 times with DMF, and 3 times with dichloromethane (DCM). Then, proceeded with normal n terminal fmoc deprotection and cleavage from the resin with normal cleavage solution with no TCEP added to preserve the disulfide bond. The peptides were then as all peptides purified using reverse phase chromatography on an HPLC using a 0-50% 20 mM ammonium acetate: acetonitrile gradient. The product was then identified from the appropriate fraction using LCMS and lyophilized to recover it from the water: acetonitrile mixture. All compounds were dissolved in sterile phosphate buffered saline (PBS) at the appropriate dose concentrations for drug delivery.

General Methods for Obtaining Test Data

[0166] The targeted conjugates were synthesized using standard Fmoc solid-phase peptide synthesis, as described above. To ensure the conjugates' activity, mouse pre-osteoblast (MCTC3-E1) cells were treated with the targeted and untargeted compounds for three days at concentrations from 1 pM to 100 nM. After three days of treatment, the cells were harvested, and the RNA was purified from the cells. Expression levels of ALP, RUNx2 (transcription factor for osteoblast differentiation), osterix (OSX), osteopontin (OPN), collagen 1A (Col-1A), OPG, RANKL, sclerostin gene (SOST), and OC were quantified via quantitative reverse transcription polymerase chain reaction (RT-qPCR). Once the biological activity of the conjugates was confirmed, they were tested in vivo in a fracture model. Aseptic surgical techniques were used to place a 23-gage needle as in intramedullary nail in the femur of anesthetized, 12-week-old Swiss Webster mice for internal fixation before fracture. Femur fractures were induced using a drop weight fracture device from RISystem. The mice received buprenorphine for three days post fracture. The mice were dosed subcutaneously each day for three weeks or 17 days. Fracture healing was assessed using microCT (Scanco Medical Ag). Morphometric parameters were quantified in the 100 widest slices of the fracture callus. Trabecular thickness (TbTh), trabecular spacing (TbSp), total volume (TV), and volume of calcified callus (BV) were calculated. Fractured femurs were tested for strength in a four-point bend to failure using an Electro Force TestBench (TA Instruments). Lower supports were 10 mm apart on the anterior face of the femur in contact with the proximal and distal diaphysis. Upper supports were 4 mm apart and spanned the entire fracture callus on the diaphysis. Force was applied from the posterior face of the femur with a displacement rate of 0.3 mm/sec. Peak load, yield load, stiffness, displacement post yield, work to fracture, and deformation data were generated. Statistical analysis was performed using a two-way analysis of variance (ANOVA) and a Tukey post-hoc analysis with significance reported at the 95% confidence level. All animal experiments were performed in accordance with protocols approved by Purdue University's Institutional Animal Care and Use Committee (IACUC).

EXAMPLES

Example 1. Targeted Delivery of Src Kinase Inhibitors to Fracture Site for Accelerated Healing

[0167] Example 1 shows representative Src kinase inhibitors Dasatinib and E738 (structures shown in FIGS. 3-4 respectively) effectively increased the bone density of the fracture callus when they are conjugated with acidic aspartic acids. See FIGS. 1-2, where bone density of the fracture callus from the targeted Dasatinib group is twice as dense as the saline group, and 50% denser than the free Dasatinib group; targeted E738 conjugate has significantly improved the bone density and trabecular thickness at the fracture callus. The structure of CBM D10 is depicted in FIG. 33.

Example 2. Representative Anabolic Peptides on Peak Load of Fractured Femurs after Two Weeks

[0168] Example 2 provides the maximum force a representative anabolic peptide induced healed femur can withstand before it refractured. As shown in FIG. 5, bone morphogenetic protein pathway signaling peptide BFP-D10 with 100 nmol/day (100.times.) treatment obtained the maximum peak load, followed by IGF derived peptide of Preptin-D10 100.times., and Osteogenic growth peptide (OGP-D10 100.times.), as compared to PBS.

