Compositions and methods for treating tendon and bone injuries

Abstract

In one embodiment, the present invention relates to synthetic molecules, compositions, and pharmaceutical compositions useful for delivering bioactive portions to the site of injury. Also disclosed are methods for treating tendon injuries, fractures, bone stress injuries, enhancing the integration of allograft tissue during or after orthopedic surgery, promoting the repair or regeneration of tendon attachment tissue, and methods for treating osteoporosis, osteoarthritis, and / or cartilage defects using the disclosed molecules, compositions, and pharmaceutical compositions.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 500,463, filed on 5 May 2023, which is incorporated herein by reference in its entirety.

[0002] Statement regarding federally funded research This invention was made with government support under IK2BX005199, granted by the U.S. Department of Veterans Affairs. The government has certain rights in this invention.

[0003] Sequence listing statement The sequence listing, created on March 3, 2024, and submitted on March 3, 2024, as a text file named "37759_0536P1_SL.xml" with a size of 5,433 bytes, is incorporated herein by reference in accordance with 37 C. FR §1.52(e)(5). [Background technology]

[0004] In the United States, there are 32 million musculoskeletal injuries annually, 45% of which involve tendons or ligaments, such as rotator cuff tears and anterior cruciate ligament (ACL) tears. 1 These two common injuries are often treated surgically and constitute a significant healthcare burden in the United States. Approximately 250,000 randomized controlled trials (RCTs) are performed annually in the U.S. alone, and it is estimated that successful rotator cuff repairs in the U.S. would result in $3.44 billion in social savings over a lifetime. 2;3 Unfortunately, on average, 12% to 40% of rotator cuff repairs fail to heal, and it has been reported that up to 94% of repairs fail if the rotator cuff tear is larger than 2 cm or occurs in patients over 60 years of age. 3~5This is a noteworthy issue because both of these factors are commonly encountered in clinical practice. A key feature associated with this observed high rate of tendon repair failure is that, currently, tendon-to-bone repair heals through the formation of unorganized fibrous tissue that fails to restore the natural tissue architecture, and this abnormal repair tissue is structurally and mechanically inferior to the natural tendon and tendon attachment tissue. 1;6~8

[0005] Generally, the biology of tissue regeneration involves cells, the extracellular matrix (ECM), and growth factors. Considering the proximity of cortical detachment bone, periosteum, bursa, and often synovial tissue during tendon-bone repair, cells are relatively abundant, and the available pool of local mesenchymal progenitor cells, when adequately exposed to various trophic factors, can reliably proliferate and produce the ECM. These include bone morphogenesis proteins (BMPs) or transforming growth factor-beta (TGFβ). 8~15 Certain growth factors have been reported to improve both the biomechanical strength and tissue organization of repaired tendon attachments, but their clinical applicability remains severely limited due to the lack of effective delivery strategies.

[0006] Delivering growth factors to the site of tendon repair is an attractive strategy for inducing local progenitor cell migration and triggering tissue regeneration. Current clinical approaches to growth factor delivery are limited to hyperphysiological doses administered at a macroscopic tissue level at a single point in time (i.e., intraoperative delivery). Unfortunately, there are no current methodologies to effectively deliver growth factors at physiologically relevant levels at the cellular level, to target delivery to the site of surgical repair, and to deliver growth factors across multiple time points.

[0007] What is needed are molecules, compositions, and pharmaceutical compositions, as well as methods for targeting molecules to the site of injury or surgical repair. Methods for treating tendon injuries, fractures, bone cuts, and bone stress injuries are also needed. [Overview of the project]

[0008] This specification describes molecules, compositions, and pharmaceutical compositions, as well as methods for targeting molecules to injury sites. Furthermore, this specification describes molecules, compositions, and pharmaceutical compositions for treating tendon injuries, fractures, and / or bone stress injuries. Also, this specification describes molecules, compositions, and pharmaceutical compositions for enhancing allograft tissue integration and promoting tendon attachment repair or regeneration during or after orthopedic surgery.

[0009] This specification discloses a molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety (BP), wherein the BP moiety has a PCP structure, and the cleavable linker links the bioactive moiety to the BP moiety.

[0010] This specification discloses a composition comprising a molecule, the molecule comprising: ii) a bioactive moiety; ii) a cleavable linker; and iii) a bisphosphonate moiety (BP), wherein the BP moiety has a PCP structure, and the cleavable linker links the bioactive moiety to the BP moiety.

[0011] This specification discloses a pharmaceutical composition comprising a molecule, the molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety (BP), wherein the BP moiety has a PCP structure, and the cleavable linker links the bioactive moiety to the BP moiety.

[0012] This specification discloses a method for targeting a molecule to a site of injury, comprising administering the molecule, wherein the molecule comprises i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety (BP), the BP moiety having a PCP structure, and the cleavable linker linking the bioactive moiety to the BP moiety.

[0013] This specification discloses a method for treating tendon injuries, fractures, and / or bone stress injuries and / or promoting the healing of soft tissue to bone, comprising administering a molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety (BP), wherein the BP moiety has a PCP structure, and the cleavable linker links the bioactive moiety to the BP moiety.

[0014] This specification discloses a method for enhancing the integration of allograft tissue during or after orthopedic surgery, comprising administering a molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety (BP), wherein the BP moiety has a PCP structure, and the cleavable linker links the bioactive moiety to the BP moiety.

[0015] This specification discloses a method for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery, comprising administering a molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety (BP), wherein the BP moiety has a PCP structure, and the cleavable linker links the bioactive moiety to the BP moiety.

[0016] This specification discloses a method for treating osteoporosis, osteoarthritis, and / or cartilage defects, comprising administering a molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety (BP), wherein the BP moiety has a PCP structure, and the cleavable linker links the bioactive moiety to the BP moiety.

[0017] The accompanying drawings incorporated herein and constituting part thereof illustrate several aspects of the disclosed methods and compositions and, together with the description, serve to illustrate the principles of the disclosed methods and compositions. [Brief explanation of the drawing]

