CN116710068A

CN116710068A - Compounds, compositions and methods for treating spinal fusion

Info

Publication number: CN116710068A
Application number: CN202180087298.2A
Authority: CN
Inventors: P·S·洛; S·洛; J·尼尔森
Original assignee: Purdue Research Foundation
Current assignee: Purdue Research Foundation
Priority date: 2020-10-26
Filing date: 2021-08-26
Publication date: 2023-09-05

Abstract

Osteogenic ligand-bone anabolic agent compounds and related compositions and methods for treating spinal fusion.

Description

Compounds, compositions and methods for treating spinal fusion

Priority

This patent application relates to and claims the benefit of priority to the following patent applications: (a) U.S. provisional patent application No. 63/105,678, filed on 26, 10, 2020, and (b) U.S. provisional patent application No. 63/193,753, filed on 27, 5, 2021. The entire contents of each of the aforementioned priority applications are hereby incorporated by reference.

Government support statement

The present invention was completed with government support under DE 028713 awarded by the national institutes of health. The government has certain rights in this invention.

Technical Field

The present disclosure relates to osteogenic ligands, bone anabolic agents, conjugates comprising the same, compositions comprising the same, and methods for treating spinal fusion.

Brief description of the sequence Listing

The sequences described herein are listed in the accompanying figures and are also provided in computer readable form, which are filed together and incorporated herein by reference. According to 37c.f.r. ≡1.821 (f), the information recorded in computer-readable form is the same as the written sequence listing provided herein.

Background

There are more than 50 tens of thousands of spinal fusion procedures per year in the united states). Spinal fusion surgery is a procedure that connects (e.g., permanently connects) two or more vertebral bodies in the spinal column such that the vertebral bodies cannot move relative to one another. In some cases, they are used to treat back pain, deformity, and/or degeneration of the vertebral disc (vertebroal disc) and degeneration of the intervertebral disc (intervertebral disk). Spinal fusion procedures can cost up to $120,000.

Spinal fusion involves techniques aimed at mimicking the normal healing process of a fracture. Thus, a significant amount of therapeutic agents (e.g., growth factors, anti-inflammatory agents, and/or synthetic substances) may be administered to a patient during or after a surgery (e.g., spinal fusion). This may cause adverse side effects, such as ectopic mineralization, which may be caused by leakage of the therapeutic agent into other tissues.

Provided herein are compounds, compositions, and methods for treating or improving spinal fusion healing (e.g., by targeted delivery of anabolic agents to a spinal fusion site). This and other objects and advantages, and inventive features, will be apparent from the description provided herein.

Disclosure of Invention

In some embodiments, provided herein is a method of treating a bone healing event (e.g., spinal fusion) in an individual (e.g., an individual in need thereof), comprising administering (e.g., a therapeutically effective amount) a compound provided herein or a pharmaceutically acceptable salt thereof, e.g., a compound comprising a bone targeting agent (e.g., a pro-bone ligand) and/or an anabolic agent (e.g., a bone anabolic agent) or a pharmaceutically acceptable salt thereof.

In some embodiments, provided herein are compounds having the structure of formula (I):

X-Y-Z。

in some embodiments, the compound having the structure of formula (I) is a pharmaceutically acceptable salt thereof.

In some embodiments, X is a bone anabolic agent. The bone anabolic agent may be any suitable bone anabolic agent. The bone anabolic agent may be parathyroid hormone (PTH), a PTH-related protein (PTHrP), a derivative of any of the foregoing having bone anabolic activity, or a fragment of any of the foregoing having bone anabolic activity. The bone anabolic agent may be abamectin, a derivative thereof having bone metabolic activity, or a fragment thereof having bone metabolic activity. In some embodiments, the bone anabolic agent may be pregabalin peptide (preptin), integrin 5β1 (ITGA), dasatinib, or a derivative or fragment of any of the foregoing having bone metabolic activity. In some embodiments, X is abamectin, ITGA, dasatinib, PTH, PTHrP, or a derivative or fragment of any of the foregoing having bone metabolic activity; y is a non-releasable oligopeptide linker; and Z is DE20. In some embodiments, X is abamectin or a derivative or fragment thereof, Y is a releasable oligopeptide linker comprising at least one protease specific amide bond, and Z is DE20.

In some embodiments, Y is absent or a linker (e.g., a releasable linker or a non-releasable linker). When Y is present, it may be a non-releasable linker, for example a non-releasable linker containing at least one carbon-carbon bond and/or at least one amide bond. Alternatively, when Y is present, it may be a releasable linker, for example, a releasable linker containing at least one disulfide bond, at least one ester bond, at least one protease-specific amide bond, or a combination of the foregoing bonds.

In some embodiments, Z is a bone-stimulating ligand (e.g., an Acidic Oligopeptide (AOP) (e.g., comprising at least 4 amino acid residues (e.g., 4 to 20 amino acid residues)). The amino acid residues may be glutamic acid amino acid residues, aspartic acid amino acid residues, or mixtures thereof.

In some embodiments, Z is a linear chain of amino acid residues. In some embodiments, Z is AOP (e.g., comprises at least 4 glutamic acid amino acid residues or 4 aspartic acid amino acid residues, or both). In some embodiments, Z is a hydroxyapatite binding molecule other than AOP, including, for example, bisphosphonates, raniates, and/or tetracyclines.

In some embodiments, Z comprises at least 4 amino acid residues (e.g., 4 or more, 10 or more, 20 or more, 30 or more, 50 or more, 75 or more, or 100 or more). In some embodiments, Z comprises 4 to 75 acidic amino acid residues (e.g., D-glutamic acid amino acid residues). In some embodiments, Z comprises up to 100 amino acid residues (e.g., 100 or fewer, 75 or fewer, 50 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, or 4 or fewer). In some embodiments, Z comprises no less than 4 and no more than 35 amino acids. In some embodiments, Z comprises no less than 4 and no more than 20 amino acids. In some embodiments, Z comprises no less than 6 and no more than 30 amino acids. In some embodiments, Z comprises no less than 8 and no more than 30 amino acids. In some embodiments, Z comprises no less than 8 and no more than 20 amino acids. In some embodiments, Z comprises a glutamic acid amino acid residue. In some embodiments, Z comprises a D-glutamic acid amino acid residue.

In some embodiments, Z comprises 4 to 75D-glutamic acid amino acid residues. In some embodiments, Z comprises 8 to 30D-glutamic acid amino acid residues. In some embodiments, Z comprises 8 to 20D-glutamic acid amino acid residues.

In some embodiments, the AOP comprises about 4 to about 20 amino acid residues (e.g., 4 to about 20 or about 4 to 20) or more amino acid residues, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In various embodiments, the AOP comprises about 20 amino acid residues, e.g., 20 amino acid residues.

In some embodiments, Z comprises at least 4 (e.g., D-) glutamic acid amino acid residues (e.g., 4 to 20D-glutamic acid amino acid residues) and/or at least 4 (e.g., D-) aspartic acid amino acid residues (e.g., 4 to 20D-aspartic acid amino acid residues).

In some embodiments, the amino acid is aspartic acid (represented by letter D), glutamic acid (represented by letter E), or a mixture thereof. The amino acid residues may have D-chirality, L-chirality or a mixture thereof. In some embodiments, the amino acid residue has D chirality. In some embodiments, the amino acid residue has L chirality. In some embodiments, Z comprises at least 4 (e.g., acidic) amino acid residues (e.g., having the same chirality (e.g., D-or L-amino acid residues)). In some embodiments, each of the at least 4 (e.g., acidic) amino acid residues has D chirality. In some embodiments, the aspartic acid is D-aspartic acid or L-aspartic acid. In some embodiments, the glutamic acid is D-glutamic acid or L-glutamic acid. In some embodiments, Z comprises no less than 4 and no more than 20D-glutamate residues or L-glutamate residues. In some embodiments, Z comprises no less than 4 and no more than 20D-aspartic acid residues or L-aspartic acid residues.

In some embodiments, Z comprises a mixture of (e.g., D-) glutamic acid amino acid residues and (e.g., D-) aspartic acid amino acid residues.

In some embodiments, Z comprises at least 4 repeated D-glutamic acid amino acid residues (DE 4) or more (e.g., 6 repeated D-glutamic acid amino acid residues (DE 6) or more, 8 repeated D-glutamic acid amino acid residues (DE 8) or more, 10 repeated D-glutamic acid amino acid residues (DE 10) or more, 15 repeated D-glutamic acid amino acid residues (DE 15) or more, 20 repeated D-glutamic acid amino acid residues (DE 20) or more, 25 repeated D-glutamic acid amino acid residues (DE 25) or more, 30 repeated D-glutamic acid amino acid residues or more, or 35 repeated D-glutamic acid amino acid residues (DE 35) or more). In some embodiments, Z is DE10 or DE20.

In some embodiments, X is abamectin or a derivative or fragment thereof (e.g., having bone anabolic activity), and Z is DE20. In some embodiments, X is abamectin, ITGA, dasatinib, PTH, PTHrP, or a derivative or fragment of any of the foregoing having bone anabolic activity, and Z is DE20.

As described above, in some embodiments, Y is a non-releasable linker. In some embodiments, Y is an unreleasable linker comprising at least one carbon-carbon bond. In some embodiments, Y is an unreleasable linker containing at least one amide bond. In some embodiments, Y is an unreleasable linker containing at least one carbon-carbon bond and at least one amide bond.

In some embodiments, Y is a non-releasable linker and comprises one or more amide linkages. In some embodiments, Y is a non-releasable linker and comprises 1-20 amide linkages. In some embodiments, Y is a non-releasable linker and comprises 1-10 amide linkages. In some embodiments, Y is a non-releasable linker and comprises 10-20 amide linkages. In some embodiments, Y is a non-releasable linker and comprises 1-5 amide linkages.

In some embodiments, Y is a non-releasable linker and comprises one or more amino acid linker groups. In some embodiments, Y is a polypeptide. In some embodiments, the polypeptide comprises 1-20 amino acid residues. In some embodiments, the polypeptide comprises 1-10 amino acid residues. In some embodiments, the polypeptide comprises 10-20 amino acid residues. In some embodiments, the polypeptide comprises 1-5 amino acid residues.

In some embodiments, Y is a non-releasable linker and comprises one or more ether linkages (C-O). In some embodiments, Y is a non-releasable linker and comprises 1-20 ether linkages (C-O). In some embodiments, Y is a non-releasable linker and comprises 1-10 ether linkages (C-O). In some embodiments, Y is a non-releasable linker and comprises 10-20 ether linkages (C-O). In some embodiments, Y is a non-releasable linker and comprises 1-5 ether linkages (C-O).

In some embodiments, Y is a non-releasable linker and comprises one or more polyethylene glycol (PEG) linker groups. In some embodiments, Y is PEG.

In some embodiments, Y is a non-releasable linker and comprises one or more thioether linkages (C-s). In some embodiments, Y is a non-releasable linker and comprises 1 thioether bond (C-S). In some embodiments, Y is a non-releasable linker and comprises 1-20 thioether linkages (C-S). In some embodiments, Y is a non-releasable linker and comprises 1-10 thioether linkages (C-S). In some embodiments, Y is a non-releasable linker and comprises 10-20 thioether linkages (C-S). In some embodiments, Y is a non-releasable linker and comprises 1-5 thioether bonds (C-S).

In some embodiments, Y is a releasable linker. In some embodiments, Y is a releasable linker containing at least one disulfide bond (S-S). In some embodiments, Y is a releasable linker containing at least one ester linkage (e.g., O (c=o)). In some embodiments, Y is a releasable linker containing at least one (e.g., protease-specific) amide bond.

In some embodiments, Y is a releasable linker and comprises one or more amide linkages. In some embodiments, Y is a releasable linker and comprises 1-20 amide linkages. In some embodiments, Y is a releasable linker and comprises 1-10 amide linkages. In some embodiments, Y is a releasable linker and comprises 10-20 amide linkages. In some embodiments, Y is a releasable linker and comprises 1-5 amide linkages.

In some embodiments, Y is a releasable linker and comprises one or more amino acid linker groups. In some embodiments, Y is a polypeptide. In some embodiments, the polypeptide comprises 1-20 amino acid residues. In some embodiments, the polypeptide comprises 1-10 amino acid residues. In some embodiments, the polypeptide comprises 10-20 amino acid residues. In some embodiments, the polypeptide comprises 1-5 amino acid residues.

In some embodiments, Y is a releasable linker and comprises one or more ether linkages (C-O). In some embodiments, Y is a releasable linker and comprises 1-20 ether linkages (C-O). In some embodiments, Y is a releasable linker and comprises 1-10 ether linkages (C-O). In some embodiments, Y is a releasable linker and comprises 10-20 ether linkages (C-O). In some embodiments, Y is a releasable linker and comprises 1-5 ether linkages (C-O).

In some embodiments, Y is a releasable linker and comprises one or more PEG linker groups.

In some embodiments, X is abamectin (e.g., or a derivative or fragment thereof (e.g., having bone anabolic activity)), Y is a releasable oligopeptide linker comprising at least one protease-specific amide bond, and Z is DE20.

In some embodiments, X is abamectin (e.g., or a derivative or fragment thereof (e.g., having bone anabolic activity)), Y is a non-releasable oligopeptide linker, and Z is DE10. In some embodiments, the compound is SEQ ID NO. 1.

In some embodiments, X is abamectin or a derivative or fragment thereof (e.g., having bone anabolic activity), Y is a non-releasable oligopeptide linker, and Z is DE20. In some embodiments, the compound is SEQ ID NO. 2.

In some embodiments, X is a peptide (e.g., a polypeptide). In some embodiments, the compound having the structure of formula (I) is a (poly) peptide.