Example 3. Preptin D10 Efficacy on Fracture Healing

[0169] Example 3 indicates Preptin D10 effect on healing fractured bone after 2 weeks of various concentrations application (1 nmol/day, 10 nmol/day and 100 nmol/day, referred as 1.times., 10.times. and 100.times. respectively). The healing was reflected as BV/TV in FIG. 6, TbTh in FIG. 7 and bone volume in FIG. 8, all in a dose dependent manner.

Example 4. OGP D10 Efficacy on Fracture Healing

[0170] Example 4 indicates osteogenic growth peptide conjugate (OGP-D10) effect on healing fractured bone after 2 weeks of various concentrations application (1 nmol/day, and 100 nmol/day, referred as 1.times., and 100.times. respectively). The healing was reflected as BV/TV in FIG. 9, TbTh in FIG. 10 and TbSp in FIG. 11, all in a dose dependent manner.

Example 5. BFP D10 Efficacy on Fracture Healing

[0171] Example 5 indicates bone forming peptide conjugate (BMP-D10) effect on healing fractured bone after 2 weeks of various concentrations application (1 nmol/day, 10 nmol/day and 100 nmol/day, referred as 1.times., 10.times. and 100.times. respectively). The healing was reflected as BV/TV in FIG. 12, and TbSp in FIG. 13 in a dose dependent manner.

Example 6. Substance P D10 Effect on Fracture Healing

[0172] Example 6 indicates substance P D10 conjugate effect on healing fractured bone after 4 weeks of various concentrations application (1 nmol/day, 10 nmol/day and 100 nmol/day, referred as 1.times., 10.times. and 100.times. respectively). The healing was reflected as BV/TV in FIG. 14A in dose dependent manner. FIG. 14B indicates the peak load of substance P D10 10.times. induced healed femur can withstand between 30-35 Newtons force.

Example 7. Ghrelin-D10 Effect on Fracture Healing

[0173] Example 7 indicates Ghrelin-D10 conjugate effect on healing fractured bone after 4 weeks of various concentrations application (1 nmol/day, 10 nmol/day and 100 nmol/day, referred as 1.times., 10.times. and 100.times. respectively). The healing was reflected as BV/TV in FIG. 15 in dose dependent manner.

Example 8. pBMP9 D10 Effect on Fracture Healing

[0174] Example 8 indicates pBMP9 D10 conjugate effect on healing fractured bone after 4 weeks of various concentrations application (1 nmol/day, 10 nmol/day and 100 nmol/day, referred as 1.times., 10.times. and 100.times. respectively). The healing was reflected as bone volume in FIG. 16 and BV/TV in FIG. 17 in a dose dependent manner.

Example 9. CNP D10 Effect on Fracture Healing

[0175] Example 9 indicates C-Type Natriuretic Peptide conjugate CNP D10 effect on healing fractured bone after 4 weeks of various concentrations application (1 nmol/day and 10 nmol/day referred as 1.times. and 10.times. respectively). The healing was reflected as BV/TV in FIG. 18 in a dose dependent manner.

Example 10. ODP D10 Effect on Fracture Healing

[0176] Example 10 indicates osteopontin derived peptide conjugate ODP D10 effect on healing fractured bone after 4 weeks of 1 nmol/day (referred as 1.times.). The healing was reflected as BV/TV in FIG. 19.

Example 11. CBM D10 Effect on Fracture Healing

[0177] Example 11 indicates collagen binding motif of osteopontin conjugate CBM D10 effect on healing fractured bone after 3 weeks of various concentrations application (0.1 nmol/day, 1 nmol/day and 10 nmol/day, referred as 0.1.times., 1.times. and 10.times. respectively). The healing was reflected as BV/TV in FIG. 20 in a dose dependent manner. It is worth noting that the lowest does of CBM D10 has the similar effect of free PTH, an anabolic drug without specific bone targeting.