[0018] [Figure 1]A and B are diagrams depicting the mechanism of action of the BP-F-Q(OSF-3) molecule. A shows that the administered BP-F-Q localizes to the bone surface exposed at the tendon (T) repair site. In the absence of Ctsk, fluorescence from the fluorophore (F) is suppressed by the covalently linked quenching dye (Q). B shows that, in the presence of locally activated osteoclasts, Ctsk at the site of tendon repair specifically cleaves the linker peptide (blue dashed line), enabling detection of the external fluorescence signal from F. [Figure 2] A - D are photographs showing the surgical method. A shows the placement of the transosseous and trans-tendon sutures. B shows a sharp transverse section of the Achilles tendon at the bone attachment. C shows the posterior superior calcaneal bar for periosteal elevation. D shows the repair of the transected tendon to the periosteal elevation calcaneus. [Figure 3A] Graph showing the mean fluorescence on-target signal (A) and off-target signal (B) over time among the systemic, local, and sham-treated groups (n = 7 per group) that received AF647-ZOL. Error bars represent the standard deviation from the mean. Both ** and * indicate significant differences between the sham group and the local group (p < 0.01, p < 0.05), respectively. Both ## and # indicate significant differences between the sham group and the systemic group (p < 0.01, p < 0.05), respectively. [Figure 3B] Graph showing the mean fluorescence on-target signal (A) and off-target signal (B) over time among the systemic, local, and sham-treated groups (n = 7 per group) that received AF647-ZOL. Error bars represent the standard deviation from the mean. Both ** and * indicate significant differences between the sham group and the local group (p < 0.01, p < 0.05), respectively. Both ## and # indicate significant differences between the sham group and the systemic group (p < 0.01, p < 0.05), respectively. [Figure 4] A - C show representative images of the fluorescence signal at POD10 among animals treated with AF647-ZOL. [Figure 5A]Graph showing the mean fluorescence on-target signal (A) and off-target signal (B) over time between the repair (whole body) and sham treatment groups (n = 3 per group) that received Cat K 680 FAST. Error bars represent the standard deviation from the mean. ** and * indicate significant differences between the sham and repair groups, respectively (p < 0.01, p < 0.05). [Figure 5B] Graph showing the mean fluorescence on-target signal (A) and off-target signal (B) over time between the repair (whole body) and sham treatment groups (n = 3 per group) that received Cat K 680 FAST. Error bars represent the standard deviation from the mean. ** and * indicate significant differences between the sham and repair groups, respectively (p < 0.01, p < 0.05). [Figure 6] A and B are representative images of the fluorescence signal at POD4 between sham (B) and Achilles tendon repair (A) animals treated with Cat K 680 FAST. [Figure 7] A - C show representative in vivo fluorescence imaging at 10 days post - surgery for the local (A), whole body (B), and sham (C) treatment groups. Note the hindfoot on - target signal in repaired local and whole body treated animals, and the forefoot off - target signal in sham animals. [Figure 8A] Graph showing the mean fluorescence on - target signal (A) and off - target signal (B) over time between the whole body, local, and sham treatment groups (n = 7 per group) that received OFS - 3. Error bars represent the standard deviation from the mean. Both ** and * indicate significant differences between the sham and local groups, respectively (p < 0.01, p < 0.05). Both ## and # indicate significant differences between the sham and whole body groups, respectively (p < 0.01, p < 0.05). [Figure 8B]This graph shows the mean on-target (A) and off-target (B) fluorescence signals over time in the systemic, local, and sham treatment groups (n=7 per group) that received OFS-3. Error bars represent the standard deviation from the mean. Both ** and * indicate significant differences between the sham and local groups, respectively (p<0.01, p<0.05). Both ## and # indicate significant differences between the sham and systemic groups, respectively (p<0.01, p<0.05). [Figure 9] A–D show hindfoot sections stained with H&E and SOFG (A) and immunohistochemistry for Ctsk (B) in normal, uninjured mice, as well as hindfoot sections stained with H&E and SOFG (C) and immunohistochemistry for Ctsk (D) in POD4 after Achilles tendon-to-bone repair, at 4x magnification. # indicates the distal Achilles tendon immediately proximal to the calcaneal tendon attachment (A, B).} indicates normal cortical and cancellous bone in the posterior superior region of the calcaneus (A, B). Arrows indicate the resected remaining proximal stump of the repaired Achilles tendon with Ctsk-positive IHC staining (C, D). * indicates the suture hole created in the posterior region of the calcaneus during repair. [] indicates the cortical detachment area of ​​the posterior superior calcaneus with prominent Ctsk-positive IHC staining (C, D). [Figure 10] A and B show mouse hindfoot sections at 4x magnification in POD7 (A) and POD28 (B) immunostained for Ctsk. Note the absence of residual Achilles tendon (RAT) and osteoclast activity in POD28 (B). Suture fragment contained within this tissue section in S=POD7 (A). Scale bar: 0.5 mm. Arrows indicate osteoclast resorption of bone at the site of tendon-bone repair. The area between [] indicates reparative tissue at the tendon-bone junction (A and B). { identifies the area of ​​osteoclast resorption in the posterior calcaneus, which may occur due to changes in hindfoot weight-bearing in the first week of recovery after Achilles tendon repair treatment. [Figure 11]This bar graph shows the activation of BMP receptors (measured by measuring bone response element (BRE) expression) in cells treated with (1) phosphate-buffered saline (control), (2) rhBMP-2, (3) variant BMP-2, (4) Ctsk-digested BP-vBMP-2, or (5) undigested BP-vBMP-2. Luciferase expression from BRE plasmids was detected and normalized to control Rinella plasmid luciferase expression. [Modes for carrying out the invention]

[0019] The methods and compositions disclosed may be more readily understood by referring to the following detailed descriptions of specific embodiments and the examples contained herein, as well as the drawings and their preceding and succeeding descriptions.

[0020] The methods and compositions disclosed are not limited to specific synthesis methods, analytical techniques, or reagents unless otherwise specified, and should therefore be understood to be subject to change. Furthermore, the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them.

[0021] A.Definition The terms used herein are for the purpose of describing specific aspects and are not intended to be limiting.

[0022] As used herein and in the appended claims, the singular forms "a," "an," and "the" can include multiple references unless the context clearly indicates otherwise. Thus, for example, a reference to "compound" includes a mixture of compounds, a reference to "pharmaceutical carrier" includes a mixture of two or more such carriers, and so on.

[0023] As used herein, the term "or" means any one member of a given list, including any combination of members of that list.

[0024] A range may be expressed herein as "approximately" from a certain value and / or "approximately" to another specific value. The term "approximately" is used herein to mean roughly, within that area, roughly, or around it. When the term "approximately" is used in conjunction with a numerical range, it modifies that range by broadening the upper and lower boundaries of the stated numerical value. Generally, the term "approximately" is used herein to modify a numerical value with a 20% variation above and below the stated value. Where such a range is expressed, another embodiment includes "from that certain value and / or to another specific value." Similarly, when a value is expressed as an approximation using the antecedent "approximately," it will be understood that a particular value forms another embodiment. It will be further understood that each endpoint of a range is important, whether related to or independent of the other endpoints.

[0025] As used herein, the term “amino acid sequence” refers to a list of abbreviations, letters, symbols, or words representing amino acid residues. The amino acid abbreviations used herein are conventional one-letter codes for amino acids and are expressed as follows: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine. All alpha-amino acids except glycine can exist as either two enantiomers, called L-amino acids or D-amino acids, which are mirror images of each other.

[0026] As used herein, the term “amino acid” refers to an organic molecule having a basic amino group (-NH2), an acidic carboxyl group (-COOH), a proton, and a variable “R” group bonded to an sp3 hybridized central carbon atom. The N-terminus of a peptide has a free amino group (-NH2). The C-terminus of a peptide has a free carboxyl group (-COOH).

[0027] As used herein, the terms “D-amino acid” or “D-type amino acid” refer to an amino acid in which the carbon alpha of the stereocenter relative to the amino group has a D configuration. D-amino acids are enantiomers of amino acids in which plane-polarized light can be rotated clockwise (to the right). D-amino acids are denoted by the prime (') symbol.

[0028] As used herein, the terms “L-amino acid” or “L-type amino acid” refer to an amino acid in which the carbon alpha of the stereocenter relative to the amino group has an L configuration. L-amino acids are enantiomers of amino acids that can rotate plane-polarized light counterclockwise (left-handed). With the exception of achiral glycine, natural amino acids have an L configuration.

[0029] As used herein, “peptide” refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A peptide is composed of a sequence of amino acids. The term “peptide” encompasses molecules that are naturally occurring or synthetic. The residues of a peptide are amino acids. As used herein, when an amino acid is said to be in the fifth position, it is the fifth amino acid from the N-terminus of the peptide.

[0030] A "fragment" means, but is not limited to, a portion of a polypeptide or nucleic acid molecule, such as a cleavage variant. This portion preferably contains at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the total length of the reference nucleic acid molecule or polypeptide. For example, a polypeptide fragment may contain about 5, about 10, about 15, about 20, about 25, or about 30 or more amino acids. In another example, a polypeptide fragment may contain about 1000, about 1500, about 2000, about 2500, or about 3000 or more amino acids.

[0031] As used herein, the term “epitope” refers to a localized area on the surface of an antigen that can trigger an immune response.

[0032] As used herein, “Sample” means a solution containing one or more molecules derived from an animal, tissue or organ from an animal, cells (either within the subject, directly collected from the subject, maintained in a culture, or from a cultured cell line), cell lysates (or lysate fractions) or cell extracts, or cells or cellular materials (e.g., polypeptides or nucleic acids), which are assayed as described herein. A sample may also be any bodily fluid or excrete containing cells or cellular components (e.g., blood, urine, feces, saliva, tears, bile, etc.).

[0033] As used herein, “subject” refers to the target of administration, for example, an animal. Therefore, the subject of the disclosed method may be a vertebrate, such as a mammal. For example, the subject may be a human. This term does not indicate a specific age or sex. “Subject” may be used interchangeably with “individual” or “patient.”