In some embodiments, provided herein are (poly) peptides (e.g., abamectin or derivatives or fragments thereof) having bone anabolic activity. In some embodiments, provided herein are substantially pure (poly) peptides (e.g., abamectin) having bone anabolic activity, wherein the (poly) peptides comprise an amino acid sequence that has at least 75%, at least 85%, at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID No. 1 or SEQ ID No. 2. In some embodiments, SEQ ID NO. 1 has bone anabolic activity (as well as, for example, bone targeting activity). In some embodiments, SEQ ID NO. 2 has bone anabolic activity (as well as, for example, bone targeting activity). In other embodiments, the (poly) peptide comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 75% or more sequence identity, at least 85% or more sequence identity, at least 90% or more sequence identity, or at least 95% or more sequence identity) to the amino acid sequence set forth in SEQ ID NO: 1. In other embodiments, the (poly) peptide comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 75% or more sequence identity, at least 85% or more sequence identity, at least 90% or more sequence identity, or at least 95% or more sequence identity) to the amino acid sequence set forth in SEQ ID NO. 2. In certain embodiments, the (poly) peptide comprises an amino acid sequence that is PTH, PTHrP, abalopeptide (Abalo), or a derivative or fragment of any of the foregoing, or that has at least 75% sequence identity (e.g., at least 75% or greater sequence identity, at least 85% or greater sequence identity, at least 90% or greater sequence identity, or at least 95% or greater sequence identity) to its nucleotide sequence.

In another embodiment, the (poly) peptide is an amino acid sequence having at least 75% or more sequence identity, at least 85% or more sequence identity, at least 90% or more sequence identity, or at least 95% or more sequence identity to the amino acid sequence depicted in figure 1. In another embodiment, the (poly) peptide is the amino acid sequence shown in figure 1.

In some embodiments, a compound provided herein comprises a payload. In some embodiments, the payload comprises abamectin (Abalo) or a derivative or fragment thereof (e.g., having bone anabolic activity)) (e.g., SEQ ID NO: 2). In some embodiments, the payload comprises a linker provided herein. In some embodiments, the payloads and linkers provided herein, wherein the payloads comprise abaclotide (Abalo) or a derivative or fragment thereof (e.g., having bone anabolic activity)) (e.g., SEQ ID NO: 2).

Provided in some embodiments herein are conjugates of formula (I) having the following structure: X-Y-Z.

In another embodiment, the linker is an amino acid sequence having at least 75% or more sequence identity, at least 85% or more sequence identity, at least 90% or more sequence identity, or at least 95% or more sequence identity to the amino acid sequence depicted in figure 1. In another embodiment, the linker comprises a portion of the amino acid sequence shown in fig. 1.

In some embodiments, Z is a osteogenic ligand, which may be an AOP comprising at least 11 to 100 amino acid residues.

The amino acid residue may be glutamic acid, aspartic acid, or a mixture thereof. The amino acid residue may have D chirality. The AOP may be a linear chain of amino acid residues. When Y is present, Y may be an unreleasable linker containing at least one carbon-carbon bond and/or at least one amide bond. Alternatively, when Y is present, Y may be a releasable linker containing at least one disulfide bond, ester bond, and/or protease-specific amide bond.

In some embodiments, the (poly) peptide is a pharmaceutically acceptable salt of any of the compounds provided herein (e.g., a compound having the structure of formula (I), SEQ ID NO:1, or SEQ ID NO: 2).

In some embodiments, provided herein are pharmaceutical compositions comprising any of the compounds provided herein (e.g., a compound having the structure of formula (I), SEQ ID NO:1 or SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof (e.g., and at least one pharmaceutically acceptable carrier or excipient).

In some embodiments, a compound provided herein (e.g., a compound having the structure of formula (I), SEQ ID NO:1, or SEQ ID NO: 2) is administered (e.g., subcutaneously) to a subject (e.g., a patient or subject in need thereof).

In some embodiments, provided herein is a method of treating (e.g., an individual (e.g., a patient or individual in need thereof)) spinal fusion. In some embodiments, the methods comprise administering (e.g., subcutaneously) to the subject (e.g., a patient or subject in need thereof) a therapeutically effective amount of any of the compounds or pharmaceutical compositions provided herein (e.g., a compound having the structure of formula (I), SEQ ID NO:1, or SEQ ID NO: 2). In some embodiments, administering (e.g., subcutaneously) a therapeutically effective amount of any of the compounds provided herein (e.g., a compound having the structure of formula (I), SEQ ID NO:1, or SEQ ID NO: 2) to the subject (e.g., a patient or subject in need thereof) treats or improves healing of spinal fusion in the subject (e.g., a patient or subject in need thereof).

In some embodiments, provided herein is a method of locating a compound provided herein (e.g., a compound having the structure of formula (I), SEQ ID NO:1, or SEQ ID NO: 2) to a spinal fusion site in an individual in need thereof (e.g., suffering from or having undergone spinal fusion), comprising administering (e.g., subcutaneously) to the individual (e.g., a patient or individual in need thereof) an amount of any compound provided herein (e.g., a compound having the structure of formula (I), SEQ ID NO:1, or SEQ ID NO: 2). In some embodiments, administering (e.g., subcutaneously) to the subject (e.g., a patient or subject in need thereof) an amount of a compound provided herein (e.g., a compound having the structure of formula (I), SEQ ID NO:1, or SEQ ID NO: 2) localizes the compound provided herein to (e.g., in) a spinal fusion.

In some embodiments, X is abamectin or a derivative or fragment thereof (e.g., having bone anabolic activity), Y is a releasable oligopeptide linker comprising at least one protease-specific amide bond, and Z is DE20.

In some embodiments, X is abamectin or a derivative or fragment thereof (e.g., having bone anabolic activity), Y is a non-releasable oligopeptide linker, and Z is DE10. In some embodiments, the compound is SEQ ID NO. 1.

A method of treating spinal fusion in a patient is provided. The method comprises administering (e.g., subcutaneously) to the patient an effective amount of one of: (i) a conjugate of formula X-Y-Z wherein:

x is an anabolic agent of bone,

when Y is present, it is a linker, which may be a releasable linker or a non-releasable linker, and

z is a osteogenic ligand (e.g., AOP comprising at least 4 amino acid residues to 20 amino acid residues), or (ii) a pharmaceutical composition comprising a conjugate of subpart (i) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In some embodiments, the compounds provided herein are administered subcutaneously (e.g., to an individual in need thereof). In some embodiments, the compound or pharmaceutical composition thereof (or a pharmaceutically acceptable salt thereof) may be administered directly to the spinal fusion site.

In some embodiments, a therapeutically effective amount of any of the compounds or pharmaceutical compositions provided herein is administered daily, weekly, biweekly, or monthly (e.g., for a period of time, such as a week, a month, a year, or longer). In some embodiments, a therapeutically effective amount of any of the compounds or pharmaceutical compositions provided herein is administered once or twice a week.

Also provided is a kit for treating spinal fusion in a patient and/or targeting a therapeutic or diagnostic agent to the spinal fusion site. The kit comprises (a) (i) a compound or conjugate of formula (i) having an X-Y-Z structure as provided herein, or a pharmaceutically acceptable salt thereof (e.g., further comprising a pharmaceutically acceptable carrier). In some embodiments, for example, X is a therapeutic agent (e.g., a bone anabolic agent) for treating spinal fusion in the patient or a diagnostic agent for identifying spinal fusion in the patient, when Y is present (e.g., Y may not be present), is a linker, which may be a releasable linker or a non-releasable linker, and Z is a osteogenic ligand (e.g., an Acidic Oligopeptide (AOP) comprising at least 4 amino acid residues to 20 amino acid residues and/or other hydroxyapatite binding molecules, including, for example, bisphosphonates, raniate, and/or tetracyclines); and (b) collagen sponge, mineralized collagen, or bone graft. The therapeutic agent may be a bone anabolic agent. In some embodiments, the therapeutic agent may be PTH, PTHrP, a derivative of any of the foregoing having bone anabolic activity, or a fragment of any of the foregoing having bone anabolic activity. The bone anabolic agent may be abamectin, a derivative thereof having bone anabolic activity, or a fragment thereof having bone anabolic activity. In some embodiments, the therapeutic agent (e.g., bone anabolic agent) may be selected from the group consisting of: abamectin, ITGA, dasatinib, PTH, PTHrP and derivatives or fragments of any of the foregoing having bone anabolic activity.

In some embodiments of the kits herein, wherein X is a therapeutic agent comprising a bone anabolic agent, X is PTH or PTHrP or a derivative or fragment thereof (e.g., having bone anabolic activity). In some embodiments, the bone anabolic agent is PTH or a derivative or fragment thereof. In some embodiments, the bone anabolic agent is PTHrP or a derivative or fragment thereof. In some embodiments, the bone anabolic agent is a (e.g., synthetic) modified PTH or derivative or fragment thereof. In some embodiments, the bone anabolic agent is a (e.g., synthetic) modified PTHrP or a derivative or fragment thereof. In some embodiments, the bone anabolic agent is abamectin or a derivative or fragment thereof (e.g., having bone anabolic activity). In some embodiments, the bone anabolic agent is abamectin. In some embodiments, the bone anabolic agent is a (e.g., synthetic) modified abamectin.

In some embodiments, wherein X comprises a therapeutic agent (e.g., a bone anabolic agent), administering to the patient a therapeutically effective amount of a compound having the structure of formula (I) to treat spinal fusion.

When X comprises a diagnostic agent, the diagnostic agent may be a fluorescent dye or any other imaging agent suitable for identifying (e.g., visually identifying) spinal fusion present in the patient. In some embodiments, wherein X comprises a diagnostic agent, the patient is administered a compound having the structure of formula (I), if present, spinal fusion is identified.

In some embodiments, Z is a linear chain of amino acid residues. In some embodiments, Z is AOP (e.g., comprises at least 4 glutamic acid amino acid residues or 4 aspartic acid amino acid residues).

In some embodiments, Z comprises at least 4 amino acid residues (e.g., 4 or more, 10 or more, 20 or more, 30 or more, 50 or more, 75 or more, or 100 or more). In some embodiments, Z comprises up to 100 amino acid residues (e.g., 100 or fewer, 75 or fewer, 50 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, or 4 or fewer). In some embodiments, Z comprises no less than 4 and no more than 35 amino acids. In some embodiments, Z comprises no less than 4 and no more than 20 amino acids. In some embodiments, Z comprises no less than 6 and no more than 30 amino acids. In some embodiments, Z comprises no less than 8 and no more than 30 amino acids. In some embodiments, Z comprises no less than 8 and no more than 20 amino acids.

In some embodiments, the amino acid is aspartic acid (represented by letter D), glutamic acid (represented by letter E), or a mixture thereof. The amino acid residues may have D-chirality, L-chirality or a mixture thereof. In some embodiments, the amino acid residue has D chirality. In some embodiments, the amino acid residue has L chirality. In some embodiments, Z comprises at least 4 (e.g., acidic) amino acid residues (e.g., D-or L-amino acid residues) having the same chirality. In some embodiments, each of the at least 4 (e.g., acidic) amino acid residues has D chirality. In some embodiments, the aspartic acid is D-aspartic acid or L-aspartic acid. In some embodiments, the glutamic acid is D-glutamic acid or L-glutamic acid. In some embodiments, Z comprises no less than 4 and no more than 20D-glutamate residues or L-glutamate residues. In some embodiments, Z comprises no less than 4 and no more than 20D-aspartic acid residues or L-aspartic acid residues.

In some embodiments, Z comprises at least DE4 or more (e.g., DE6 or more, DE8 or more, DE10 or more, DE15 or more, or DE20 or more, DE25 or more, DE30 or more, or DE35 or more). In some embodiments, Z comprises at least 10 repeated D-glutamic acid amino acid residues (e.g., DE10 or more, DE15 or more, or DE20 or more, DE25 or more, DE30 or more, or DE35 or more). In some embodiments, X is abamectin or a derivative or fragment thereof (e.g., having bone anabolic activity), and Z is DE20.

In some embodiments, Y is a non-releasable linker. In some embodiments, Y is an unreleasable linker containing at least one carbon-carbon bond. In some embodiments, Y is an unreleasable linker containing at least one amide bond. In some embodiments, Y is an unreleasable linker containing at least one carbon-carbon bond and at least one amide bond.

In some embodiments, Y is a linker as described elsewhere herein (e.g., above).

In some embodiments, Z is a osteogenic ligand as described elsewhere herein (e.g., above).

In some embodiments, X is abamectin or a derivative or fragment thereof (e.g., having bone anabolic activity), Y is a non-releasable oligopeptide linker, and Z is DE20.

In some embodiments, the compound is an imaging agent (e.g., dye).

In some embodiments, the compound is any of the compounds described herein.

In some embodiments, the compound is SEQ ID NO. 1. In some embodiments, the compound is SEQ ID NO. 2.

In some embodiments, provided herein is a pharmaceutical composition comprising any of the compounds provided herein (e.g., a compound having the structure of formula (I), SEQ ID NO:1, or SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof (e.g., and at least one pharmaceutically acceptable carrier or excipient). In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration to a patient. In some embodiments of the pharmaceutical composition, X is a therapeutic agent that is a bone anabolic agent. In some embodiments, X is a diagnostic agent that is an imaging agent (e.g., a fluorescent dye).

Methods of locating a therapeutic or diagnostic agent to a spinal fusion site in a patient are also provided. In some embodiments, such methods comprise administering to the patient a therapeutically effective amount of any of the compounds or pharmaceutical compositions described herein.

Further embodiments and full scope of applicability of the present disclosure will become apparent from the detailed description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments, are given by way of illustration only. Various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art.