Example 12. P4 D10 Effect on Fracture Healing

[0178] Example 12 indicates P4 D10 conjugate effect on healing fractured bone after 4 weeks of various concentrations application (1 nmol/day and 10 nmol/day, referred as 1.times. and 10.times. respectively). The healing was reflected as BV/TV in FIG. 21 in a dose dependent manner and bone volume in FIG. 22.

Example 13. MGF D10 Effect on Fracture Healing

[0179] Example 13 indicates mechano growth factor conjugate MGF D10 effect on healing fractured bone after 4 weeks of various concentrations application (1 nmol/day and 10 nmol/day, referred as 1.times. and 10.times. respectively). The healing was reflected as BV/TV in FIG. 23 in a dose dependent manner.

Example 14. TP508 D10 Effect on Fracture Healing

[0180] Example 14 indicates thrombin fragment TP508 conjugate TP508 D10 effect on healing fracture after 4 weeks of various concentrations application (1 nmol/day and 10 nmol/day, referred as 1.times. and 10.times. respectively). The healing was reflected as BV/TV in FIG. 24 in a dose dependent manner.

Example 15. VIP D10 Effect on Fracture Healing

[0181] Example 15 indicates vasoactive intestinal peptide conjugate VIP D10 effect on healing fracture. Referring to FIGS. 25a and 25b, healing was reflected as BV/TV (FIG. 25a) and TbTh (FIG. 25b) after 4 weeks of 1 nmol/day application (1.times.). Referring to FIGS. 26a, 26b, and 26c, in vivo fracture healing efficacy of VIP_mp4_(D)E10 conjugate on Swiss Webster fracture-bearing mice (n=5) after 3 weeks was reflected as BV/TV, Max Load, and Work to Fracture respectively. BV/TV represents the bone volume divided by total volume of the 100 thickest micro CT slices of the fracture callus and is a measure of how dense the bone is at the site of fracture repair. Max load represents the maximum force the healed femur withstood before it refractured in a postmortem 4 point bend analysis. Peak load is a measure of how strong the bone is at the site of fracture repair. Work to fracture represents the total amount of energy absorbed by the healed femur before it refractured in a postmortem 4 point bend analysis. Work to fracture is a measure of how strong the bone is at the site of fracture repair. 0.05.times. and 0.1.times. and 0.5.times. represent that a dose of 0.05 nmol, 0.1 nmol and 0.5 nmol of the conjugate was delivered daily by subcutaneous injection. VIP_mp4_(D)E10 conjugate raises bone density and bone strength at the fracture calluses three weeks post fracture.

Example 16. D.sub.10-DOP Effect on Fracture Healing

[0182] Example 16 indicates sphinogosine-1-phosphate lyase inhibitor conjugate DOP in vivo fracture healing efficacy after 3 weeks of 3 .mu.mol/kg every other day application (03Q), 10 .mu.mol/kg every other day application (1Q), three doses of 0.3 .mu.mol/kg in the third week application (003W3), three doses of 1.0 .mu.mol/kg in the third week application (01W3), three doses of 3 .mu.mol/kg in the third week application (03W3), and three doses of 10 .mu.mol/kg in the third week application (1W3). The in vivo fracture healing efficacy of D.sub.10-DOP conjugate on fracture-bearing mice was reflected as BV/TV in FIG. 39. D.sub.10-DOP conjugate raises bone density at the fracture calluses three weeks post fracture. The conjugate remains active with various dosing schedules.

Example 17. Ozanimod In Vitro Effect on Pre-Osteoblastic Cell Line MC3T3-E1

[0183] Example 17 indicates the in vitro efficacy of ozanimod on pre-osteoblastic cell line MC3T30E1. Specifically, example 17 shows a boost in alkaline phosphatase (ALP) activity of pre-osteoblastic cell line MC3T3-E1 following exposure to ozanimod of various concentrations in the culture media for 7 days. Upregulated ALP activity indicates promoted osteoblast differentiation. Referring to FIG. 40, the ALP activity in mOD/min/ug protein is shown at 0.1 nM, 0.2 nM, 0.5 nM, 1 nM, and 5 nM concentrations of ozanimod and dimethyl sulfoxide (DMSO) alone.