[0034] Substantial changes in functional or immunological identity are achieved by selecting substitutions that are less conserved than those in Table 1, i.e., by selecting residues that have a more pronounced difference in (a) the structure of the polypeptide backbone in the region of the substitution as a sheet or helix, (b) the molecular charge or hydrophobicity at the target site, or (c) their effect on maintaining the bulk of the side chains. Generally, the substitutions expected to bring about the greatest change in protein properties are as follows: (a) a hydrophilic residue, e.g., ceryl or threonyl, is replaced by (or thereby) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl, tryptophan, or tyrosinyl; (b) cysteine ​​or proline is replaced by (or thereby) any other residue; (c) a residue with an electronegative side chain, e.g., lysyl, arginyl, or histidyl, is replaced by (or thereby) an electronegative residue, e.g., glutamyl or aspartyl; (d) a residue with a bulky side chain, e.g., phenylalanine, is replaced by (or thereby) a residue without a side chain, e.g., glycine in this case; or (e) an increase in the number of sites for sulfated and / or glycosylated residues. [Table 1] [Table 2]

[0035] It should be understood that one way of defining protein variants and derivatives disclosed herein is to define them in terms of homology / identity to a particular known sequence. Specifically disclosed are peptide variants disclosed herein that have at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to the peptides specifically enumerated herein. Those skilled in the art will readily understand how to determine the identity of two proteins.

[0036] Polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques well known in the art. Modifications can occur anywhere on the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. The same type of modification may be present at several sites on a given polypeptide, to the same or varying degrees. Furthermore, a given polypeptide can have many different types of modifications. Modifications include, without limitation, the transfer of amino acids to proteins—RNA-mediated additions—such as acetylation, acylation, ADP-ribosylation, amidation, covalent crosslinking or cyclization, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine ​​or pyroglutamates, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristolyation, oxidation, PEGylation, proteolysis, phosphorylation, prenylation, racemization, selenoylation, sulfated, and arginylation. (See *Proteins-Structure and Molecular Properties*, 2nd Ed., TECreighton, WH Freeman and Company, New York (1993); and *Posttranslational Covalent Modification of Proteins*, BC Johnson, Ed., Academic Press, New York, pp. 1-12 (1983).)

[0037] As used herein, “osteochondrogenic factors” refers to biological compounds that promote the repair and / or regeneration of bone and / or cartilage. Osteochondrogenic factors include, but are not limited to, NELL-1, TGF-β1, TGF-β2, TGF-β3, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-11, BMP-15, or rhBMP.

[0038] As used herein, “osteogenesis factors” refers to biological compounds that promote the formation of new bone and / or new cartilage. Buser Z et al. Eur Spine J. 2017 Nov;26(11):2763-2772. Osteogenesis factors include, but are not limited to, oxy-133 and oxysterol-133.

[0039] As used herein, “tendon morphogenetic factors” refers to biological compounds that promote the repair and / or regeneration of tendons. Tendon morphogenetic factors include, but are not limited to, TGFβ-1, TGFβ-2, TGF-β3, FGF, CTGF, BMP-12, BMP-13, BMP-14, CCN1, or WISP-1.

[0040] As used herein, “ECM proteins” refers to proteins present in the extracellular matrix. The extracellular matrix is ​​a three-dimensional network of extracellular macromolecules and minerals (including hydroxyapatite) that provide structural and biochemical support to the surrounding cells. ECM proteins include, but are not limited to, collagen, elastin, fibrin, fibronectin, gelatin, laminin, integrin, CCN1, WISP-1, or CTGF.

[0041] As used herein, “tendon injury” refers to damage or inflammation of the connective tissue that connects muscles to bone. It may be caused by overuse, degeneration, or trauma. As used herein, treating a tendon injury includes promoting the healing of soft tissue into bone.

[0042] As used herein, “fracture” refers to a break in a bone.

[0043] As used herein, “bone stress injury” refers to an injury of overuse characterized by the progressive onset of pain due to activity. Examples of bone stress injuries include, but are not limited to, stress reactions and stress fractures. Bone stress injuries occur in response to repeated loading on the bone. Bone stress injuries are often seen as high signal intensity on MRI.

[0044] As used herein, “osteointegration of an implantable prosthesis” includes enhancing bone healing to an implant when the implant material (such as metal or ceramic) is embedded in bone or for dental purposes.

[0045] As used herein, “treating allograft bone integration” includes, but is not limited to, osteochondral allografts and osteoallografts.

[0046] As used herein, “osteoarthritis” includes, but is not limited to, degenerative cartilage damage within the joint or intervertebral disc space, including damage from abnormal bone remodeling. Treatment of osteoarthritis includes, but is not limited to, treatment of bone abnormalities in osteoarthritis, including subchondral bone and bone marrow edema, as well as cartilage abnormalities in osteoarthritis, because these abnormalities are associated with the bone and cartilage of osteoarthritis.

[0047] As used herein, “cartilage defect” includes, but is not limited to, an area of ​​damaged cartilage surrounded by relatively healthier cartilage. In some embodiments, a cartilage defect may be a defect or injury to joint or hyaline cartilage, fibrocartilage, or elastomer. A cartilage defect may be the result of traumatic mechanical damage or progressive mechanical damage. In some embodiments, a cartilage defect may be in a specific, localized area of ​​damage to cartilage.

[0048] As used herein, “dosage” or “amount of medication” refers to a specific amount of a therapeutic agent, such as a molecule, composition, or pharmaceutical composition, taken at a specific time.

[0049] As used herein, “to treat” means to administer one of the disclosed compositions to a subject such as a human or other mammal (e.g., an animal model) having atherosclerosis in order to prevent or delay the worsening of the effects of a disease or condition, or to partially or completely reverse the effects of a disease.

[0050] As used herein, “effective dose” means one or more of the molecules, compositions, or pharmaceutical compositions disclosed herein in an amount sufficient to produce the desired effect. For example, one or more effective doses of the molecules, compositions, or pharmaceutical compositions disclosed herein may be an amount that produces a therapeutic effect and a sustained therapeutic effect after discontinuation of treatment. One or more effective doses of the molecules, compositions, or pharmaceutical compositions disclosed herein may be an amount that can produce benefits exemplified by, for example, promoting the healing of tendon injuries, accelerating recovery time, improving the biomechanical properties (such as strength or toughness) of bone, cartilage, and / or tendons, and / or improving the organization of bone, cartilage, and / or tendon tissues, as well as an amount that enables a sustained therapeutic effect after discontinuation of the molecule, composition, or pharmaceutical composition. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease (or underlying genetic defect) being treated, the specific compound used, and its mode of administration. Therefore, it is impossible to determine an exact “effective dose.” However, a suitable “effective dose” can be determined by a person skilled in the art using only routine experiments.

[0051] As used herein, the term “nucleic acid” refers to naturally occurring or synthetic oligonucleotides or polynucleotides that enable hybridization to complementary nucleic acids by Watson-Crick base pairing, whether DNA or RNA, or DNA-RNA hybrids, single-stranded or double-stranded, sense or antisense. The nucleic acids of the present invention may also include nucleotide analogs (e.g., BrdU) and non-phosphodiester nucleoside linkages (e.g., peptide nucleic acids (PNA) or thiodiester links). In particular, nucleic acids may include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0052] The terms “vector” or “construct” refer to a nucleic acid sequence capable of transporting another nucleic acid to a cell, to which the vector sequence is ligated. The term “expression vector” includes any vector (e.g., plasmid, cosmid, or phage chromosome) containing a gene construct in a form suitable for expression by cells (e.g., ligated to a transcriptional regulatory element). “Plasmid” and “vector” are used interchangeably because plasmids are the commonly used form of vectors. Furthermore, the present invention is intended to include other vectors that perform equivalent functions.

[0053] The term “expression vector” as used herein refers to a vector capable of directing the expression of a gene to which it is operably linked. Common expression vectors of utility in recombinant DNA techniques are often in plasmid form. Recombinant expression vectors may contain nucleic acids, such as those disclosed herein, in a form suitable for expression in host cells. In other words, a recombinant expression vector may contain one or more regulatory elements or promoters, which can be selected based on the host cell used for expression, operably linked to the nucleic acid sequence to be expressed.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the disclosed methods and compositions belong. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the methods and compositions, but particularly useful methods, devices, and materials are those described herein. The documents cited herein and the materials they cite are incorporated herein specifically by reference. Nothing herein should be construed as an admission that the present invention does not have prior rights to such disclosure by prior art. No reference is permitted to constitute prior art. The consideration of references describes what the authors assert, and the applicant reserves the right to object to the accuracy and appropriateness of the cited documents. While several documents are mentioned herein, it should be clearly understood that such references do not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0055] B. Composition 1. Molecules Non-limiting examples of the molecules of this disclosure are provided herein.