Drawings

FIG. 1 shows the amino acid sequences of SEQ ID NO. 1 and SEQ ID NO. 2.

Figure 2 shows the biodistribution of the compounds provided herein after 2 weeks post-surgical injection with a collagen scaffold.

Figure 3 shows the biodistribution of the compounds provided herein after 2 weeks post-surgical injection with mineralized collagen scaffolds.

Fig. 4 shows the biodistribution of the compounds provided herein after 2 weeks post-surgical injection with a bone graft scaffold.

Figure 5 shows the biodistribution of the compounds provided herein after 8 weeks post-surgical injection.

Fig. 6 shows a microscopic CT scan three-dimensional reconstruction of spinal fusion of rats treated with collagen matrix and a compound provided herein for 8 weeks, wherein panel a is a left medial view, panel B is a dorsal view, and panel C is a right medial view.

FIG. 7 shows a three-dimensional reconstruction of a microscopic CT scan of spinal fusion in rats treated with rhBMP-2 infiltrated collagen matrix for 8 weeks, wherein panel A is a left medial view, panel B is a dorsal view, and panel C is a right medial view.

Fig. 8 shows a three-dimensional reconstruction of a microscopic CT scan of rat spinal fusion after 8 weeks of treatment with collagen matrix and physiological saline, wherein panel a is left medial view, panel B is dorsal view, and panel C is right medial view.

FIG. 9 shows a three-dimensional reconstruction of a microscopic CT scan of a rat spinal fusion treated with bone graft matrix and rhBMP-2 (10 μg) for 8 weeks, wherein panel A is a left medial view, panel B is a dorsal view, and panel C is a right medial view.

Fig. 10 shows a three-dimensional reconstruction of a microscopic CT scan of a rat spinal fusion procedure treated with bone graft matrix and a compound provided herein for 8 weeks, wherein panel a is a left medial view, panel B is a dorsal view, and panel C is a right medial view.

Fig. 11 shows a three-dimensional reconstruction of a microscopic CT scan of a rat spinal fusion after 8 weeks of treatment with bone graft matrix and physiological saline, wherein panel a is a left medial view, panel B is a dorsal view, and panel C is a right medial view.

Figure 12 shows a three-dimensional reconstruction of a microscopic CT scan of spinal fusion of rats treated with mineralized collagen scaffolds and physiological saline one week after implantation.

Figure 13 shows a three-dimensional reconstruction of a microscopic CT scan of spinal fusion in rats after 8 weeks of treatment with mineralized collagen scaffolds and different treatments. Panels A and B show three-dimensional reconstructions of microscopic CT scans of spinal fusion after 8 weeks of treatment with mineralized collagen and BMP-2. Panels C and D show microscopic CT scan three-dimensional reconstructions of spinal fusion after 8 weeks of treatment with mineralized collagen and a compound provided herein. Panels E and F show microscopic CT scan three-dimensional reconstructions of spinal fusion after 8 weeks of treatment with mineralized collagen and physiological saline.

Figure 14 shows a three-dimensional reconstruction of a microscopic CT scan of rat spinal fusion after 5 weeks of treatment with mineralized collagen scaffolds and different treatments. Panel A shows a three-dimensional reconstruction of a microscopic CT scan of spinal fusion after 5 weeks of treatment with mineralized collagen and a compound provided herein. Panel B shows a three-dimensional reconstruction of a microscopic CT scan of spinal fusion after 5 weeks of treatment with mineralized collagen and rhBMP-2. Panel C shows a three-dimensional reconstruction of a microscopic CT scan of spinal fusion after 5 weeks of treatment with mineralized collagen and physiological saline.

Figure 15 shows a graph of weeks of BMP and the compounds provided herein versus total fusion score.

FIG. 16 is a graph of week number versus total fusion score for physiological saline, BMP, and compounds provided herein.

FIG. 17 is a cycle number and Bone Mineral Density (BMD) (HA mg/cm) of a compound provided herein and physiological saline ³ ) Is a graph of the relationship of (1).

FIG. 18 shows the total area (weeks versus Bone Volume (BV) (mm) of mineralized tissue in the bone bridge formed by treatment of spinal fusion with a compound provided herein or physiological saline ³ ) Is a graph of the relationship).

Detailed Description

The present disclosure relates to the preparation and use of compounds and compositions for treating spinal fusion and/or facilitating spinal fusion treatment. In some embodiments, the compounds, compositions, and methods utilize strategies (e.g., selectively) to localize a therapeutic agent to a spinal fusion site. For example, the provided compounds, compositions, and methods can comprise osteogenic ligands that localize the compounds and compositions to a spinal fusion site in a patient. In some embodiments, the compounds and compositions are formulated to exhibit an extended shelf life (e.g., due to increased resistance to degradation), thus reducing the frequency of reapplication of the compound or composition to maintain a therapeutically effective concentration at a target site (e.g., spinal fusion site). Kits for treating and/or targeting therapeutic or diagnostic agents to spinal fusions in a patient are also provided.

The compounds, compositions and methods described herein have significant advantages over conventional spinal fusion therapies. In some embodiments, the non-invasive targeted drug administration method for spinal fusion is capable of specifically repeating drug administration to spinal fusion, and thus, is capable of achieving therapeutic levels of drug to stimulate repair of spinal fusion over a longer period of time than conventional methods. Such an extension of the duration of the therapeutic level of the compound and/or composition may increase the efficacy and effect of the anabolic drug relative to topical drug administration (e.g., administration at a large dose), and may ultimately result in faster repair of spinal fusion. The targeting properties of the compounds, compositions and methods also reduce systemic side effects and side effects caused by leakage of the compounds from the implantation site (e.g., as in the case of bone morphogenic protein-2 (BMP 2) frequently occurring treatments). Furthermore, the compounds, compositions and methods are non-invasive, which enables time control of drug administration. This allows the physician more control over when and how long the compounds and compositions are administered so that they can be easily tailored to different phases of bone healing and take into account patient-to-patient variability in healing response.

The requirement for topical application of approved osteogenic drugs during surgery (e.g., spinal fusion surgery) limits its usefulness. Furthermore, the metabolic switching of approved bone anabolic agents is relatively rapid, which limits the duration of their therapeutic effect to within a short window following topical administration during surgery. However, the application of large amounts of therapeutic agents at the time of surgery can lead to undesirable side effects, such as ectopic bone growth. In addition to ectopic mineralization due to leakage of topically applied anabolic drugs to surrounding tissues, systemic administration can also stimulate unwanted anabolic processes in healthy tissues (e.g., nerves, muscles, and vasculature). Indeed, hypercalcemia, hypertension, immunosuppression, and even cancer are all problems arising from systemic administration of bone anabolic drugs.

One possible solution is bone targeting. Bone targeting has so far focused mainly on delivering payloads to bone pathologies unrelated to fractures, such as osteoporosis, osteomyelitis and bone metastases. Most of these treatments are bisphosphonates to selectively deliver the compound to bone. However, in treating bone fractures, the compounds must be selectively delivered to the fracture site to avoid ectopic ossification that may occur when the drug is non-specifically delivered to all bones. Although tetracycline may be moderately more selective than healthy bone, it is toxic to bone, liver and kidneys and is therefore not an ideal solution.

There are also limitations to using bisphosphonates for fracture targeting, including that they inhibit osteoclasts, which are critical to normal bone remodeling and transformation of fracture callus from woven bone to lamellar bone. Another problem with the use of bisphosphonates as targeting ligands is that their half-life in bone is as long as 20 years, depending on the stability of their therapeutic cargo (cargo), may lead to an excessive stimulation time of their molecular targets, which is undesirable.

Similar targeting of the ranilate can also be observed. These compounds can be used as targeting molecules for many bone diseases and can be attached to anabolic agents to accelerate bone growth and healing. However, as with bisphosphonates, their skeletal half-life is long.

In view of the problems of bisphosphonates, raniates and polyphosphates (including but not limited to cumbersome synthetic procedures and poor solubility), there remains a need for osteogenic ligands that do not suffer from these drawbacks. Desirably, the osteogenic ligand may deliver an attached peptide, therapeutic or diagnostic agent to a fracture, particularly a fracture callus.

Abamectin is a synthetic analogue of the anabolic, 34 amino acid, parathyroid hormone-related protein (PTHrP). It helps promote bone growth and maintain bone density, and can be used for treating osteoporosis. The action of abaclotide is similar to PTHrP, in that it targets, binds to and activates parathyroid hormone 1 (PTH 1) receptor (PTH 1R).

PTH1R is a G protein-coupled receptor (GPCR) expressed in osteoblasts and bone matrix cells. PTH1R in turn activates cyclic adenosine monophosphate (cAMP) signaling pathway and bone anabolic signaling pathway, allowing bone growth and increasing bone mineral density and volume. The increase in bone mass and strength helps prevent/treat osteoporosis and reduces the risk of fracture.

Compounds of formula (I)

Provided in some embodiments herein are compounds comprising an Acidic Oligopeptide (AOP) (e.g., 10-and 20-mers of acidic amino acids, 10-and 20-mers of aspartic acid or glutamic acid, or various combinations of the foregoing amino acids). In some embodiments, AOP is effective to target spinal fusion. In some embodiments, 20 polymer than 10 polymer more effective. In some embodiments, AOP is highly selective compared to bisphosphonates and tetracyclines. In some embodiments, the glutamate polymer and aspartate polymer have similar residence times at the delivery site. In some embodiments, although the oligoaspartic acid shortens non-specific retention in the kidney, a slight extension in the time of retention of the oligoglutamic acid is observed to be short. In some embodiments, the aspartic acid oligopeptide and the glutamic acid oligopeptide (e.g., almost quantitatively) are cleared from the kidney after 18 hours. In some embodiments, AOP targets peptides of all chemical classes (e.g., hydrophobic peptides, neutral peptides, cationic peptides, anionic peptides, short oligopeptides, and long polypeptides), which may be particularly advantageous in the case of targeting spinal fusion sites, as many other therapies of chemical classes can be developed that target the spinal fusion via AOP. While many bone anabolic agents are peptides, their physical properties may vary widely, and thus, many are unsuitable and/or incapable of targeting a desired bone site without conjugation to a targeting ligand.

Provided in some embodiments herein are compounds comprising the unnatural D enantiomer of AOP, in some cases with prolonged retention on fracture surfaces (e.g., due to increased resistance to degradation) as compared to the corresponding L enantiomer. This may be due, for example, to an enhanced resistance to degradation compared to other compounds. In some embodiments, the extended residence time affects the frequency with which the therapeutic agent needs to be reapplied to maintain a therapeutically effective concentration at the surgical site (e.g., spinal fusion site). In some embodiments, the prolonged duration of residence affects the amount of therapeutic agent that needs to be administered in order to elicit a targeted response (e.g., therapeutic response). In some embodiments, linear AOP is preferred over branched AOP (e.g., due to reduced or absent steric interference).

In some embodiments, targeted delivery of the anabolic agent enables localization of the therapeutic agent to the fracture (e.g., by injection, e.g., subcutaneous injection at a distal site). In some embodiments, a compound provided herein is repeatedly administered to a patient (e.g., a patient in need thereof). In some embodiments, the compounds provided herein are administered to a patient (e.g., a patient in need thereof) at a relatively low dose. In some embodiments, a compound provided herein is administered to a patient (e.g., a patient in need thereof) in a safe dose. In some embodiments, a compound provided herein is administered to a patient (e.g., a patient in need thereof) in a therapeutic dose. In some embodiments, targeted delivery minimizes (e.g., if not eliminates) drift of anabolic agents (e.g., into other tissues and cause unwanted mineralization). In some embodiments, bone growth in the region is stimulated over a relatively long period of time (e.g., to achieve a relatively rapid efficacy (e.g., such that the patient may resume post-operative mobility more quickly than in non-targeted delivery methods)).

X-Y-Z type (I)

Or a pharmaceutically acceptable salt thereof, wherein:

x is a bone anabolic agent;

y is absent or, when present, a linker, which may be a releasable linker or a non-releasable linker; and is also provided with

Z is a osteogenic ligand (e.g., an AOP comprising at least 4 amino acid residues to 20 amino acid residues).

In some embodiments, the compound is any of the compounds provided herein, or a pharmaceutically acceptable salt thereof (e.g., a compound having the structure of formula (I), SEQ ID NO:1, or SEQ ID NO:2 (see fig. 1)).

In some embodiments, targeted delivery of an agent (e.g., a therapeutic agent (e.g., an anabolic agent) or a diagnostic agent, such as an imaging agent (e.g., a fluorescent dye)) (e.g., targeted delivery by the osteogenic ligand (Z)) localizes the agent to spinal fusion (e.g., by injection, e.g., subcutaneous injection, e.g., at a distal site). In some embodiments, a compound provided herein is repeatedly administered to a patient (e.g., a patient in need thereof). In some embodiments, the compounds provided herein are administered to a patient (e.g., a patient in need thereof) at a relatively low dose. In some embodiments, a compound provided herein is administered to a patient (e.g., a patient in need thereof) in a safe dose. In some embodiments, a compound provided herein is administered to a patient (e.g., a patient in need thereof) in a therapeutic dose. In some embodiments, targeted delivery minimizes (e.g., if not eliminates) drift of anabolic agents (e.g., into other tissues and cause unwanted mineralization). In some embodiments, bone growth in the region is stimulated over a relatively long period of time (e.g., to achieve a relatively fast efficacy (e.g., the patient may resume post-operative mobility faster than a non-targeted delivery method)).