Example 18. D.sub.10-Ozanimod Effect on Fracture Healing

[0184] Example 18 indicates S1PR1 agonist conjugate D.sub.10-ozanimod effect on healing fracture after 3 week of 0.01 .mu.mol/kg every day application (0.001.times.), 0.03 .mu.mol/kg every day application (0.003.times.), 0.1 .mu.mol/kg every day application (0.01.times.), 0.3 .mu.mol/kg every day application (0.3.times.), and 1 .mu.mol/kg every day application (0.1.times.). The in vivo fracture healing efficacy of D.sub.10-ozanimod conjugate on fracture-bearing mice is reflected as BV/TV in FIG. 41. Daily dosages of D.sub.10-ozanimod conjugate raises bone density at the fracture calluses three weeks post fracture. The conjugate is effective over a wide dosage range.

Example 19. PACAP-(D)E.sub.10 Effect on Fracture Healing

[0185] Example 19 indicates PACAP-(D)E.sub.10 conjugate effect on healing fracture after 3 weeks of 0.1 nmol per day application (0.1.times.), 1 nmol per day application (1.times.), and 10 nmol per day application (10.times.) delivered by subcutaneous injection. The in vivo fracture healing efficacy of PACAP-(D)E.sub.10 conjugate on Swiss Webster fracture-bearing mice (n=5) after 3 weeks is reflected as BV/TV in FIG. 42, as Max Load (Newtons) in FIG. 43, and Stiffness (N/mm) in FIG. 44. PACAP-(D)E.sub.10 raises bone density, improves bone strength, and improves bone stiffness at the fracture calluses three weeks post fracture.

Example 20. Amylin(1-8)-D10 Effect on Fracture Healing

[0186] Example 20 indicates Amylin(1-8)-D.sub.10 effect on healing fracture after 3 weeks of 1 nmol per day application (1.times.), 10 nmol per day application (10.times.), and 100 nmol per day application (100.times.) delivered by subcutaneous injection. The in vivo fracture healing efficacy of Amylin (1-8)-D.sub.10 on Swiss Webster fracture-bearing mice (n=5) after 3 weeks is reflected as BV/TV in FIG. 46, as Max Load (Newtons) in FIG. 47, and as Work to Fracture (mJ) in FIG. 48. Amylin(1-8)-D.sub.10 conjugate raises bone density, and improves bone strength at the fracture calluses three weeks post fracture.

Example 21. CGRP (D)E.sub.10 Effect on Fracture Healing

[0187] Example 21 indicates CGRP (D)E.sub.10 conjugate effect on healing fracture after 3 weeks of 0.1 nmol per day application (0.1.times.), 1 nmol per day application (1.times.), and 10 nmol per day application (10.times.) delivered by subcutaneous injection. The in vivo fracture healing efficacy of CGRP (D)E.sub.10 conjugate on Swiss Webster fracture-bearing mice (n=5) after 3 weeks is reflected as BV/TV in FIG. 50, as Bone Volume (BV) in FIG. 51, as Max Load (Newtons) in FIG. 52, and as Work to Fracture (mJ) in FIG. 53. CGRP (D)E.sub.10 conjugate raises bone density, stimulates more bone to be generated, and raises bone strength at the fracture calluses three weeks post fracture.