[0056] This specification discloses a molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety, wherein the BP moiety has a PCP structure, and the cleavable linker links the bioactive moiety to the BP moiety.

[0057] i. Biologically active moiety As used herein, “bioactive moiety” means a portion of a molecule that has or can induce an action in the body of the subject, or a physiological or pharmacological response in the body of the subject.

[0058] In some embodiments, the bioactive moiety is a bone chondrocyte factor, bone morphogenetic factor, tendon morphogenetic factor, or extracellular matrix (ECM) protein. In some embodiments, the ECM protein is collagen, elastin, fibrin, fibronectin, gelatin, laminin, integrin, CCN1, WISP-1, or CTGF.

[0059] In some embodiments, the bioactive moiety is an osteochondral factor, which is NELL-1, TGF-β1, TGF-β2, TGF-β3, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-11, BMP-15, or rhBMP. In some embodiments, the osteochondral factor is NELL-1 or BMP-2.

[0060] In some embodiments, the bioactive moiety is a bone morphogenetic factor, which is oxy-133 or oxysterol-133.

[0061] In some embodiments, the bioactive moiety is a tendinogenesis factor, which is TGFβ-1, TGFβ-2, TGFβ3, FGF, CTGF, BMP-12, BMP-13, BMP-14, CCN1, or WISP-1. In some embodiments, the tendinogenesis factor is TGFβ-1 or TGFβ-2.

[0062] In some embodiments, the bioactive portion is a hormone. In some embodiments, the hormone is parathyroid hormone (e.g., teriparatide, Natpara). In some embodiments, the bioactive portion is a parathyroid hormone-related protein.

[0063] In some embodiments, the bioactive portion is a growth differentiation factor. In some embodiments, the growth differentiation factor is bone morphogenetic protein 11 (BMP-11).

[0064] In some embodiments, the bioactive moiety is activin receptor-like kinase-1-Fc (ALK1-Fc, dalantercept). ALK1-Fc is a chimeric protein having an ALK1 receptor domain combined with the Fc portion of human IgG, and it acts as a ligand trap for ALK1 ligands BMP9 and BMP10.

[0065] In some embodiments, the bioactive moiety is activin receptor-like kinase-4-Fc (ALK4-Fc). ALK4-Fc is a chimeric protein having an ALK4 receptor domain combined with the Fc portion of human IgG, and it acts as a ligand trap for the ALK4 ligands activin, GDF8, and GDF11.

[0066] ii. Cuttable linker As used herein, “cleavable linker” means a peptide linker that can be cleaved. In some embodiments, a cleavable linker can be enzymatically cleaved (i.e., an enzymatically cleavable linker). In some embodiments, a cleavable linker can be a protease-sensitive peptide linker, an acid-sensitive hydrazone linker, or a glutathione-sensitive disulfide linker. Examples of cleavable linkers include, but are not limited to, peptides that can be cleaved by the enzyme cathepsin K (cathepsin K-sensitive linker) or the matrix metalloproteinase 2 enzyme (MMP linker). Cleavable linkers may also include peptide linkers that are substrates for other matrix metalloproteinase enzymes such as MMP-1, MMP-3, MMP-9, MMP-10, and MMP-13. Examples of cleavable linkers include, but are not limited to, the cleavable linkers listed in Table 3. [Table 3]

[0067] Also disclosed herein are variants or derivatives of the cleavable linkers disclosed herein. Where used herein, the term “analog” is interchangeable with “variant” and “derivative.” Variants and derivatives are well understood by those skilled in the art and may involve amino acid sequence modifications. Such amino acid sequence modifications typically fall into one or more of three classes: substantial, insertion, or deletion variants. Insertions include fusions of amino-terminuses and / or carboxyl-terminuses, as well as intrasequence insertions of single or multiple amino acid residues. Insertions are usually smaller than insertions of amino-terminuses or carboxyl-terminuses, typically consisting of, for example, 1 to 4 residues. These variants are usually prepared by site-directed mutagenesis of nucleotides in protein-coding DNA, thereby producing variant-coding DNA, which is then expressed in recombinant cell cultures. Techniques for producing substitutional mutations at predetermined sites in DNA with known sequences, such as M13 primer mutagenesis and PCR mutagenesis, are well known. Amino acid substitutions are typically single-residue, but can occur at several different positions simultaneously. Substitutions, deletions, insertions, or any combination thereof can lead to the final derivative or analogue. A substituted variant is one in which at least one residue is removed and a different residue is inserted in its place. Such substitutions are generally made according to Tables 1 and 2 and are referred to as conservative substitutions.

[0068] In some embodiments, the cleavable linker is a cathepsin K-sensitive peptide linker. In some embodiments, the cathepsin K-sensitive peptide linker is GHPGGPQG (SEQ ID NO: 1) or GGGMGPSGPWGGK (SEQ ID NO: 2).

[0069] In some embodiments, the severable linker is an MMP severable linker. In some embodiments, the MMP severable linker is PLGLAG (SEQ ID NO: 3), or Ac-GCRD-GPQGIWGQ-DRCG (SEQ ID NO: 4), or Ac-GCRD-GPQGIAGQ-DRCG (SEQ ID NO: 5).

[0070] In some embodiments, the cleavable linker is a pH-sensitive linker. A pH-sensitive linker may be one found in US8,063,209, US11,219,697, and US11,840,549, all of which are incorporated herein by reference in their entirety. A pH-sensitive linker may also be found, for example, in US10,383,912 and US10,688,193, both of which are incorporated herein by reference in their entirety.

[0071] iii. Bisphosphonate portion As used herein, “bisphosphonate moiety” refers to a molecule characterized by two CP bonds. When the two CP bonds share a single carbon atom (PCP), they are considered analogs of pyrophosphates (POPs) and are called geminal bisphosphonates (so named because the carbon is in a central or geminal position). The PCP bonds of geminal bisphosphonates are stable to heat and most chemical reagents and are completely resistant to enzymatic hydrolysis. In some embodiments, “bisphosphonate moiety” refers to a molecule (PCP structure) having two phosphate ions linked by a carbon atom. Bisphosphonates are analogs of pyrophosphates containing carbon instead of oxygen atoms. A single PCP structure can allow for a number of possible deformations, particularly by altering the two lateral chains on the carbon atom. In some embodiments, “bisphosphonate moiety” will exhibit high affinity for hydroxyapatite (HAP) calcium-phosphate minerals exposed in the ECM of bone and will preferentially bind to newly reabsorbed bone surfaces. Some bisphosphonates, such as etidronate and methylhydroxyl diphosphonate, do not inhibit osteoclast function. Generally, nitrogen-containing bisphosphonates inhibit osteoclasts, while nitrogen-free bisphosphonates do not. See USPN 11,400,104 and Hokugo A et al. Bone. 2019 Jun;123:115-128. In some embodiments, the BP portion does not sufficiently or completely inhibit osteoclast function, or inhibits it only minimally. In some embodiments, BP only minimally inhibits farnesyl pyrophosphate synthase (FPSS).In some embodiments, the BP portion is 2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-PyrEBP), 1-hydroxy-2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS), methylene bisphosphonate (MBP), methylene hydroxyl bisphosphonate (MHDP), etidronate (EHDP), methyl hydroxyl diphosphonate, clodronate, isclodronate, tildronate, 2-hydroxy-2-phosphono-3-(pyridine-3-yl)propanoic acid (3-PEHPC), or 2-hydroxy-3-(imidazo[1,2-c]pyridine-3-yl)-2-phosphonopropanoic acid (3-IP-EHPC).

[0072] 2. Composition This specification discloses a composition comprising a molecule, the molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety, wherein the BP moiety has a PCP structure, and the cleavable linker links the bioactive moiety to the BP moiety.

[0073] i. Biologically active moiety In some embodiments, the bioactive moiety is a bone chondrocyte factor, bone morphogenetic factor, tendon morphogenetic factor, or extracellular matrix (ECM) protein. In some embodiments, the ECM protein is collagen, elastin, fibrin, fibronectin, gelatin, laminin, integrin, CCN1, WISP-1, or CTGF.

[0074] In some embodiments, the bioactive moiety is an osteochondral factor, which is NELL-1, TGF-β1, TGF-β2, TGF-β3, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-11, BMP-15, or rhBMP. In some embodiments, the osteochondral factor is NELL-1 or BMP-2.