Returning to the compound having the structure of formula (I) or a pharmaceutically acceptable salt thereof, Z may be any suitable osteogenic ligand. In some embodiments, the osteogenic ligand has a bone affinity, such as hydroxyapatite. In some embodiments, the osteogenic ligand aids in directing the compound (or derivative or fragment thereof) to the bone. In some embodiments, the osteogenic ligand has the potential to target the bone anabolic agent to a spinal fusion site. In some embodiments, the osteogenic ligand is a ligand having affinity for hydroxyapatite. In some embodiments, the osteogenic ligand is a raninate, a bisphosphonate (e.g., alendronate), a trilosponate, a tetracycline, a polyphosphate, an acidic molecule (e.g., a molecule having two or more carboxylic acids), a calcium chelator, a metal chelator, or an AOP. In some embodiments, the osteogenic ligand is AOP.

In some embodiments, Z comprises at least 4 amino acid residues (e.g., 4 or more, 10 or more, 20 or more, 30 or more, 50 or more, 75 or more, or 100 or more). In some embodiments, Z comprises up to 100 amino acid residues (e.g., 100 or fewer, 75 or fewer, 50 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, or 4 or fewer). In some embodiments, Z comprises no less than 4 and no more than 30 amino acids. In some embodiments, Z comprises no less than 4 and no more than 20 amino acids. In some embodiments, the AOP comprises about 4 to about 20 amino acid residues (e.g., 4 to about 20 or about 4 to 20) or more amino acid residues, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In various embodiments, the AOP comprises about 20 amino acid residues, e.g., 20 amino acid residues. In other embodiments, the AOP may comprise more than 20 amino acid residues, for example 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or up to 100 amino acid residues.

In some embodiments, the amino acid may be aspartic acid (represented by letter D), glutamic acid (represented by letter E), or a mixture thereof. The amino acid residues may have D-chirality, L-chirality or a mixture thereof. In some embodiments, the amino acid residue has D chirality. In some embodiments, the amino acid residue has L chirality. In some embodiments, the aspartic acid is D-aspartic acid or L-aspartic acid. In some embodiments, the glutamic acid is D-glutamic acid or L-glutamic acid. In some embodiments, Z comprises no less than 4 and no more than 20D-glutamate residues or L-glutamate residues. In some embodiments, Z comprises no less than 4 and no more than 20D-aspartic acid residues or L-aspartic acid residues.

In some embodiments, the AOP comprises one or more neutral or basic amino acids (e.g., so long as the AOP effectively functions as a osteogenic ligand). In some embodiments, the AOP comprises one or more synthetic amino acids (e.g., which may be acidic, neutral, or basic amino acids).

In some embodiments, the AOP is linear (straight chain) or branched (branched). In various embodiments, a straight chain is used. In some embodiments, the AOP may be cyclized.

The osteogenic ligand (Z) may be a single unit, a polymer, a dendrimer or a plurality of units. In some embodiments, the osteogenic ligand is a polymer.

In some embodiments, X is a diagnostic agent for identifying a target site. In some embodiments, X is any suitable imaging agent, such as a fluorescent dye. Because of the targeting properties of the compound's osteogenic ligand, the compound will target and concentrate at the patient's spinal fusion (e.g., if present) when administered, and where X is a diagnostic agent, a physician or other health care provider can readily image and/or identify the compound's imaging agent.

In some embodiments, X is a therapeutic agent for treating spinal fusion in the patient. In some embodiments, X is any suitable bone anabolic agent. In some embodiments, the bone anabolic agent is neutral, anionic, cationic or hydrophobic. In some embodiments, the bone anabolic agent is an oligopeptide (e.g., comprising less than or equal to about 10 (or less than 10) amino acid residues, such as 10, 9, 8, 7, 6, 5, or 4 amino acid residues). In some embodiments, the bone anabolic agent comprises greater than or equal to about 10 (or greater than 10) amino acid residues, e.g., 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, or 40 amino acid residues.

Examples of anabolic agents include, but are not limited to, abamectin, pranoplatin (preptin, e.g., a 34 residue peptide hormone secreted by islet beta cells; asp69-Leu102 corresponding to the E peptide of insulin-like growth factor II (pro-IGF-II)), integrin 5β1 (ITGA), dasatinib, PTH, PTHrP, or derivatives of any of the foregoing having bone anabolic activity (e.g., one or more amino acid mutations, e.g., insertions, deletions, and substitutions with naturally occurring amino acids or non-naturally occurring amino acids), or fragments of any of the foregoing having bone anabolic activity.

In some embodiments, a bone anabolic agent, followed by the name d# (e.g., D20), means that the bone anabolic agent is attached (or linked) to a osteogenic ligand (e.g., an orthotropic ligand having, e.g., 20 aspartic acid residues), e.g., at the N-terminus or C-terminus. In some embodiments, a bone anabolic agent, followed by the name e# (e.g., E20), means that the bone anabolic agent is attached (or linked) to a osteogenic ligand (e.g., an orthotropic ligand having 20 glutamic acid residues), e.g., at the N-terminus or C-terminus.

In some embodiments of formula (I), Y is a non-releasable linker. In some embodiments, Y is an unreleasable linker comprising at least one carbon-carbon bond, at least one amide bond, or at least one carbon-carbon bond and at least one amide bond. In some embodiments, Y is a releasable linker. In some embodiments, Y is a releasable linker comprising at least one disulfide bond (S-S), at least one ester bond (e.g., -O (c=o) -), at least one protease-specific amide bond, or a combination of one or more of the foregoing bonds.

In some embodiments, the targeting molecule (i.e., osteogenic ligand (Z)) does not cleave from the drug/anabolic agent (X) to render the compound therapeutically effective in vivo. This may be advantageous because osteogenic ligands and compositions comprising anabolic agents may be used because only a negligible amount (e.g., if any) of anabolic agent is released (e.g., systemic release) prior to targeted delivery of the compound to the spinal fusion site. In some embodiments, modulating the release profile of an active ingredient is a difficulty in preparing an effective pharmaceutical composition. In some embodiments, compounds comprising the non-releasable linkers provided herein avoid difficulties in preparing effective pharmaceutical compositions (e.g., by eliminating the necessity of timed release). In some embodiments, anabolic agents of the compounds provided herein are active upon binding (e.g., conjugation to the osteogenic ligand). Thus, in some embodiments, a compound comprising a targeting molecule (e.g., Z) conjugated to a non-releasable linker (e.g., Y) may reduce systemic exposure and/or systemic adverse effects of an anabolic agent (X) attached thereto.

In some embodiments, conjugates comprising non-releasable linkers reduce or eliminate toxicity of components released from the conjugates in free form (e.g., free form of compounds and/or ligands provided herein).

Both releasable and non-releasable linkers can be engineered according to methods well known in the art or developed below (e.g., by pegylation, etc.) to optimize the biodistribution (e.g., of the compound), bioavailability, and PK/PD and/or increase absorption by the targeted tissue (e.g., of the compound). In some embodiments, the linker is configured to avoid substantial release of the pharmaceutically active amount of the anabolic agent into the circulation prior to capture by a cell (e.g., bone cells at a spinal fusion site).

In some embodiments, the linker may comprise one or more spacers (e.g., to facilitate achieving a specific release time, to facilitate enhanced absorption by the target tissue, and/or to optimize the biodistribution, bioavailability, and/or PK/PD of the compound). The spacer may comprise one or more of an alkyl chain, polyethylene glycol (PEG), a peptide, a saccharide, a peptidoglycan, a clickable linker (e.g., triazole), a rigid linker (e.g., polyproline and polypiperidine), and the like.

In some embodiments, the one or more linkers of the compounds provided herein may comprise PEG, PEG derivatives, or any other linker known in the art or developed below that can achieve the purposes described herein. In some embodiments, the linker is repeated n times, where n is a positive integer.

In some embodiments, X is abamectin, ITGA, dasatinib, PTH, PTHrP, or a derivative or fragment of any of the foregoing having bone anabolic activity; y is a non-releasable oligopeptide linker; and Z is DE20. In some embodiments, X is abamectin or a derivative or fragment thereof having bone anabolic activity; y is a releasable oligopeptide linker comprising at least one protease specific amide bond; and Z is DE20.

In some embodiments, the compound is any of the compounds provided herein or a pharmaceutically acceptable salt thereof (e.g., a compound having the structure of formula (I), SEQ ID NO:1, or SEQ ID NO: 2). The letter "H" at the beginning of the peptide sequence indicates the N-terminus. For example, SEQ ID NO. 1, is:

HAVSEHQLLHDKGKSIQDLRRRELLEKLLxKLHTAEIRATSEVSPNSeeeeeeeee, wherein x = alpha-aminoisobutyric acid (Aib), "e" means D-glutamic acid, H means that the adjacent alanine residue is the N-terminal alanine residue, such that SEQ ID NO:1 can be understood as H-AVSEHQLLQLLQLLQDLRRRELLEKLLxKLHTAEIRATSEVSSeeeeeee (or, as shown in the computer readable forms of the simultaneous submission sequence Listing according to Table 1 and Table 3 of WIPO Standard ST.25 (1998) appendix 2, SEQ ID NO:1 is

Avsehqllhdkgksiqdlrrelllekllxklhtaeirsetseeeeeeeeee, wherein alanine is an N-terminal residue, and "x" and "e" are as defined herein.

Similarly, SEQ ID NO. 2, is

HAVSEHQLLHDKGKSIQDLRRRELLEKLLxKLHTAEIRATSEVSPNSeeeeeeeeeeeeeeeeee ee where x=Aib, "e" denotes D-glutamic acid, the beginning H denotes the N-terminus of the sequence, such that SEQ ID NO:2 can be understood as H-AVSEHQLLHDKGKSIQRRRELLEKLxKLHTAEIRATSEVSPNSeeeeeeeeeeeeeeeeeeeee (or, as shown in the computer readable forms of Table 1 and Table 3 of appendix 2 of WIPO Standard ST.25 (1998), the sequence listing is submitted simultaneously with SEQ ID NO:2

AVSEHQLLHDKGKSIQDLRRRELLEKLxKLHTAEIRSEVSSEEeEeEeEeEeEeEeEeEeEee wherein the N-terminal amino acid is alanine, and "x" and "e" are as defined herein). In some embodiments, "E" corresponds to L-glutamic acid and "E" corresponds to D-glutamic acid.

In some embodiments, the compound has at least 75% or more sequence identity, at least 85% or more sequence identity, at least 90% or more sequence identity, or at least 95% or more sequence identity to SEQ ID No. 1. In some embodiments, the compound has at least 75% or more sequence identity, at least 85% or more sequence identity, at least 90% or more sequence identity, or at least 95% or more sequence identity to SEQ ID NO. 2.

The compounds may be synthesized according to methods known in the art and exemplified herein (e.g., solid-phase polypeptide synthesis methods).

Pharmaceutical composition

The compounds described herein may be administered alone or formulated into pharmaceutical compositions comprising the compounds and one or more pharmaceutically acceptable excipients. The term "composition" generally refers to any product comprising more than one ingredient, including the compounds described herein. It is to be understood that the compositions described herein may be prepared from isolated compounds or from salts, solutions, hydrates, solvates and other forms of the compounds. Certain functional groups (e.g., hydroxyl, amino, and the like) may form complexes with water and/or various solvents, in various physical forms of the compounds. It is also understood that the compositions may be prepared from various amorphous, non-amorphous, partially crystalline, and/or other morphological forms of the compounds, and that the compositions may be prepared from various hydrates and/or solvates of the compounds. Thus, such references to pharmaceutical compositions of the compounds include each of the various morphological forms and/or solvate or hydrate forms of the compounds, or any combination, or individual forms.

One embodiment provides a pharmaceutical composition comprising a compound of formula (I) or any compound encompassed by the formula, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier and/or excipient.

One embodiment provides a pharmaceutical composition comprising an effective amount of a therapeutically (or prophylactically) effective compound of formula (I) or any compound encompassed by the formula, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier and/or excipient.

In some embodiments, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of any of the compounds provided herein, which can be administered (e.g., subcutaneously) to a patient in need thereof. In various embodiments, the composition is an injectable composition, such as a composition suitable for subcutaneous injection.

The compounds and/or compositions described herein may be administered in unit dosage forms and/or compositions containing one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients and/or vehicles, and combinations thereof.

The term "administration" generally refers to any and all means of introducing a compound described herein into a host subject, including, but not limited to, oral, intravenous, intramuscular, subcutaneous, transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, and similar routes of administration.

It may be appropriate to administer the compound as a salt. Examples of acceptable salts include, but are not limited to, alkali metal (e.g., sodium, potassium, or lithium) salts or alkaline earth metal (e.g., calcium) salts; however, any salt that is generally non-toxic and effective for the subject being treated upon administration is acceptable. In at least one embodiment, the salt may be an ammonium acetate salt. Similarly, "pharmaceutically acceptable salts" refer to those salts with a counterion that can be used in the pharmaceutical. Such salts may include, but are not limited to: (1) Acid addition salts can be obtained by reacting the free base of the parent compound with an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, perchloric acid, and the like) or with an organic acid (e.g., acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid, malonic acid, and the like); or (2) a salt formed when the acidic proton present in the parent compound is replaced with a metal ion (e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion), or coordinated with an organic base (e.g., ethanolamine, diethanolamine, triethanolamine, tricarboxymethylaminomethane, N-methylglucamine, etc.). Pharmaceutically acceptable salts are well known to those skilled in the art, and any such pharmaceutically acceptable salt is contemplated.