Sequence CWU 1

1

22125PRTArtificial SequenceBFP D10 1Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Gly Gln Gly Phe Ser Tyr1 5 10 15Pro Tyr Lys Ala Val Phe Ser Thr Gln 20 25224PRTArtificial SequenceOGP D10 2Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Ala Leu Lys Arg Gln Gly1 5 10 15Arg Thr Leu Tyr Gly Phe Gly Gly 20327PRTArtificial SequencePreptin D10 3His Asp Val Ser Thr Ser Gln Ala Val Leu Pro Asp Asp Phe Pro Arg1 5 10 15Tyr Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp 20 25421PRTArtificial Sequencesubstance P 4 mini PEG D10 withMISC_FEATURE(10)..(11)4 mini PEG linker 4Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Arg Pro Lys Pro Gln Gln1 5 10 15Phe Phe Gly Leu Met 20538PRTArtificial SequenceGhrelin D10 with Ser-3 replacementMISC_FEATURE(3)..(3)Ser-3 modified with diaminopropionic acid 5His Gly Ser Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys1 5 10 15Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro Arg Asp Asp Asp Asp 20 25 30Asp Asp Asp Asp Asp Asp 35633PRTArtificial SequenceBMP9-D10MOD_RES(23)..(24)4 MINI PEG LINKER 6Cys Gly Gly Lys Val Gly Lys Ala Cys Cys Val Pro Thr Lys Leu Ser1 5 10 15Pro Ile Ser Val Leu Tyr Lys Asp Asp Asp Asp Asp Asp Asp Asp Asp 20 25 30Asp733PRTArtificial SequenceCNP D10DISULFID(17)..(33) 7His Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Gly Leu Ser Lys Gly1 5 10 15Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly 20 25 30Cys839PRTArtificial SequenceVasoactive intestinal peptide with D10 (VIP D10) 8His His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys1 5 10 15Gln Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn Asp Asp Asp 20 25 30Asp Asp Asp Asp Asp Asp Asp 35939PRTArtificial SequenceCBM D10 9His Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Gly Leu Arg Ser Lys1 5 10 15Ser Lys Lys Phe Arg Arg Pro Asp Ile Gln Tyr Pro Asp Ala Thr Asp 20 25 30Glu Asp Ile Thr Ser His Met 351031PRTArtificial SequenceP4 D10-a BMP2 fragment 10His Lys Ile Pro Lys Ala Ser Ser Val Pro Thr Glu Leu Ser Ala Ile1 5 10 15Ser Thr Leu Tyr Leu Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp 20 25 301134PRTArtificial SequenceMechano-growth factor (MGF) D10, an IGF-I fragment 11Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Tyr Gln Pro Pro Ser Thr1 5 10 15Asn Lys Asn Thr Lys Ser Gln Arg Arg Lys Gly Ser Thr Phe Glu Glu 20 25 30His Lys1233PRTArtificial SequenceTP 508_D10 12Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Ala Gly Tyr Lys Pro Asp1 5 10 15Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe 20 25 30Val1325PRTArtificial SequenceODP-D10MISC_FEATURE(10)..(11)4 mini PEG linker 13Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Val Asp Val Pro Asp1 5 10 15Gly Arg Gly Asp Ser Leu Ala Tyr Gly 20 251431PRTArtificial SequenceBMP9MISC_FEATURE(21)..(22) 14Gly Gly Lys Val Gly Lys Ala Cys Cys Val Pro Thr Lys Leu Ser Pro1 5 10 15Ile Ser Val Leu Tyr Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp 20 25 301535PRTArtificial SequenceGhrelin D10 15Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys Glu Ser Lys1 5 10 15Lys Pro Pro Ala Lys Leu Gln Pro Arg Asp Asp Asp Asp Asp Asp Asp 20 25 30Asp Asp Asp 351630PRTArtificial SequenceCNP-D10.MISC_FEATURE(15)..(16) 16Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Gly Leu Ser Lys Gly Phe1 5 10 15Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly 20 25 301739PRTArtificial SequenceVIP D10 17His His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Thr Lys1 5 10 15Gln Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn Asp Asp Asp 20 25 30Asp Asp Asp Asp Asp Asp Asp 351811PRTArtificial Sequence4 mini PEG D10 18His Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp1 5 101926PRTArtificial SequenceODPD10MISC_FEATURE(4)..(5)MISC_FEATURE(16)..(17) 19His Ala Ile Ser Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys1 5 10 15Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu 20 252016PRTArtificial SequencePACAP-(D)E10MISC_FEATURE(1)..(2)MISC_FEATURE(5)..(6)MISC_FEATURE(- 6)..(7) 20Lys Asn Thr Ala Thr Ala Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp1 5 10 152146PRTArtificial SequenceAmylin 1-8MISC_FEATURE(2)..(3)MISC_FEATURE(6)..(7)MISC_FEATURE(36)..(37) 21His Ser Asn Thr Ala Thr Val Thr His Arg Leu Ala Gly Leu Leu Ser1 5 10 15Arg Ser Gly Gly Val Val Lys Asp Asn Phe Val Pro Thr Asn Val Gly 20 25 30Ser Glu Ala Phe Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu 35 40 452249PRTArtificial SequenceCGRP-E10 22His His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys1 5 10 15Gln Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu Gly Lys Arg Tyr 20 25 30Lys Gln Arg Val Lys Asn Lys Glu Glu Glu Glu Glu Glu Glu Glu Glu 35 40 45Glu