[0075] In some embodiments, the bioactive moiety is a bone morphogenetic factor, which is oxy-133 or oxysterol-133.

[0076] In some embodiments, the bioactive moiety is a tendinogenesis factor, which is TGFβ-1, TGFβ-2, TGFβ3, FGF, CTGF, BMP-12, BMP-13, BMP-14, CCN1, or WISP-1. In some embodiments, the tendinogenesis factor is TGFβ-1 or TGFβ-2.

[0077] In some embodiments, the bioactive moiety is a bioactive moiety and is a parathyroid hormone, a parathyroid hormone-related protein, a growth factor 11 (GDF11), an activin receptor-like kinase-1-Fc (ALK1-Fc), or an activin receptor-like kinase-4-Fc (ALK4-Fc).

[0078] ii. Cuttable linker In some embodiments, the cleavable linker is a cathepsin K-sensitive peptide linker. In some embodiments, the cathepsin K-sensitive peptide linker is GHPGGPQG (SEQ ID NO: 1) or GGGMGPSGPWGGK (SEQ ID NO: 2).

[0079] In some embodiments, the severable linker is an MMP severable linker. In some embodiments, the MMP severable linker is PLGLAG (SEQ ID NO: 3), or Ac-GCRD-GPQGIWGQ-DRCG (SEQ ID NO: 4), or Ac-GCRD-GPQGIAGQ-DRCG (SEQ ID NO: 5).

[0080] In some embodiments, the cleavable linker is a pH-sensitive linker. A pH-sensitive linker may be one found in US8,063,209, US11,219,697, and US11,840,549, all of which are incorporated herein by reference in their entirety. A pH-sensitive linker may also be found, for example, in US10,383,912 and US10,688,193, both of which are incorporated herein by reference in their entirety.

[0081] iii. Bisphosphonate portion In some embodiments, the BP portion does not sufficiently or completely inhibit osteoclast function, or inhibits it only minimally. In some embodiments, BP only minimally inhibits farnesyl pyrophosphate synthase (FPSS). In some embodiments, the BP portion is 2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-PyrEBP), 1-hydroxy-2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS), methylene bisphosphonate (MBP), methylene hydroxyl bisphosphonate (MHDP), etidronate (EHDP), methyl hydroxyl diphosphonate, clodronate, isoclodronate, tildronate, 2-hydroxy-2-phosphono-3-(pyridine-3-yl)propanoic acid (3-PEHPC), or 2-hydroxy-3-(imidazo[1,2-c]pyridine-3-yl)-2-phosphonopropanoic acid (3-IP-EHPC).

[0082] 3. Pharmaceutical composition This specification discloses a pharmaceutical composition comprising a molecule, the molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety, wherein the BP moiety has a PCP structure, and the cleavable linker links the bioactive moiety to the BP moiety.

[0083] i. Biologically active moiety In some embodiments, the bioactive moiety is a bone chondrocyte factor, bone morphogenetic factor, tendon morphogenetic factor, or extracellular matrix (ECM) protein. In some embodiments, the ECM protein is collagen, elastin, fibrin, fibronectin, gelatin, laminin, integrin, CCN1, WISP-1, or CTGF.

[0084] In some embodiments, the bioactive moiety is an osteochondral factor, which is NELL-1, TGF-β1, TGF-β2, TGF-β3, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-11, BMP-15, or rhBMP. In some embodiments, the osteochondral factor is NELL-1 or BMP-2.

[0085] In some embodiments, the bioactive moiety is a bone morphogenetic factor, which is oxy-133 or oxysterol-133.

[0086] In some embodiments, the bioactive moiety is a tendinogenesis factor, which is TGFβ-1, TGFβ-2, TGFβ3, FGF, CTGF, BMP-12, BMP-13, BMP-14, CCN1, or WISP-1. In some embodiments, the tendinogenesis factor is TGFβ-1 or TGFβ-2.

[0087] In some embodiments, the bioactive moiety is a bioactive moiety and is a parathyroid hormone, a parathyroid hormone-related protein, a growth factor 11 (GDF11), an activin receptor-like kinase-1-Fc (ALK1-Fc), or an activin receptor-like kinase-4-Fc (ALK4-Fc).

[0088] ii. Cuttable linker In some embodiments, the cleavable linker is a cathepsin K-sensitive peptide linker. In some embodiments, the cathepsin K-sensitive peptide linker is GHPGGPQG (SEQ ID NO: 1) or GGGMGPSGPWGGK (SEQ ID NO: 2).

[0089] In some embodiments, the severable linker is an MMP severable linker. In some embodiments, the MMP severable linker is PLGLAG (SEQ ID NO: 3), or Ac-GCRD-GPQGIWGQ-DRCG (SEQ ID NO: 4), or Ac-GCRD-GPQGIAGQ-DRCG (SEQ ID NO: 5).

[0090] In some embodiments, the cleavable linker is a pH-sensitive linker. A pH-sensitive linker may be one found in US8,063,209, US11,219,697, and US11,840,549, all of which are incorporated herein by reference in their entirety. A pH-sensitive linker may also be found, for example, in US10,383,912 and US10,688,193, both of which are incorporated herein by reference in their entirety.

[0091] iii. Bisphosphonate portion In some embodiments, the BP portion does not sufficiently or completely inhibit osteoclast function, or inhibits it only minimally. In some embodiments, BP only minimally inhibits farnesyl pyrophosphate synthase (FPSS). In some embodiments, the BP portion is 2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-PyrEBP), 1-hydroxy-2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS), methylene bisphosphonate (MBP), methylene hydroxyl bisphosphonate (MHDP), etidronate (EHDP), methyl hydroxyl diphosphonate, clodronate, isoclodronate, tildronate, 2-hydroxy-2-phosphono-3-(pyridine-3-yl)propanoic acid (3-PEHPC), or 2-hydroxy-3-(imidazo[1,2-c]pyridine-3-yl)-2-phosphonopropanoic acid (3-IP-EHPC).

[0092] "Pharmacologically acceptable," as is well known to those skilled in the art, means a material or carrier selected to minimize any degradation of the active ingredient and any adverse side effects in the subject. Examples of carriers include dimyristoyl phosphatidyl (DMPC), phosphate-buffered saline, or polybuoyant liposomes. For example, in the present invention, PG:PC:cholesterol:peptide or PC:peptide can be used as a carrier. Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. ARGennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make it isotonic. Other examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution can be about 5 to about 8 or about 7 to about 7.5. Further carriers include sustained-release preparations such as a semipermeable matrix of a solid hydrophobic polymer containing the composition, the matrix being in the form of molded articles, e.g., films, stents (implanted in blood vessels during angioplasty), liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferred, for example, depending on the route of administration and the concentration of the composition being administered. These are most typically standard carriers for drug administration to humans, including solutions such as sterile water, saline, and buffer solutions at physiological pH.

[0093] The pharmaceutical composition may also contain carriers, thickeners, diluents, buffers, preservatives, etc., as long as the intended activity of the molecules, compositions, or pharmaceutical compositions of the present invention is not impaired. The pharmaceutical composition may also contain one or more active ingredients (in addition to the compositions of the present invention), such as antimicrobial agents, anti-inflammatory agents, anesthetics, etc. The pharmaceutical composition may be administered in several ways, depending on whether topical or systemic treatment is desired and the area to be treated.

[0094] Parenteral administration preparations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffer media. Parenteral vehicles include sodium chloride solutions, ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixative oils. Intravenous vehicles include fluids and nutritional supplements, and electrolyte supplements such as those based on ringer's dextrose. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0095] Compositions for oral administration include powders or granules, suspensions or solutions in water or a non-aqueous medium, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersants, or binders may be desirable. Some compositions may potentially be administered as pharmaceutically acceptable acids or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, as well as organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, as well as organic bases such as mono, di, trialkyl, and arylamines, and substituted ethanolamines.

[0096] C. Method 1. Methods for targeting molecules to the site of injury This specification discloses a method for targeting a molecule to a site of injury in a subject requiring such targeting, comprising administering one of the disclosed molecules, compositions, or pharmaceutical compositions to the subject.

[0097] This specification discloses a method for targeting a molecule to a site of injury, comprising administering a molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety, wherein the BP moiety has a PCP structure and the cleavable linker links the bioactive moiety to the BP moiety.