Acceptable salts may be obtained using standard procedures known in the art, including, but not limited to, reacting a sufficiently acidic compound with a suitable base to provide a physiologically acceptable anion. Suitable acid addition salts are formed from acids that form non-toxic salts. Illustrative, but non-limiting examples include acetates, aspartates, benzoates, benzenesulfonates, bicarbonates/carbonates, bisulphates/sulphates, borates, camphorsulphonates, citrates, ethanedisulfonates, ethanesulphonates, formates, fumarates, glucoheptonates, gluconate, glucuronate, hexafluorophosphates, oxybenzoates, hydrochlorides/chlorides, hydrobromides/bromides, hydroiodides/iodides, isethionates, lactates, malates, maleates, malonates, methanesulfonates, methylsulfates, naphthanates, 2-naphthanates, nicotinates, nitrates, orotate, oxalates, palmates, pamonates, phosphates/hydrogen phosphate/dihydrogen phosphate, glycoproteins, stearates, succinates, tartrates, tosylates and trifluoroacetates. Suitable base salts of the compounds described herein are formed from bases that form non-toxic salts. Illustrative, but non-limiting examples include arginine salts, benzathine salts, calcium salts, choline salts, diethylamine salts, diethanolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, ethanolamine salts, potassium salts, sodium salts, tromethamine salts, and zinc salts. Semi-salts of acids and bases, such as hemisulfate and hemicalcium salts, may also be formed.

The compounds may be formulated into pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms suitable for the chosen route of administration. For example, the pharmaceutical compositions may be formulated for and administered by intra-osseous, intravenous, intra-arterial, intraperitoneal, intracranial, intramuscular, topical, inhalation, and/or subcutaneous routes. In at least one embodiment, the compounds and/or compositions can be administered directly (by injection, placement, or other means) to the spinal fusion site. In at least one embodiment, the compounds may be administered systemically in combination with a pharmaceutically acceptable vehicle (e.g., an inert diluent or an assimilable edible carrier). For oral therapeutic administration, the active compounds may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. The percentage of the compositions and formulations may vary and may be from about 1% to about 99% by weight of the active ingredient and binders, excipients, disintegrants, lubricants and/or sweeteners (as known in the art). The amount of active compound in such therapeutically useful compositions will be such that an effective dosage level is achieved.

The preparation of parenteral compounds/compositions under sterile conditions (e.g., by lyophilization) can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art. In at least one embodiment, the solubility of the compounds used to prepare the parenteral compositions can be increased by using appropriate formulation techniques (e.g., adding solubility enhancers).

As previously mentioned, the compounds/compositions may also be administered by infusion or injection (e.g., using needle (including microneedle) syringes and/or needleless syringes). The solution of the active composition may be an aqueous solution, optionally mixed with a non-toxic surfactant and/or containing carriers or excipients such as salts, carbohydrates and buffers (preferably pH 3 to 9), but for some applications they may be more suitable to be formulated as a sterile non-aqueous solution or in dry form for use with a suitable vehicle such as sterile, pyrogen-free water or phosphate buffered saline. For example, dispersions can be prepared in glycerol, liquid PEG, glyceryl triacetate and mixtures thereof, and oils. Under ordinary conditions of storage and use, these formulations may also contain a preservative to prevent microbial growth.

Pharmaceutical dosage forms suitable for injection or infusion may comprise sterile aqueous solutions or dispersions or sterile powders containing the active ingredient which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the final dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be a solvent or liquid dispersion medium including, for example, but not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid PEG, etc.), vegetable oils, non-toxic glycerides, and/or suitable mixtures thereof. In at least one embodiment, proper fluidity may be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. The action of microorganisms can be prevented by adding various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like). In some cases, it may be desirable to include one or more isotonic agents, for example, sugars, buffers, or sodium chloride. The absorption of the injectable composition may be prolonged by the addition of agents formulated to delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable or infusible solutions can be prepared by mixing the active compounds and/or compositions with one or more of the other ingredients described above in the required amount of the appropriate solvent(s) as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient in a previously sterile-filtered solution thereof.

For topical application, the compounds are preferably applied to the bone as a composition or formulation in combination with an acceptable carrier (which may be a solid, liquid or gel matrix). For example, in certain embodiments, a useful liquid carrier may comprise water, an alcohol, or a glycol or a water-alcohol/glycol blend, wherein the compound may be dissolved or dispersed at an effective level, optionally with the aid of a non-toxic surfactant. In some embodiments, the compounds and/or compositions may be topically applied to a collagen sponge, mineralized collagen, or bone graft.

Additionally or alternatively, adjuvants (e.g., antimicrobial agents) may be added to optimize the characteristics of a particular use. The resulting liquid composition may be applied from an absorbent pad for impregnating bandages and/or other dressings, sprayed onto a target area using a pump or aerosol sprayer, or applied directly only to a desired area of the subject (e.g., spinal fusion site).

Thickeners, such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials, may also be used with the liquid carrier to form spreadable pastes, gels, ointments, soaps, and the like for direct application to the subject's skin.

As used herein, the term "therapeutically effective dose" refers to (unless specifically indicated otherwise) an amount of a compound that, when administered once or over a treatment period, affects the health, well-being, or mortality of a subject (e.g., without limitation, supporting or promoting healing and/or bone growth or fusion at a spinal fusion site).

In some embodiments, a therapeutically effective amount of any of the compounds or pharmaceutical compositions provided herein is determined according to methods known in the art (e.g., animal models, human data, and human data for compounds exhibiting similar pharmacological activity). The useful dosage of the compound can be determined by comparing its in vitro activity to the in vivo activity of an animal model. Methods of extrapolating effective dosages of mice and other animals to human subjects are known in the art. In fact, the dosage of the compounds may vary greatly depending on the condition of the host subject, the fracture being treated, the route of administration and tissue distribution of the compounds, and the possibility of co-using other therapeutic means (e.g., in combination with administration of other injectable compositions that promote bone growth such as growth factors, stem cells, natural grafts, biological and synthetic based tissue engineering scaffolds, etc., hardware implantation and/or ultrasound therapy, etc.). In some embodiments, a therapeutically effective amount of any of the compounds or pharmaceutical compositions provided herein is determined by considering, for example, the potency of X of formula (I) (e.g., the type of therapeutic agent used), body weight, mode of administration (e.g., subcutaneous administration), disease or condition being treated, severity of disease or condition, and the like, or any combination thereof. The amount of composition required for treatment (e.g., therapeutically effective amount or dose) will vary not only with the particular application, but also with the salt selected (if applicable) and the subject characteristics (e.g., age, condition, sex, surface area and/or weight of the subject, tolerance to drugs), and will ultimately be at the discretion of the attendant physician, clinician or other personnel.

In some embodiments, a therapeutically effective amount of any of the compounds or pharmaceutical compositions provided herein is about 0.01 mg/kg/day to about 1,000 mg/kg/day. For example, a therapeutically effective amount or dose may range from about 0.05mg/kg patient weight to about 30.0mg/kg patient weight, or from about 0.01mg/kg patient weight to about 5.0mg/kg patient weight, including but not limited to 0.01mg/kg, 0.02mg/kg, 0.03mg/kg, 0.04mg/kg, 0.05mg/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 1.0mg/kg, 1.5mg/kg, 2.0mg/kg, 2.5mg/kg, 3.0mg/kg, 3.5mg/kg, 4.0mg/kg, 4.5mg/kg, and 5.0mg/kg, all in kg of patient weight. Intravenous doses can be several orders of magnitude lower. In some embodiments, a therapeutically effective amount of any of the compounds or pharmaceutical compositions provided herein is administered daily, weekly, biweekly, monthly, or bi-monthly (e.g., subcutaneously).

In some embodiments, a therapeutically effective amount (e.g., administered to an individual) of any of the compounds or pharmaceutical compositions provided herein (e.g., SEQ ID NO:1 or SEQ ID NO: 2) is from about 0.01 mg/kg/day to about 1,000 mg/kg/day. In some embodiments, a therapeutically effective amount (e.g., administered to an individual) of any of the compounds or pharmaceutical compositions provided herein (e.g., SEQ ID NO:1 or SEQ ID NO: 2) is from about 1 μg/dose to about 10 mg/dose. In some embodiments, a therapeutically effective amount (e.g., administered to an individual) of any of the compounds or pharmaceutical compositions provided herein (e.g., SEQ ID NO:1 or SEQ ID NO: 2) is from about 50 μg/dose to about 5 mg/dose. In some embodiments, a therapeutically effective amount (e.g., administered to a subject) of any of the compounds or pharmaceutical compositions provided herein (e.g., SEQ ID NO:1 or SEQ ID NO: 2) is from about 0.01 nmol/kg/dose to about 10 ng/kg/dose. In some embodiments, a therapeutically effective amount (e.g., administered to a subject) of any of the compounds or pharmaceutical compositions provided herein (e.g., SEQ ID NO:1 or SEQ ID NO: 2) is from about 0.1 nmol/kg/dose to about 5 ng/kg/dose.

The total therapeutically effective amount of the compounds may be administered in a single or multiple administrations and, as determined by the physician, is outside of the typical ranges set forth herein. In some embodiments, a therapeutically effective amount of any of the compounds or pharmaceutical compositions provided herein is administered once or twice a week. In some embodiments, a therapeutically effective amount of any of the compounds or pharmaceutical compositions provided herein is administered once a week. In some embodiments, a therapeutically effective amount of any of the compounds or pharmaceutical compositions provided herein is administered twice weekly.

An effective amount of the X-Y-Z conjugate, e.g., a pharmaceutical composition comprising an effective amount of the conjugate, may be administered by any suitable route (e.g., subcutaneously). Examples of suitable routes are injection, e.g. subcutaneous injection.

Kit for detecting a substance in a sample

In some embodiments, provided herein is a kit for treating and/or targeting a therapeutic or diagnostic agent to a spinal fusion in a patient. In some embodiments, the kit comprises:

(a) One of the following:

(i) A compound of formula X-Y-Z wherein:

x is a therapeutic agent (e.g., a bone anabolic agent) for treating spinal fusion in the patient or a diagnostic agent for identifying spinal fusion in the patient,

Y is absent, or when present is a linker, and may be releasable or non-releasable, and

z is a osteogenic ligand (e.g., AOP comprising at least 4 amino acid residues to 20 amino acid residues), or

(ii) Pharmaceutical compositions comprising a compound of subpart (i) or a pharmaceutically acceptable salt thereof and

a pharmaceutically acceptable carrier; and

(b) Collagen sponges, mineralized collagen or bone grafts. In some embodiments, the patient is administered a therapeutically effective amount of the compound having the structure of formula (I) or a pharmaceutically acceptable salt thereof to treat spinal fusion.

The compounds of the kit may be any of the compounds or pharmaceutical compositions described herein (e.g., a compound of formula (I), SEQ ID NO:1, or SEQ ID NO: 2). In some embodiments where X is a therapeutic agent, the therapeutic agent may be a bone anabolic agent that is PTH, PTHrP, a derivative of any of the foregoing having bone anabolic activity, or a fragment of any of the foregoing having bone anabolic activity. In some embodiments where X is a therapeutic agent (e.g., a bone anabolic agent), X may be abaclotide, a derivative thereof having bone anabolic activity, or a fragment thereof having bone anabolic activity, or any other suitable bone anabolic agent. In some embodiments, X is a therapeutic agent selected from the group consisting of: abamectin, ITGA, dasatinib, PTH, PTHrP and derivatives or fragments of any of the foregoing having bone anabolic activity.

In some embodiments, X of the compound or pharmaceutical composition is a diagnostic agent. Such diagnostic agents may be any suitable imaging agent such that administration of a compound having the structure of formula (I) or a pharmaceutically acceptable salt thereof (or a pharmaceutical composition comprising such compound or a pharmaceutically acceptable salt thereof) may recognize spinal fusion, if present. In some embodiments, the diagnostic agent is a fluorescent dye. In some embodiments, the diagnostic agent is a near infrared fluorescent molecule.

Therapeutic method

Provided in some embodiments herein are methods of treating spinal fusion (e.g., in an individual in need thereof). Provided in some embodiments herein are methods of treating spinal fusion in a patient (e.g., a patient in need thereof) using the provided compounds and/or compositions. The compounds, compositions, and methods can utilize strategies (e.g., selectively) to target spinal fusion sites to prevent off-target effects of therapeutic agents (e.g., anabolic agents) present in the compounds or compositions.

In some embodiments, the methods comprise administering (e.g., subcutaneously) to a patient in need thereof a therapeutically effective amount of any of the compounds or pharmaceutical compositions described herein, including, for example:

(i) Compounds provided herein, for example, compounds having the structure of formula (I):

X-Y-Z，

or a pharmaceutically acceptable salt thereof, wherein:

x is an anabolic agent of bone,

z is a osteogenic ligand, which may be, for example, AOP (e.g., comprising at least 4 amino acid residues to 20 amino acid residues), or

(ii) A pharmaceutical composition comprising a compound of subsection (i) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

Methods of locating a therapeutic or diagnostic agent to spinal fusion in a patient are also described. In some embodiments, a method for locating a therapeutic or diagnostic agent to a spinal fusion site in a patient comprises administering to the patient a therapeutically effective amount of any of the compounds described herein, or a pharmaceutically acceptable salt thereof.

As shown in fig. 2, provided herein is biodistribution in bilateral posterolateral lumbar fusion of collagen scaffold treatment at 24 hours post-operative 2 weeks, targeting near Infrared (IR) fluorescent molecule S0456 (see, e.g., scheme 1 in example 9 below). Animals were imaged on an AMI spectrometer at 5% excitation power for 1 second with excitation wavelength of 745nM and emission wavelength of 810nM (see example 4 and example 7 below).

Figure 3 shows the biodistribution in bilateral posterolateral lumbar fusion of mineralized collagen scaffold treatment at 24 hours post-operative 2 weeks after injection of targeted near infrared fluorescent molecule S0456. Animals were imaged on an AMI spectrometer at 5% excitation power for 1 second with excitation wavelength of 745nM and emission wavelength of 810nM (see examples 4 and 7 below).