Claims

1-18. (canceled)

19. A compound having a structure of: X-Y-Z wherein: X is a Src kinase inhibitor; Y is absent or a linker; and Z is a bone-targeting molecule, or a pharmaceutically acceptable salt thereof.

20. The compound of claim 19, wherein Z comprises a polypeptide.

21. The compound of claim 19, wherein Z comprises not less than 4 and not more than 40 amino acid residues.

22. The compound of claim 21, wherein at least one amino acid is aspartic acid or glutamic acid.

23. The compound of claim 19, wherein Z comprises not less than 6 and not more than 20 glutamic acid residues.

24. The compound of claim 23, wherein Z is 10 D-glutamic acid residues.

25. The compound of claim 19, wherein Z comprises not less than 6 and not more than 20 aspartic acid residues.

26. The compound of claim 25, wherein Z is 10 D-aspartic acid residues.

27. The compound of claim 19, wherein Y is a releasable linker or non-releasable linker.

28. The compound of claim 27, wherein the releasable linker comprises at least one releasable linker group, each releasable linker group being independently selected from the group consisting of a disulfide (S-S), an ester, and a protease specific amide bond.

29. The compound of claim 27, wherein the non-releasable linker comprises at least one non-releasable linker group, each non-releasable linker group being independently selected from the group consisting of a carbon-carbon bond, ether, and an amide.

30. The compound of claim 27, wherein Y comprises one or more ethylene glycol unit.

31. The compound of claim 30, wherein Y comprises 2-8 oxyethylene units.

32. The compound of claim 19, wherein the Src inhibitor is selected from the group consisting of Dasatinib and E739.

33. A compound having a structure of: X-Y-Z wherein: X is an agent that activates sphingosine-1-phosphate (S1P); Y is absent or a linker; and Z is a bone-targeting molecule, or a pharmaceutically acceptable salt thereof.

34. The compound of claim 33, wherein the S1P is selected from the group consisting of a Sphingosine-1-phosphate receptor 1 (S1P1R) agonist and a S1P lyase inhibitor.

35. The compound of claim 34, wherein the SIP1R agonist is Ozanimod.

36. The compound of claim 34, wherein the S1P lyase inhibitor is 4-deoxypyridoxine (DOP).

37. A compound having a structure of: X-Y-Z wherein: X is a neuropeptide; Y is absent or a linker; and Z is a bone-targeting molecule, or a pharmaceutically acceptable salt thereof.

38. The compound of claim 37, wherein the neuropeptide is selected from the group consisting of Substance P, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), amylin, and calcitronin gene-like related peptide (CGRP).