[0098] In some embodiments of the methods disclosed herein for targeting molecules to a site of injury in subjects requiring such targeting, the site of injury is tendon attachment tissue. In some embodiments of the methods disclosed herein for targeting molecules to a site of injury in subjects requiring such targeting, the site of injury is the rotator cuff, distal biceps tendon, pectoralis major, patellar tendon, quadriceps tendon, or triceps tendon.

[0099] In some embodiments of methods disclosed herein for performing molecular targeting of injury sites in subjects requiring such targeting, the injury site is the surgical site, and the surgery is ACL reconstruction, PCL reconstruction, LCL or MCL repair / reconstruction, medial patellofemoral ligament reconstruction, meniscal root repair, meniscal allograft, or osteochondral allograft.

[0100] In some embodiments of methods disclosed herein for targeting molecules to the site of injury, the molecules are administered systemically. In some embodiments of methods disclosed herein for targeting molecules to the site of injury, the molecules are administered topically.

[0101] i. Biologically active moiety In some embodiments of methods disclosed herein for targeting molecules to the site of injury, the bioactive moiety is an osteochondral factor, osteogenic factor, tendon morphogenetic factor, or extracellular matrix (ECM) protein. In some embodiments of methods disclosed herein for targeting molecules to the site of injury, the bioactive moiety is an ECM protein, which is collagen, elastin, fibrin, fibronectin, gelatin, laminin, integrin, CCN1, WISP-1, or CTGF.

[0102] In some embodiments of methods disclosed herein for targeting molecules to the site of injury, the bioactive moiety is an osteochondral factor, which is NELL-1, TGF-β1, TGF-β2, TGF-β3, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-11, BMP-15, or rhBMP. In some embodiments of methods disclosed herein for targeting molecules to the site of injury, the osteochondral factor is NELL-1 or BMP-2.

[0103] In some embodiments of methods disclosed herein for targeting molecules to the site of injury, the bioactive moiety is an osteogenic factor, which is oxy-133 or oxysterol-133.

[0104] In some embodiments of the methods disclosed herein for targeting molecules to the site of injury, the bioactive moiety is a tendinogenic factor, which is TGFβ-1, TGFβ-2, TGFβ3, FGF, CTGF, BMP-12, BMP-13, BMP-14, CCN1, or WISP-1. In some embodiments, the tendinogenic factor is TGFβ-1 or TGFβ-2.

[0105] In some embodiments of methods disclosed herein for targeting molecules to the site of injury, the bioactive moiety is parathyroid hormone, parathyroid hormone-related protein, growth factor 11 (GDF11), activin receptor-like kinase-1-Fc (ALK1-Fc), or activin receptor-like kinase-4-Fc (ALK4-Fc).

[0106] ii. Cuttable linker In some embodiments of methods disclosed herein for targeting molecules to the site of injury, the cleavable linker is a cathepsin K-sensitive peptide linker. In some embodiments, the cathepsin K-sensitive peptide linker is GHPGGPQG (SEQ ID NO: 1) or GGGMGPSGPWGGK (SEQ ID NO: 2).

[0107] In some embodiments of methods disclosed herein for targeting molecules to the site of injury, the cleavable linker is an MMP cleavable linker. In some embodiments of methods disclosed herein for targeting molecules to the site of injury, the MMP cleavable linker is PLGLAG (SEQ ID NO: 3), or Ac-GCRD-GPQGIWGQ-DRCG (SEQ ID NO: 4), or Ac-GCRD-GPQGIAGQ-DRCG (SEQ ID NO: 5).

[0108] In some embodiments, the cleavable linker is a pH-sensitive linker. A pH-sensitive linker may be one found in US8,063,209, US11,219,697, and US11,840,549, all of which are incorporated herein by reference in their entirety. A pH-sensitive linker may also be found, for example, in US10,383,912 and US10,688,193, both of which are incorporated herein by reference in their entirety.

[0109] iii. Bisphosphonate portion In some embodiments of methods disclosed herein for targeting molecules to the site of injury, the BP portion does not sufficiently or completely inhibit osteoclast function, or inhibits it only minimally. In some embodiments, BP only minimally inhibits farnesyl pyrophosphate synthase (FPSS). In some embodiments of methods disclosed herein for targeting molecules to the site of injury, the BP portion is 2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-PyrEBP), 1-hydroxy-2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS), methylene bisphosphonate (MBP), methylene hydroxyl bisphosphonate (MHDP), etidronate (EHDP), methyl hydroxyl diphosphonate, clodronate, isochlordronate, tildronate, 2-hydroxy-2-phosphono-3-(pyridine-3-yl)propanoic acid (3-PEHPC), or 2-hydroxy-3-(imidazo[1,2-c]pyridine-3-yl)-2-phosphonopropanoic acid (3-IP-EHPC).

[0110] 2. Methods for treating tendon injuries and / or promoting the healing of soft tissues into bone. A method for treating a tendon injury in a subject requiring treatment is disclosed, comprising administering to the subject an effective amount of one of the molecules, compositions, or pharmaceutical compositions disclosed herein. Also disclosed is a method for promoting the healing of soft tissue to bone, comprising administering to the subject an effective amount of one of the molecules, compositions, or pharmaceutical compositions disclosed herein.

[0111] This specification discloses a method for treating tendon injury, comprising administering a molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety, wherein the BP moiety has a PCP structure and the cleavable linker links the bioactive moiety to the BP moiety.

[0112] In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the tendon injury is the rotator cuff, distal biceps tendon, pectoralis major, patellar tendon, quadriceps tendon, or triceps tendon.

[0113] In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the molecule is administered systemically. In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the molecule is administered topically.

[0114] i. Biologically active moiety In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the bioactive moiety is an osteochondral factor, osteogenic factor, tendon morphogenetic factor, or extracellular matrix (ECM) protein. In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the bioactive moiety is an ECM protein, which is collagen, elastin, fibrin, fibronectin, gelatin, laminin, integrin, CCN1, WISP-1, or CTGF.

[0115] In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the bioactive portion is an osteochondral factor, which is NELL-1, TGF-β1, TGF-β2, TGF-β3, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-11, BMP-15, or rhBMP. In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the osteochondral factor is NELL-1 or BMP-2.

[0116] In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the bioactive portion is an osteogenic factor, which is oxy-133 or oxysterol-133.

[0117] In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the bioactive portion is a tendinogenesis factor, which is TGFβ-1, TGFβ-2, TGF-β3, FGF, CTGF, BMP-12, BMP-13, BMP-14, CCN1, or WISP-1. In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the tendinogenesis factor is TGFβ-1 or TGFβ-2.

[0118] In some embodiments of methods for treating tendon injury in subjects requiring treatment, as disclosed herein, the bioactive moiety is a bioactive moiety which is parathyroid hormone, parathyroid hormone-related protein, growth differentiation factor 11 (GDF11), activin receptor-like kinase-1-Fc (ALK1-Fc), or activin receptor-like kinase-4-Fc (ALK4-Fc).

[0119] ii. Cuttable linker In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the cleavable linker is a cathepsin K-sensitive peptide linker. In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the cathepsin K-sensitive peptide linker is GHPGGPQG (SEQ ID NO: 1) or GGGMGPSGPWGGK (SEQ ID NO: 2).

[0120] In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the severable linker is an MMP severable linker. In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the MMP severable linker is PLGLAG (SEQ ID NO: 3), or Ac-GCRD-GPQGIWGQ-DRCG (SEQ ID NO: 4), or Ac-GCRD-GPQGIAGQ-DRCG (SEQ ID NO: 5).

[0121] In some embodiments, the cleavable linker is a pH-sensitive linker. A pH-sensitive linker may be one found in US8,063,209, US11,219,697, and US11,840,549, all of which are incorporated herein by reference in their entirety. A pH-sensitive linker may also be found, for example, in US10,383,912 and US10,688,193, both of which are incorporated herein by reference in their entirety.