Fig. 4 shows the biodistribution in bilateral posterolateral lumbar fusion of bone graft stent treatment 24 hours after 2 weeks post-surgery, targeting near infrared fluorescent molecule S0456 injection. In some embodiments, animals are imaged on an AMI spectrometer at 5% excitation power for 1 second with excitation wavelength of 745nM and emission wavelength of 810nM (see examples 4 and 7 below).

In some embodiments, as shown in fig. 4, the localization (of the targeted near infrared fluorescent molecules) is on the outside both at the paw and the head, as the compound is present in the urine (e.g., the animal walks in the urine and then rubs on its head as it combs).

Figure 5 shows biodistribution in bilateral posterolateral lumbar fusion of collagen scaffold treatment at 8 weeks post-surgery, 24 hours post injection of targeted near infrared fluorescent molecule S0456. At this point, the animals were subjected to a full necropsy to examine the accumulation in other (non-targeted) tissues. Animals were imaged on an AMI spectrometer at 5% excitation power for 1 second with excitation wavelength of 745nM and emission wavelength of 810nM.

In some cases, localization of a compound provided herein (e.g., a targeting compound) to a spinal fusion site is observed in all stent types. In some embodiments, the whole body anatomy (see fig. 5) is shown positioned only within the kidneys and spine. Since the kidneys are the excretion pathway for certain embodiments of the compounds provided herein, these results indicate that acidic oligopeptides can be used to localize therapeutic drugs at spinal fusion. In some embodiments, (the compound) is positioned on the paw and head as the compound is expelled through the urine (e.g., the rat steps into the urine during combing and then rubs on its head).

Figure 12 shows a three-dimensional reconstruction of a microscopic CT scan of posterolateral spinal fusion of rats L4-L5 treated with mineralized collagen scaffolds and physiological saline one week after implantation. In some embodiments, mineralization is inherent to the scaffold (e.g., as seen early in the spinal fusion process).

In some embodiments, the scaffold absorbs too rapidly, so after 8 weeks, all treatments are unfused. For example, FIG. 13 shows a three-dimensional reconstruction of a microscopic CT scan of posterolateral spinal fusion of rats L4-L5 after 8 weeks of treatment with a mineralized collagen scaffold and its compounds with different therapeutic means (e.g., physiological saline, BMP2, and abaclotide DE 20). In some embodiments, the scaffold resorbs too fast to bridge. The three-dimensional reconstructive image of the micro-CT scan shown in panels a and B of fig. 13 is a spinal fusion image after 8 weeks of treatment with mineralized collagen and BMP 2.

The three-dimensional reconstructions of the micro CT scan shown in panels C and D of FIG. 13 are spinal fusion maps after 8 weeks of treatment with mineralized collagen and SEQ ID NO 2. The three-dimensional reconstructive image of the micro CT scan shown in panels E and F of fig. 13 is a spinal fusion image after 8 weeks of treatment with mineralized collagen and physiological saline.

In some embodiments, the transverse processes heal after being peeled from the cortex. In some embodiments, no bone is formed between the L4 and L5 vertebral bodies. However, most promising and consistent are collagen scaffolds, in which only the treated spine of abamectin (e.g., the compound of SEQ ID NO: 2) is fused (e.g., FIG. 6).

Panels A-C in FIG. 6 show microscopic CT scan three-dimensional reconstructions of lateral spinal fusion of rats L4-L5 after 8 weeks of treatment with uncolored collagen matrix and the compound of SEQ ID NO: 2. In some embodiments, SEQ ID NO. 2 is administered at 33nmol/kg (e.g., for rodents) twice a week. The fusion was confirmed by manual palpation.

Panels A-C in FIG. 7 show three-dimensional reconstructive images of microscopic CT scans of lateral spinal fusion following treatment of 8 week rats L4-L5 with rhBMP-2 (10 μg) infiltrated non-pigmented collagen matrix. In some embodiments, the collagen matrix is applied during surgery. Panels A-C in FIG. 7 show that BMP-treated spines (e.g., positive controls) did ossify, but were not as controlled as spines treated with the compounds of SEQ ID NO:2 (e.g., possibly due to ectopic leakage or poor cortical stripping). In some embodiments, the BMP-treated spine is not fused at all (artificial palpation confirmed unfused).

In some embodiments, collagen scaffolds alone and surgery are insufficient to form bone. In some embodiments, the anabolic composition is sufficient to mineralize the collagen sponge. In some embodiments, the bone graft is fully fused (e.g., this may be an artifact of difficulty in continuously filling collagen sponges with sufficient bone particles) (e.g., fig. 8).

Panels A-C in FIG. 8 show three-dimensional reconstructive images of microscopic CT scan of posterolateral spinal fusion of rats L4-L5 treated with uncolored collagen matrix and physiological saline for 8 weeks. In some embodiments, phosphate buffered saline is administered twice a week at a 1x concentration at the same dose as SEQ ID NO. 2. In some embodiments, manual palpation confirmed that phosphate buffered saline treated rats were unfused.

In some embodiments, BMP fuses some bone particles (e.g., fig. 9, panels a-C).

FIG. 9 shows three-dimensional reconstructions of microscopic CT scans of lateral spinal fusion following treatment of rats L4-L5 with bone graft matrix and rhBMP-2 (10 μg) for 8 weeks. rhBMP-2 was infiltrated into the collagen spongy bone graft matrix applied during surgery. In some embodiments, manual palpation confirms unfused.

In some embodiments, the bone particles resorb in abamectin treated rats (fig. 10, panels a-C).

In some embodiments, provided herein are three-dimensional reconstructive images of microscopic CT scan of spinal fusion outside the posterior lateral side of rats L4-L5 treated with bone graft matrix and SEQ ID NO:2 for 8 weeks. In some embodiments, SEQ ID NO 2 is allowed to permeate into the collagen spongy bone graft matrix applied during surgery. In some embodiments, SEQ ID NO. 2 is administered twice weekly (for rodents) at a dose of 33 nmol/kg. In some embodiments, manual palpation confirms unfused.

In some embodiments, a physiological saline treated rodent with a bone graft has no change in the number or shape of bone particles (e.g., indicating no change) (e.g., fig. 11).

Panels A-C in FIG. 11 show three-dimensional reconstructions of microscopic CT scan of posterolateral spinal fusion of rats L4-L5 treated with bone graft matrix and physiological saline for 8 weeks. In some embodiments, physiological saline is infiltrated into the collagen spongy bone graft matrix applied during surgery. In some embodiments, phosphate buffered saline is administered twice a week (for rodents) at a concentration of 1x at the same dose as SEQ ID NO. 2.

In some embodiments, a collagen scaffold is used. In some cases, for example, such collagen scaffolds that produce consistent results are commercially available and are commonly used clinically. Although mineralized collagen achieves targeting at the fastest rate in terms of how much time after spinal fusion the targeting compound can be detected, its performance is not as expected in some cases. In some cases, mineralized collagen treated spinal fusion takes about 5 days, but in some cases, collagen scaffold treated spinal fusion takes approximately 12 days. In some cases, spinal fusion for bone graft treatment requires approximately 10 days. In some embodiments, for targeting, a blood supply and hydroxyapatite are required.

Figure 14 shows a three-dimensional reconstruction of a microscopic CT scan of posterolateral spinal fusion of rats L4-L5 after 5 weeks of treatment with mineralized collagen scaffolds and various treatments with compounds of the present disclosure (see example 8 below). Panel A in FIG. 14 shows a three-dimensional reconstruction of a microscopic CT scan taken from a rat spinal fusion site 5 weeks after treatment with mineralized collagen and SEQ ID NO: 2. Panel B in FIG. 14 shows a three-dimensional reconstruction of a microscopic CT scan taken from a rat spinal fusion site 5 weeks after treatment with mineralized collagen and rhBMP-2 (10 μg). Panel C in FIG. 14 shows a three-dimensional reconstruction of a microscopic CT scan taken from a rat spinal fusion site 5 weeks after treatment with mineralized collagen and physiological saline.

As shown in FIG. 14, administration of the compound of SEQ ID NO. 2 enhanced mineralization of the scaffold more than BMP2 or physiological saline.

FIG. 15 shows a graph of the weeks of BMP and SEQ ID NO. 2 versus total fusion score. In a microscopic CT scan of rats, two cases of bilateral spinal fusion were scored for osseointegration, density and bridging, scoring 0-5 points, determined by individuals blinded to treatment and examined for random samples. The total score is the sum of all five scores added together.

FIG. 16 provides a plot of week number versus total fusion score for physiological saline, BMP, and SEQ ID NO. 2. In a microscopic CT scan of rats, two cases of bilateral spinal fusion were scored for osseointegration, density and bridging, scoring 0-5 points, determined by individuals blinded to treatment and examined for random samples. The total score is the sum of all five scores added together. In some cases, the degree of fusion of physiological saline is artificially high, because more bone is removed during cortical stripping, inducing a stronger osteogenic response. .

FIG. 17 shows the weeks of SEQ ID NO. 2 and physiological saline versus BMD (bone mineral density; HA mg/cm) ³ ) For example, which shows BMD forming a bone bridge during fusion. In some cases, BMP does not produce enough mineralized tissue to be quantified. The scan density is specified based on a standard curve generated from the motif hydroxyapatite standard set.

FIG. 18 provides the number of weeks versus BV (bone volume; mm) ³ ) For example, which shows the total area of mineralized tissue in a bone bridge formed by treatment of spinal fusion with SEQ ID NO:2 or physiological saline. In some cases, this was quantified by ImageJ analysis of a microscopic CT scan of rats.

All patents, patent application publications, journal articles, textbooks, and other publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All of these publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

While certain embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The claimed invention is not intended to be limited to the specific examples provided in the specification.

While the invention has been described with reference to the above description, the description and illustration of the embodiments herein is not meant to be construed in a limiting sense. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, depending on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the present invention shall also cover any such alternatives, modifications, variations or equivalents. The following claims are intended to define the scope of the invention and methods and structures within the scope of these claims and their equivalents are covered thereby.

Certain definitions

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of such compounds. When ranges of physical properties (e.g., molecular weight) or chemical properties (e.g., formula) are used herein, all combinations and subcombinations of ranges, as well as specific embodiments thereof, are intended to be included. When referring to a number or range of values, the term "about" means that the number or range of values referred to is an approximation within experimental variation (or statistical experimental error), and thus the number or range of values may vary from 1% to 15% of the stated number or range of values. The term "comprising" (and related terms such as "comprises" or "comprising") or "having" or "including") is not intended to exclude any embodiment of a compound, composition, method, process, etc., which may "consist of" or "consist essentially of the recited feature. The invention illustratively described herein suitably may be practiced in the absence of any element or limitations which is not specifically disclosed herein.

"percent (%) sequence identity" is defined as the percentage of amino acid residues or nucleic acid residues in a candidate sequence that are identical to the residues in a reference sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not taking into account any conservative substitutions as part of the sequence identity, relative to the referenced sequence. The alignment used to determine the percent sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software. For example, the percent identity or similarity between sequences may be determined by using the GAP program (Genetics Computer Group, software; now available through Accelrys website http:// www.accelrys.com), and the alignment may be performed using, for example, the ClustalW algorithm (VNTI software, inforMax Inc., gaithersburg, MD). In addition, the sequence database may be searched using the nucleic acid or amino acid sequence of interest. Algorithms for database searches are typically based on BLAST software (Altschul et al, 1990), but one skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. In some embodiments, the percent identity may be determined along the entire length of the nucleic acid or amino acid sequence.

As used herein, the terms "patient," "subject," and "individual" are used interchangeably. None of these terms require supervision by medical personnel. For example, administering to an individual includes the individual administering the therapeutic agent to himself, and a medical professional administering the therapeutic agent to the individual.

The term "free radical" as used herein refers to a fragment of a molecule, wherein the fragment has an open valency and is the point of attachment for bond formation. The monovalent radical has an open valence such that it can form a bond with another chemical group. In some embodiments, the free radical of a molecule used herein is generated by removing a hydrogen atom from the molecule to generate a monovalent radical having an open valence at the site of removal of the hydrogen atom. Where appropriate, the radicals may be divalent, trivalent, etc., wherein two, three or more hydrogen atoms have been removed to generate radicals that may be bonded to two, three or more chemical groups. The radical open valence may be generated by removing atoms other than hydrogen atoms (e.g., halogen atoms) or by removing two or more atoms (e.g., hydroxyl groups), as long as the removed atoms are a fraction (about 20% of the number of atoms or less) of the total atoms in the molecule forming the radical, where appropriate.

The terms "treatment", "treatment" or "treatment" include reducing, alleviating, reducing, ameliorating, alleviating or attenuating the symptoms associated with bone fractures, diabetes, osteoporosis in the case of chronic or acute treatment.

The terms and expressions which have been employed are used as terms of description and not of limitation. In this regard, if a certain term is defined in "certain definition" and is defined, described, or discussed elsewhere in "detailed description," all such definitions, descriptions, and discussions are intended to be attributed to that term. There is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof. Moreover, although sub-headings, such as "certain definitions," are used in the detailed description, such use is for convenience only and is not intended to limit any disclosure in a section to that section only; rather, any disclosure under one subtitle is intended to constitute the disclosure under each subtitle and all other subtitles.

Examples

The following examples serve to illustrate the disclosure. These examples are not intended to limit the scope of the claimed invention in any way.