[0122] iii. Bisphosphonate portion In some embodiments of methods disclosed herein for treating tendon injuries in subjects requiring treatment, the BP portion does not sufficiently or completely inhibit osteoclast function, or inhibits it only minimally. In some embodiments, BP only minimally inhibits farnesyl pyrophosphate synthase (FPSS). In some embodiments of methods for treating tendon injuries in subjects requiring treatment, as disclosed herein, the BP portion is 2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-PyrEBP), 1-hydroxy-2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS), methylene bisphosphonate (MBP), methylene hydroxyl bisphosphonate (MHDP), etidronate (EHDP), methyl hydroxyl diphosphonate, clodronate, isoclodronate, tildronate, 2-hydroxy-2-phosphono-3-(pyridine-3-yl)propanoic acid (3-PEHPC), or 2-hydroxy-3-(imidazo[1,2-c]pyridine-3-yl)-2-phosphonopropanoic acid (3-IP-EHPC).

[0123] 3. How to treat a fracture A method for treating a fracture in a subject requiring treatment is disclosed, comprising administering to the subject an effective amount of one of the molecules, compositions, or pharmaceutical compositions disclosed herein. In some embodiments, the fracture is a bone stress injury, such as a stress reaction or stress fracture.

[0124] This specification discloses a method for treating a fracture, comprising administering a molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety, wherein the BP moiety has a PCP structure and the cleavable linker links the bioactive moiety to the BP moiety.

[0125] In some embodiments of the methods disclosed herein for treating fractures in subjects requiring such treatment, the molecule is administered systemically. In some embodiments, the molecule is administered topically.

[0126] i. Biologically active moiety In some embodiments of methods disclosed herein for treating fractures in subjects requiring treatment, the bioactive moiety is an osteochondral factor, osteogenic factor, tendon morphogenetic factor, or extracellular matrix (ECM) protein. In some embodiments of methods disclosed herein for treating fractures in subjects requiring treatment, the bioactive moiety is an ECM protein, which is collagen, elastin, fibrin, fibronectin, gelatin, laminin, integrin, CCN1, WISP-1, or CTGF.

[0127] In some embodiments of methods disclosed herein for treating fractures in subjects requiring treatment, the bioactive moiety is an osteochondral factor, which is NELL-1, TGF-β1, TGF-β2, TGF-β3, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-11, BMP-15, or rhBMP. In some embodiments of methods disclosed herein for treating fractures in subjects requiring treatment, the osteochondral factor is NELL-1 or BMP-2.

[0128] In some embodiments of methods disclosed herein for treating fractures in subjects requiring such treatment, the bioactive portion is an osteogenic factor, which is oxy-133 or oxysterol-133.

[0129] In some embodiments of the methods disclosed herein for treating fractures in subjects requiring treatment, the bioactive portion is a tendinogenesis factor, which is TGFβ-1, TGFβ-2, TGF-β3, FGF, CTGF, BMP-12, BMP-13, BMP-14, CCN1, or WISP-1. In some embodiments of the methods disclosed herein for treating fractures in subjects requiring treatment, the tendinogenesis factor is TGFβ-1 or TGFβ-2.

[0130] In some embodiments of methods disclosed herein for treating fractures in subjects requiring treatment, the bioactive moiety is a bioactive moiety which is parathyroid hormone, parathyroid hormone-related protein, growth differentiation factor 11 (GDF11), activin receptor-like kinase-1-Fc (ALK1-Fc), or activin receptor-like kinase-4-Fc (ALK4-Fc).

[0131] ii. Cuttable linker In some embodiments of methods disclosed herein for treating fractures in subjects requiring such treatment, the cleavable linker is a cathepsin K-sensitive peptide linker. In some embodiments of methods disclosed herein for treating fractures in subjects requiring such treatment, the cathepsin K-sensitive peptide linker is GHPGGPQG (SEQ ID NO: 1) or GGGMGPSGPWGGK (SEQ ID NO: 2).

[0132] In some embodiments of methods disclosed herein for treating fractures in subjects requiring treatment, the severable linker is an MMP severable linker. In some embodiments of methods disclosed herein for treating fractures in subjects requiring treatment, the MMP severable linker is PLGLAG (SEQ ID NO: 3), or Ac-GCRD-GPQGIWGQ-DRCG (SEQ ID NO: 4), or Ac-GCRD-GPQGIAGQ-DRCG (SEQ ID NO: 5).

[0133] In some embodiments, the cleavable linker is a pH-sensitive linker. A pH-sensitive linker may be one found in US8,063,209, US11,219,697, and US11,840,549, all of which are incorporated herein by reference in their entirety. A pH-sensitive linker may also be found, for example, in US10,383,912 and US10,688,193, both of which are incorporated herein by reference in their entirety.

[0134] iii. Bisphosphonate portion In some embodiments of methods disclosed herein for treating fractures in subjects requiring such treatment, the BP portion does not sufficiently or completely inhibit osteoclast function, or inhibits it only minimally. In some embodiments, BP only minimally inhibits farnesyl pyrophosphate synthase (FPSS). In some embodiments of methods for treating fractures in subjects requiring treatment as disclosed herein, the BP portion is 2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-PyrEBP), 1-hydroxy-2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS), methylene bisphosphonate (MBP), methylene hydroxyl bisphosphonate (MHDP), etidronate (EHDP), methyl hydroxyl diphosphonate, clodronate, isochlordronate, tildronate, 2-hydroxy-2-phosphono-3-(pyridine-3-yl)propanoic acid (3-PEHPC), or 2-hydroxy-3-(imidazo[1,2-c]pyridine-3-yl)-2-phosphonopropanoic acid (3-IP-EHPC).

[0135] 4. Methods for treating bone stress injuries A method for treating bone stress injury in a subject requiring treatment is disclosed, comprising administering to the subject an effective amount of one of the molecules, compositions, or pharmaceutical compositions disclosed herein.

[0136] This specification discloses a method for treating bone stress injury, comprising administering a molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety, wherein the BP moiety has a PCP structure and the cleavable linker links the bioactive moiety to the BP moiety.

[0137] In some embodiments of methods disclosed herein for treating bone stress injuries in subjects requiring treatment, the molecule is administered systemically. In some embodiments of methods disclosed herein for treating bone stress injuries in subjects requiring treatment, the molecule is administered topically.

[0138] i. Biologically active moiety In some embodiments of methods disclosed herein for treating bone stress injuries in subjects requiring treatment, the bioactive moiety is an osteochondral factor, osteogenic factor, tendon morphogenetic factor, or extracellular matrix (ECM) protein. In some embodiments of methods disclosed herein for treating bone stress injuries in subjects requiring treatment, the bioactive moiety is an ECM protein, which is collagen, elastin, fibrin, fibronectin, gelatin, laminin, integrin, CCN1, WISP-1, or CTGF.

[0139] In some embodiments of methods disclosed herein for treating bone stress injuries in subjects requiring treatment, the bioactive moiety is an osteochondral factor, which is NELL-1, TGF-β1, TGF-β2, TGF-β3, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-11, BMP-15, or rhBMP. In some embodiments of methods disclosed herein for treating bone stress injuries in subjects requiring treatment, the osteochondral factor is NELL-1 or BMP-2.

[0140] In some embodiments of methods disclosed herein for treating bone stress injury in subjects requiring treatment, the bioactive moiety is a bone morphogenetic factor, which is oxy-133 or oxysterol-133.

[0141] In some embodiments of methods disclosed herein for treating bone stress injuries in subjects requiring treatment, the tendinogenesis factor is an osteochondral factor, and the tendinogenesis factor is TGFβ-1, TGFβ-2, TGF-β3, FGF, CTGF, BMP-12, BMP-13, BMP-14, CCN1, or WISP-1. In some embodiments of methods disclosed herein for treating bone stress injuries in subjects requiring treatment, the tendinogenesis factor is TGFβ-1 or TGFβ-2.

[0142] In some embodiments of methods disclosed herein for treating bone stress injury in subjects requiring treatment, the bioactive moiety is a bioactive moiety which is parathyroid hormone, parathyroid hormone-related protein, growth differentiation factor 11 (GDF11), activin receptor-like kinase-1-Fc (ALK1-Fc), or activin receptor-like kinase-4-Fc (ALK4-Fc).

[0143] ii. Cuttable linker In some embodiments of methods disclosed herein for treating bone stress injury in subjects requiring treatment, the cleavable linker is a cathepsin K-sensitive peptide linker. In some embodiments of methods disclosed herein for treating bone stress injury in subjects requiring treatment, the cathepsin K-sensitive peptide linker is GHPGGPQG (SEQ ID NO: 1) or GGGMGPSGPWGGK (SEQ ID NO: 2).