Example 1: synthesis of peptide payloads

All payloads (e.g., fig. 1A) were synthesized in solid-phase polypeptide synthesis vials under argon flow. Wang resin (0.6 mmol/g) was reacted with a catalytic amount of 4-Dimethylaminopyridine (DMAP) at 9:1v/v CH ₂ Cl ₂ 3-fold excess of the first amino acid (cysteine), HOBt-Cl and DIC were loaded in Dimethylformamide (DMF) for 4 hours. The resin was then capped with two equivalents of acetic anhydride and pyridine for 30 minutes to block any unreacted hydroxyl groups on the resin. After these steps, three successive washes with Dichloromethane (DCM) and DMF were performed.

After each coupling reaction, the 9-fluorenylmethoxycarbonyl (Fmoc) group was removed by incubation with 20% (v/v) piperidine in DMF twice for 10 minutes each. The resin was then washed twice with DMF before the next amino acid was added. Each amino acid was reacted in a 3-fold excess of 2- (1H-benzotriazol-1-yl) -1, 3-tetramethyluronyl Hexafluorophosphate (HBTU)/N-methylmorpholine (NMM) for 30 minutes and then double coupled with a 3-fold excess of benzotriazol-1-yl-oxy-tripyrrolidinylphosphine (PyBOP)/N-methylmorpholine (NMM) for 30 minutes. All amino acids were added as described above. Standard Fmoc protected amino acids with acid sensitive side chain protecting groups were used unless otherwise indicated. Thereafter, tyrosine or the peptide sequences shown in table 1 were added to the peptides using the solid phase procedure listed above using a peptide autosynthesizer (Focus XC, AAPPTec). After completion of the synthesis, the terminal Fmoc was removed using the conditions described above, after which the resin was washed three times with DMF, three times with DCM, twice with methanol, and then dried with argon.

The dried resin with peptide was cleaved for 2 hours using 95:2.5:2.5 trifluoroacetic acid/water/triisopropylsilane and excess tris (2-carboxyethyl) phosphine (TCEP). The peptide was then precipitated from the lysis solution using 10 volumes of cold diethyl ether. The solution was spun at 2000 Relative Centrifugal Force (RCF) for 5 minutes and then decanted. The particles were then dried and subjected to analytical liquid chromatography-mass spectrometry (1220LC; 6130MS, agilent) to confirm synthesis. The crude peptide was dissolved in a mixture of DMF and water and purified by preparative reverse phase high performance liquid chromatography (1290,Agilent,Santa Clara,CA). 2, 6-Tetramethylpiperidine (TMP) was purified using a C-18 column with 0-50% ammonium acetate in acetonitrile as mobile phase for 40 minutes. Fractions containing only the pure payload as assessed by analytical liquid chromatography-mass spectrometry (1220LC;6130MS,Agilent,Santa Clara,CA) were lyophilized (FreeZone, LABCONCO, kansas City, MO) and stored as lyophilized powder at-20 ℃ until it was coupled to the targeting ligand.

The following substitutions were introduced into residues 1-46 of parathyroid hormone-related protein (PTHrP) (SEQ ID NO: 1) according to methods known in the art: glu22, glu25, leu23, leu28, leu31, lys26, lys30 and Aib29. These substitutions enhance the stability of the peptide, induce increased bone density in osteoporotic patients, and expand the window of strongest anabolic activity without increasing toxicity. In order to maximize the signaling effect of anabolic peptides upon adsorption to exposed hydroxyapatite, the C-terminus of the PTHrP fragment was conjugated to a linear peptide of 20D-glutamic acid (E) residues (D-Glu) using standard solid phase peptide chemistry ₂₀ Or DE 20) to give the final fusion protein (SEQ ID NO: 2) in a total yield of 19% and a final purity of 94%, as indicated by High Pressure Liquid Chromatography (HPLC) and mass spectrometry.

Example 2: synthesis of (Linear) osteogenic peptides

The targeting ligand peptides were all synthesized according to the solid phase synthesis method described above to achieve the appropriate length, amino acid composition and enantiomeric stereochemical configuration, as the name suggests. While the N-terminal amine was still on the resin, deprotection was performed as described above and the resin was reacted with 3-fold maleimide propionic acid, 3-fold excess benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate (PYBOP), HOBt-Cl and 5-fold excess N, N-Diisopropylethylamine (DIPEA) in DMF for 4 hours. The peptide was then coupled to cysteine-containing peptide in Phosphate Buffered Saline (PBS) containing a 10-fold excess of TCEP using maleimide chemistry at room temperature for 24 hours. The targeted payload conjugate is then cleaved, deprotected, and purified as described above.

Example 3: synthesis of (branched) osteogenic peptides

Briefly, branched targeting ligands were synthesized using solid phase polypeptide synthesis methods under argon flow. 2-chlorotrityl resin (0.6 mmol/g) was loaded with Nalpha, nalpha-di-Fmoc-L-lysine in DCM and DIPEA at a concentration of 0.6mmol/g for 60 min. The resin was then capped with MeOH four times followed by three consecutive washes with DCM and DMF. The branches are then synthesized as described above. N-terminal Fmoc was retained and the peptide was soft cleaved in a 1:1:8 mixture of acetic acid/Tetrafluoroethylene (TFE)/DCM for 30 min. The cleavage solution was evaporated under reduced pressure and the terminal carboxylic acid was conjugated with a 3-fold excess of N- (2-aminoethyl) maleimide, a 3-fold excess of PYBOP and HOBt-Cl, and a 5-fold excess of DIPEA in DCM for 4 hours. The acid-sensitive protecting group was then deprotected by incubation in 95:2.5:2.5 trifluoroacetic acid/water/triisopropylsilane for two hours. The peptide was then precipitated with 10 volumes of cold diethyl ether and the terminal Fmoc was deprotected by incubation with 20% (v/v) piperidine in DMF for 15 min, followed by precipitation in cold diethyl ether. The crude product obtained was purified by preparative reverse phase high performance liquid chromatography (1290, agilent) as described above. Finally, the purified targeting ligand was conjugated to a different payload by maleimide coupling, also as described above.

Example 4: acidic oligopeptide targeting

Rats are the smallest animals that can simulate spinal fusion surgery. The posterolateral lumbar fusion model of rats is the most useful and most easy site for surgery. This model is also the easiest model to measure bone growth over time.

Female Sprague-Dawley rats were subjected to bilateral spinal fusion of the L4 and L5 transverse processes of the vertebral bodies by insertion of mineralized collagen sponges. Two weeks after surgery, rats were injected with S0456 conjugated to an Acidic Oligopeptide (AOP) targeting moiety, a near Infrared (IR) fluorescent dye. Rats were imaged with a spectroscopic imaging system (AMI) for 1 second 24 hours after injection with excitation wavelength of 745nM and emission wavelength of 810nM. Imaging confirmed the localization of the S0456 conjugate to the spine and kidneys, which is the excretory pathway of the compound (see, e.g., fig. 3).

Example 5: bilateral posterolateral lumbar vertebra operation

Bilateral posterolateral lumbar fusion was performed under sterile conditions using isoflurane as anesthetic. The skin surrounding the area of the back L2-L6 was shaved and disinfected with Betadine solution followed by an alcohol pad. A 3cm incision was made in the skin of the prepared area extending from L3 to the L6 vertebral body. The muscles on the vertebral body between the spinous process and the transverse process are passively stripped from the vertebral body, exposing the transverse process.

Using a low-speed bone drill, L4 and L5 transverse process cortical bone was removed in addition to the fascial junction (fascian joint) between the two vertebral bodies. The bone drill removes the surface layer of the bone. This process is called cortical stripping, in order to allow direct regeneration of bone tissue. After the cortex is stripped, the drug-soaked collagen sponge is placed on both sides of the vertebral body. A 5x 7.5mm collagen sponge was soaked in the drug for 10 minutes to saturation. The collagen sponge (RCM 6 membrane of ACE surgical supplies) was used as a scaffold to aid in fusion of the vertebral bodies. Mineralized collagen scaffolds (supplied by Houston Methodist, houston, TX) and collagen scaffolds encapsulating inorganic bone particles (inters bone particles; sigmaGraft inc. The muscles are sutured with absorbable sutures. The external wound was then sutured with nylon suture. Buprenorphine (0.05-0.1 mg/kg) was administered subcutaneously directly after surgery and repeated every 12 hours for 3 days post-surgery.

Example 6: micro CT and AMI scanning

Fracture healing was assessed using micro CT (Scanco Medical Ag or PerkinElmer). The anesthetized animals were scanned at high resolution for two minutes using PerkinElmer Quantum FX microtct, at 90kV and 88uA using an al0.5mm+cu0.06 mm filter, at a voxel size of 10 μm. The rate of scanning the detector was 117fps. Images were analyzed in ImageJ using BoneJ software package. Morphometric parameters were quantified in ROIs that included only fracture callus. The trabecular thickness (tb.th.), trabecular spacing (tb.sp.), total Volume (TV) and calcified callus volume (BV) were calculated. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Dunnett post hoc analysis, reporting significance at a P value of 0.05. All animal experiments were performed according to the protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the university of ferry.

Rats were anesthetized with isoflurane and scanned in AMI.

Example 7: spinal fusion targeting

For three main spinal fusion approaches: collagen sponges, mineralized collagen and bone grafts demonstrate the ability to selectively localize compounds to spinal fusion sites by subcutaneous injection rather than to other parts of the body. The results are shown in FIGS. 2-4.

Example 8: spinal fusion targeting

Rats were assigned to one of the following 3 treatments: physiological saline as a negative control, 10 μg BMP2 (clinically approved drug; current standard of care) as a positive control, or AbaloDE20 (a compound of the present disclosure). Preliminary results at week 5 are shown in FIGS. 14A-C.

Example 9: synthesis of Near Infrared (NIR) dye conjugates

Maleimide derivative S0456 of NIR fluorescent dye was prepared for labeling the above-mentioned fracture targeting ligand. Its synthesis is shown in scheme I (below). For this, S0456, N-Boc-tyramine and potassium hydroxide (KOH) were mixed in a flask containing dimethyl sulfoxide (DMSO) to dissolve the solids, and the solution was stirred under argon at 60 ℃ for 1.2 hours. The resulting solution was precipitated with cold ethyl acetate and, after vigorous stirring, centrifuged at 3000rpm for 3 minutes. The dark green solid was dried overnight in a vacuum desiccator and deprotected in 40% trifluoroacetic acid (TFA)/DCM for 30 min, then concentrated in vacuo to remove all TFA and DCM. The crude solid is then dissolved in water and subjected to preparative reverse phase high performance liquid chromatography (1290, Agilent, santa Clara, CA). The pure fractions were concentrated in vacuo and freeze-dried. For derivatization with maleimide, the solid was dissolved in DMSO together with N-hydroxysuccinimide 3-maleimide propionate and DIPEA and stirred under argon atmosphere for 1 hour, and then purified by preparative reverse phase high performance liquid chromatography (1290,Agilent,Santa Clara,CA) as described above. The N-terminal cysteine-bearing deca-aspartic acid (L and D) targeting ligands were prepared and purified as described previously. In order to conjugate decaaspartic acid cysteine with S0456-maleimide, S0456-maleimide was dissolved in DMSO in a flask degassed with argon, then Asp was added to the solution with stirring ₁₀ -Cys. The mixture was stirred at room temperature for 2.5 hours, and then purified by preparative reverse phase high performance liquid chromatography (1290,Agilent,Santa Clara,CA). The purified and freeze-dried product was a green fluffy solid. (D) Asp (Asp) ₁₀ The synthesis of the-S0456 conjugate follows with (L) Asp ₁₀ The same procedure as described in S0456, except (D) Asp ₁₀ D-aspartic acid was used for the synthesis of (C).

Scheme I: (L) Asp ₁₀ -synthesis of S0456 conjugate. Reagents and conditions: a) S0456-Cl, DIPEA, DMSO,60 ℃; b) 40% TFA/DCM, room temperature; c) 3-maleimide propionic acid N-hydroxysuccinimide ester, DIPEA, DMSO, room temperature; d) Asp (Asp) ₁₀ -Cys、DMSO。

It should be appreciated that various modifications may be made within the scope of the claimed invention. Thus, it should be understood that although the present invention has been specifically disclosed in the context of preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art. Such modifications and variations are considered to be within the scope of the invention as claimed herein.

Sequence listing

<110> general research Foundation

<120> Compounds, compositions and methods for treating spinal fusion

<130> 69249-03

<150> 63/105,678

<151> 2020-10-26

<150> 63/193,753

<151> 2021-05-27

<160> 2

<170> patent in version 3.5

<210> 1

<211> 56

<212> PRT

<213> artificial sequence

<220>

<223> a compound for targeting and treating spinal fusion sites

<220>

<221> site

<222> (29)..(29)

<223> wherein "X" at position 29 is alpha-aminoisobutyric acid

<220>

<221> site

<222> (47)..(56)

<223> wherein the glutamic acid residue at positions 47-56 has D chirality

<400> 1

Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln

1 5 10 15

Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Xaa Lys Leu His

20 25 30

Thr Ala Glu Ile Arg Ala Thr Ser Glu Val Ser Pro Asn Ser Glu Glu

35 40 45

Glu Glu Glu Glu Glu Glu Glu Glu

50 55

<210> 2

<211> 66

<212> PRT

<213> artificial sequence

<220>

<223> a compound for targeting and treating spinal fusion sites, the compound having a payload comprising abamectin

<220>

<221> site

<222> (29)..(29)

<223> wherein "X" at position 29 is alpha-aminoisobutyric acid

<220>

<221> site

<222> (47)..(66)

<223> wherein "X" at position 29 is alpha-aminoisobutyric acid having D chirality

<400> 2

Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln

1 5 10 15

Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Xaa Lys Leu His

20 25 30

Thr Ala Glu Ile Arg Ala Thr Ser Glu Val Ser Pro Asn Ser Glu Glu

35 40 45

Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu

50 55 60

Glu Glu

65

Claims

1. A method of treating spinal fusion in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound having the structure of formula (I):

X-Y-Z formula (I),

or a pharmaceutically acceptable salt thereof,

wherein:

x is a bone anabolic agent;

y is absent or a linker; and

z is a bone-promoting ligand which is a ligand,

thereby treating spinal fusion in the patient.

2. The method of claim 1, wherein Y is a releasable linker or a non-releasable linker.

3. The method of claim 1, wherein X is abamectin, pranoplatin (preptin), integrin 5β1 (ITGA), dasatinib, parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), or a derivative or fragment of any of the foregoing having bone anabolic activity.

4. A method according to any one of claims 1-3, wherein X is abamectin.

5. The method of claim 1, wherein Z is an Acidic Oligopeptide (AOP) or another hydroxyapatite binding molecule.

6. The method of claim 1, wherein Z is an AOP comprising at least 4 amino acid residues.

7. The method of any one of claims 1-3, 5, and 6, wherein Z is an AOP comprising 4 to 20 amino acid residues.

8. The method of any one of claims 1-3, 5, and 6, wherein the bone anabolic agent is parathyroid hormone (PTH), a PTH-related protein (PTHrP), or a derivative or fragment of any of the foregoing that has bone anabolic activity.

9. The method of any one of claims 1-3, 5, and 6, wherein the bone anabolic agent is abaclotide or a derivative or fragment thereof having bone anabolic activity.

10. The method of any one of claims 1-3, 5, and 6, wherein Z is a linear chain of amino acid residues.

11. The method of claim 1, wherein Z is an AOP comprising at least 4 glutamic acid amino acid residues or 4 aspartic acid amino acid residues.

12. The method of any one of claims 1-3, 5, 6, and 11, wherein Z comprises at least 4 amino acid residues having D or L chirality.

13. The method of any one of claims 1-3, 5, 6, and 11, wherein Z comprises at least 4 amino acid residues with D chirality.

14. The method of any one of claims 1-3, 5, 6, and 11, wherein Z comprises at least 4 glutamic acid amino acid residues, at least 4 aspartic acid amino acid residues, or at least 4 glutamic acid amino acid residues and at least 4 aspartic acid amino acid residues.

15. The method of any one of claims 1-3, 5, 6, and 11, wherein Z comprises 4 to 20D-glutamic acid amino acid residues and/or 4 to 20D-aspartic acid amino acid residues.

16. The method of any one of claims 1-3, 5, 6, and 11, wherein Z comprises a mixture of glutamic acid amino acid residues and aspartic acid amino acid residues.

17. The method of claim 1, wherein Z comprises at least 10 repeated D-glutamic acid amino acid residues (DE 10).

18. The method of any one of claims 1-3, 5, 6, 11, and 17, wherein Z comprises at least 15 repeated D-glutamic acid amino acid residues (DE 15) or at least 20 repeated D-glutamic acid amino acid residues (DE 20).

19. The method of any one of claims 1-3, 5, 6, 11, and 17, wherein Z is DE10 or DE20.

20. The method of any one of claims 1-3, 5, 6, 11, and 17, wherein X is abamectin, ITGA, dasatinib, PTH, PTHrP, or a derivative or fragment of any of the foregoing having bone anabolic activity; and Z is DE20.

21. The method of any one of claims 1-3, 5, 6, and 11, wherein Z comprises 4 to 75 acidic amino acid residues.

22. The method of any one of claims 1-3, 5, 6, and 11, wherein Z comprises 4 to 75D-glutamic acid amino acid residues.

23. The method of any one of claims 1-3, 5, 6, and 11, wherein Z comprises 8 to 30 acidic amino acid residues.

24. The method of any one of claims 1-3, 5, 6, and 11, wherein Z comprises 8 to 30D-glutamic acid amino acid residues.

25. The method of any one of claims 1-3, 5, 6, 11, and 17, wherein Y is an unreleasable linker containing at least one carbon-carbon bond, at least one amide bond, or at least one carbon-carbon bond and at least one amide bond.

26. The method of any one of claims 1-3, 5, 6, 11, and 17, wherein Y is a releasable linker.

27. The method of any one of claims 1-3, 5, 6, 11, and 17, wherein Y is a releasable linker comprising at least one disulfide bond, at least one ester bond, at least one protease-specific amide bond, or a combination of the foregoing.

28. The method according to claim 1, wherein:

x is abamectin, ITGA, dasatinib, PTH, PTHrP, or a derivative or fragment of any of the foregoing having bone anabolic activity;

Y is a non-releasable oligopeptide linker; and is also provided with

Z is DE20.

29. The method of claim 1, wherein X is abamectin or a derivative or fragment thereof, Y is a releasable oligopeptide linker comprising at least one protease specific amide bond, and Z is DE20.

30. The method of claim 1, wherein the compound has at least 75% or more sequence identity, at least 85% or more sequence identity, at least 90% or more sequence identity, or at least 95% or more sequence identity to SEQ ID No. 1.

31. The method of claim 1, wherein the compound has at least 75% or more sequence identity, at least 85% or more sequence identity, at least 90% or more sequence identity, or at least 95% or more sequence identity to SEQ ID No. 2.

32. A kit for treating spinal fusion and/or targeting a therapeutic or diagnostic agent to spinal fusion in a patient in need thereof, the kit comprising:

(a) A compound having the structure of formula (I):

X-Y-Z formula (I),

or a pharmaceutically acceptable salt thereof,

wherein:

x is a therapeutic or diagnostic agent for treating spinal fusion in said patient,

Y is absent or is a linker, and

z is a osteogenic ligand; and

(b) Collagen sponges, mineralized collagen or bone grafts.

33. The kit of claim 32, wherein X comprises a bone anabolic agent, and wherein the patient is administered a therapeutically effective amount of a compound having the structure of formula (I) or a pharmaceutically acceptable salt thereof to treat the spinal fusion.

34. The kit of claim 33, wherein X is a therapeutic agent selected from the group consisting of: abamectin, integrin 5β1 (ITGA), dasatinib, parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP) and derivatives or fragments of any of the foregoing that have bone anabolic activity.

35. The kit of claim 32, wherein X comprises a diagnostic agent and a therapeutically effective amount of a compound having the structure of formula (I), or a pharmaceutically acceptable salt thereof, if present, is administered to the patient, spinal fusion is identified.

36. The kit of any one of claims 32-35, wherein Y is a releasable linker or a non-releasable linker.

37. The kit of any one of claims 32-35, wherein Y is a non-releasable linker comprising at least one carbon-carbon bond, at least one amide bond, or at least one carbon-carbon bond and at least one amide bond.

38. The kit of any one of claims 32-35, wherein Z is an Acidic Oligopeptide (AOP).

39. The kit of any one of claims 32-35, wherein Z is an AOP comprising at least 4 amino acid residues.

40. The kit of any one of claims 32-35, wherein the AOP comprises 4 to 20 amino acid residues.

41. The kit of any one of claims 32-35, wherein X is a bone anabolic agent that is parathyroid hormone (PTH), PTH receptor protein (PTHrP), or a derivative or fragment of any of the foregoing that has bone anabolic activity.

42. The kit of claim 32, wherein X is a therapeutic agent that is abaclotide or a derivative or fragment thereof having bone anabolic activity.

43. A pharmaceutical composition comprising a compound having the structure of formula (I):

X-Y-Z formula (I),

or a pharmaceutically acceptable salt thereof,

wherein:

x is a therapeutic or diagnostic agent and,

y is absent or is a linker, and

z is a osteogenic ligand.

44. The pharmaceutical composition according to claim 43, further comprising at least one pharmaceutically acceptable carrier or excipient.

45. The pharmaceutical composition of claim 43 or 44, formulated for subcutaneous administration to the patient.

46. The pharmaceutical composition of claim 43, wherein X is a therapeutic agent that is a bone anabolic agent.

47. The pharmaceutical composition of claim 43, wherein X is a diagnostic agent that is an imaging agent.

48. The pharmaceutical composition of claim 43, wherein Y is a releasable linker or a non-releasable linker.

49. The pharmaceutical composition according to any one of claims 43, 44 and 46, wherein X is abamectin, pranoplatin peptide (preptin), integrin 5β1 (ITGA), dasatinib, parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), or a derivative or fragment of any of the foregoing having bone anabolic activity.

50. The pharmaceutical composition according to any one of claims 43, 44 and 46, wherein Z is an Acidic Oligopeptide (AOP) or another hydroxyapatite binding molecule.

51. The pharmaceutical composition of any one of claims 43, 44 and 46, wherein Z is AOP comprising at least 4 amino acid residues.

52. The pharmaceutical composition of any one of claims 43, 44 and 46, wherein Z is AOP comprising 4 to 20 amino acid residues.

53. The pharmaceutical composition of any one of claims 43, 44 and 46, wherein the bone anabolic agent is abaclotide or a derivative or fragment thereof having bone anabolic activity.

54. The pharmaceutical composition of any one of claims 43, 44 and 46, wherein Z is a linear chain of amino acid residues.

55. The pharmaceutical composition of any one of claims 43, 44 and 46, wherein Z comprises 4 to 20D-glutamic acid amino acid residues and/or 4 to 20D-aspartic acid amino acid residues.

56. The pharmaceutical composition according to claim 43, wherein Z comprises at least 10 repeats of D-glutamic acid amino acid residue (DE 10).

57. The pharmaceutical composition of any one of claims 43, 44 and 46, wherein Z comprises at least 15 repeated D-glutamic acid amino acid residues (DE 15) or at least 20 repeated D-glutamic acid amino acid residues (DE 20).

58. The pharmaceutical composition of any one of claims 43, 44, 46 and 56, wherein:

y is a non-releasable linker; and is also provided with

Z is DE20.

59. The pharmaceutical composition of any one of claims 43, 44, 46 and 56, wherein Y is an unreleasable linker comprising at least one carbon-carbon bond, at least one amide bond, or at least one carbon-carbon bond and at least one amide bond.

60. The pharmaceutical composition of any one of claims 43, 44, 46 and 56, wherein Y is a releasable linker.

61. The pharmaceutical composition of any one of claims 43, 44, 46 and 56, wherein Y is a releasable linker comprising at least one disulfide bond, at least one ester bond, at least one protease-specific amide bond, or a combination of the foregoing.

62. The pharmaceutical composition of claim 43, wherein the compound has at least 75% or more sequence identity, at least 85% or more sequence identity, at least 90% or more sequence identity, or at least 95% or more sequence identity to SEQ ID NO. 1.

63. The pharmaceutical composition of claim 43, wherein the compound has at least 75% or more sequence identity, at least 85% or more sequence identity, at least 90% or more sequence identity, or at least 95% or more sequence identity to SEQ ID NO. 2.

64. A method of locating a therapeutic or diagnostic agent at a spinal fusion site in a patient, the method comprising administering to the patient a therapeutically effective amount of a compound having the structure of formula (I):

X-Y-Z

the compound of formula (I),

or a pharmaceutically acceptable salt thereof,

wherein:

x is a therapeutic or diagnostic agent for treating spinal fusion in the patient;

y is absent or a linker; and is also provided with

Z is a osteogenic ligand.

65. The method of claim 64, wherein Y is a releasable linker or a non-releasable linker.

66. The method of claim 64, wherein Y is a non-releasable linker.

67. The method of claim 64 or 65, wherein X is a diagnostic agent that is a fluorescent dye.

68. The method of claim 64, wherein X is a therapeutic agent for treating spinal fusion, said therapeutic agent being a bone anabolic agent.

69. The method of claim 64, wherein Z is an Acidic Oligopeptide (AOP) or another hydroxyapatite binding molecule.

70. The method of claim 64, wherein Z is an AOP comprising at least 4 amino acid residues.

71. The method of any one of claims 64-66 and 68-70, wherein X is a therapeutic agent that is abaclotide, integrin 5β1 (ITGA), dasatinib, parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), or a derivative or fragment of any of the foregoing that has bone anabolic activity.

72. The method of any one of claims 64-66 and 68-70, wherein X is a therapeutic agent that is PTH, PTHrP, or a derivative or fragment of any of the foregoing that has bone anabolic activity.

73. The method of any one of claims 64-66 and 68-70, wherein X is a therapeutic agent that is abaclotide or a derivative or fragment thereof having bone anabolic activity.

74. The method of any one of claims 64-66 and 68-70, wherein Z is a linear chain of amino acid residues.

75. The method of any one of claims 64-66 and 68-70, wherein Z comprises 4 to 20D-glutamic acid amino acid residues, 4 to 20D-aspartic acid amino acid residues, or 4 to 20D-glutamic acid amino acid residues and 4 to 20D-aspartic acid amino acid residues.

76. The method of claim 64, wherein Z comprises at least 10 repeated D-glutamic acid amino acid residues (DE 10).

77. The method of any one of claims 64-66, 68-70 and 76, wherein Z comprises at least 15 repeated D-glutamic acid amino acid residues (DE 15) or at least 20 repeated D-glutamic acid amino acid residues (DE 20).

78. The method of any one of claims 64-66, 68-70 and 76, wherein X is a therapeutic agent that is abaclotide or a derivative or fragment thereof having bone anabolic activity, and Z is DE20.

79. The method of claim 64, wherein the compound has at least 75% or more sequence identity, at least 85% or more sequence identity, at least 90% or more sequence identity, or at least 95% or more sequence identity to SEQ ID No. 1.

80. The method of claim 64, wherein the compound has at least 75% or more sequence identity, at least 85% or more sequence identity, at least 90% or more sequence identity, or at least 95% or more sequence identity to SEQ ID No. 2.