[0144] In some embodiments of methods disclosed herein for treating bone stress injuries in subjects requiring treatment, the cleavable linker is an MMP cleavable linker. In some embodiments of methods disclosed herein for treating bone stress injuries in subjects requiring treatment, the MMP cleavable linker is PLGLAG (SEQ ID NO: 3), or Ac-GCRD-GPQGIWGQ-DRCG (SEQ ID NO: 4), or Ac-GCRD-GPQGIAGQ-DRCG (SEQ ID NO: 5).

[0145] In some embodiments, the cleavable linker is a pH-sensitive linker. A pH-sensitive linker may be one found in US8,063,209, US11,219,697, and US11,840,549, all of which are incorporated herein by reference in their entirety. A pH-sensitive linker may also be found, for example, in US10,383,912 and US10,688,193, both of which are incorporated herein by reference in their entirety.

[0146] iii. Bisphosphonate portion In some embodiments of methods disclosed herein for treating bone stress injuries in subjects requiring treatment, the BP portion does not sufficiently or completely inhibit osteoclast function, or inhibits it only minimally. In some embodiments, BP only minimally inhibits farnesyl pyrophosphate synthase (FPSS). In some embodiments, the BP portion is 2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-PyrEBP), 1-hydroxy-2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS), methylene bisphosphonate (MBP), methylene hydroxyl bisphosphonate (MHDP), etidronate (EHDP), methyl hydroxyl diphosphonate, clodronate, isoclodronate, tildronate, 2-hydroxy-2-phosphono-3-(pyridine-3-yl)propanoic acid (3-PEHPC), or 2-hydroxy-3-(imidazo[1,2-c]pyridine-3-yl)-2-phosphonopropanoic acid (3-IP-EHPC).

[0147] 5. Methods to enhance the integration of allogeneic tissue during or after orthopedic surgery. A method is disclosed for a subject requiring enhancement of allograft tissue integration during or after orthopedic surgery, comprising administering to the subject an effective amount of one of the molecules, compositions, or pharmaceutical compositions disclosed herein.

[0148] This specification discloses a method for enhancing the integration of allograft tissue during or after orthopedic surgery, comprising administering a molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety, wherein the BP moiety has a PCP structure and the cleavable linker links the bioactive moiety to the BP moiety.

[0149] In some aspects of the methods disclosed herein for performing augmentation of allograft tissue integration during or after an orthopedic surgery in subjects requiring such augmentation, the orthopedic surgery is bone cutting, ACL reconstruction, PCL reconstruction, LCL or MCL repair / reconstruction, medial patellofemoral ligament reconstruction, meniscal root repair, meniscal allograft, and / or osteochondral allograft.

[0150] In some embodiments of the methods disclosed herein for performing augmentation of allograft tissue integration during or after orthopedic surgery in subjects requiring such augmentation, the orthopedic surgery is bone integration of an implantable prosthesis. In some embodiments, the implantable prosthesis is, but is not limited to, an implantable material such as metal, ceramic, or plastic that is embedded in bone or for dental purposes.

[0151] In some embodiments of the methods disclosed herein for performing augmentation of allograft tissue integration in subjects requiring such augmentation during or after orthopedic surgery, the orthopedic surgery is bone integration of allograft tissue. In some embodiments, the allograft tissue is osteochondral allograft and / or bone allograft.

[0152] In some embodiments of the methods disclosed herein for enhancing the integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the molecule is administered immediately before the orthopedic surgery. In some embodiments of the methods disclosed herein for enhancing the integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the molecule is administered during the orthopedic surgery. In some embodiments of the methods disclosed herein for enhancing the integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the molecule is administered after the orthopedic surgery.

[0153] In some embodiments of the methods disclosed herein for enhancing the integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the molecule is administered systemically. In some embodiments, the molecule is administered topically.

[0154] i. Biologically active moiety In some embodiments of methods disclosed herein for enhancing the integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the bioactive moiety is an osteochondral factor, osteogenic factor, tendon morphogenetic factor, or extracellular matrix (ECM) protein. In some embodiments of methods disclosed herein for enhancing the integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the bioactive moiety is an ECM protein, which is collagen, elastin, fibrin, fibronectin, gelatin, laminin, integrin, CCN1, WISP-1, or CTGF.

[0155] In some embodiments of the methods disclosed herein for achieving enhanced integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the bioactive portion is an osteochondral factor, which is NELL-1, BMP-1, BMP-2, TGF-β1, TGF-β2, TGF-β3, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-11, BMP-15, or rhBMP. In some embodiments of the methods disclosed herein for achieving enhanced integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the osteochondral factor is NELL-1 or BMP-2.

[0156] In some embodiments of the methods disclosed herein for achieving enhancement of allograft tissue integration during or after orthopedic surgery in subjects requiring such enhancement, the bioactive portion is an osteogenic factor, which is oxy-133 or oxysterol-133.

[0157] In some embodiments of the methods disclosed herein for achieving enhanced integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the bioactive portion is a tendinogenic factor, which is TGFβ-1, TGFβ-2, TGF-β3, FGF, CTGF, BMP-12, BMP-13, BMP-14, CCN1, or WISP-1. In some embodiments of the methods disclosed herein for achieving enhanced integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the tendinogenic factor is TGFβ-1 or TGFβ-2.

[0158] In some embodiments of methods disclosed herein for achieving enhancement of allograft tissue integration during or after orthopedic surgery in subjects requiring such enhancement, the bioactive moiety is a bioactive moiety which is parathyroid hormone, parathyroid hormone-related protein, growth differentiation factor 11 (GDF11), activin receptor-like kinase-1-Fc (ALK1-Fc), or activin receptor-like kinase-4-Fc (ALK4-Fc).

[0159] ii. Cuttable linker In some embodiments of methods disclosed herein for enhancing the integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the cleavable linker is a cathepsin K-sensitive peptide linker. In some embodiments of methods disclosed herein for enhancing the integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the cathepsin K-sensitive peptide linker is GHPGGPQG (SEQ ID NO: 1) or GGGMGPSGPWGGK (SEQ ID NO: 2).

[0160] In some embodiments of methods disclosed herein for enhancing the integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the cleavable linker is an MMP cleavable linker. In some embodiments of methods disclosed herein for enhancing the integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the MMP cleavable linker is PLGLAG (SEQ ID NO: 3), or Ac-GCRD-GPQGIWGQ-DRCG (SEQ ID NO: 4) or Ac-GCRD-GPQGIAGQ-DRCG (SEQ ID NO: 5).

[0161] In some embodiments, the cleavable linker is a pH-sensitive linker. A pH-sensitive linker may be one found in US8,063,209, US11,219,697, and US11,840,549, all of which are incorporated herein by reference in their entirety. A pH-sensitive linker may also be found, for example, in US10,383,912 and US10,688,193, both of which are incorporated herein by reference in their entirety.

[0162] iii. Bisphosphonate portion In some embodiments of the methods disclosed herein for enhancing the integration of allograft tissue during or after orthopedic surgery in subjects requiring such enhancement, the BP portion does not sufficiently or completely inhibit osteoclast function, or inhibits it only minimally. In some embodiments, BP only minimally inhibits farnesyl pyrophosphate synthase (FPSS). In some embodiments of methods disclosed herein for achieving enhancement of allograft tissue integration during or after orthopedic surgery in subjects requiring such enhancement, the BP portion is 2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-PyrEBP), 1-hydroxy-2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS), methylene bisphosphonate (MBP), methylene hydroxyl bisphosphonate (MHDP), etidronate (EHDP), methyl hydroxyl diphosphonate, clodronate, isochlordronate, tildronate, 2-hydroxy-2-phosphono-3-(pyridine-3-yl)propanoic acid (3-PEHPC), or 2-hydroxy-3-(imidazo[1,2-c]pyridine-3-yl)-2-phosphonopropanoic acid (3-IP-EHPC).

[0163] 6. Methods to promote the repair or regeneration of tendon attachments during or after orthopedic surgery. A method is disclosed for a subject requiring the repair or regeneration of a tendon attachment during or after orthopedic surgery, the method comprising administering to the subject an effective amount of one of the molecules, compositions, or pharmaceutical compositions disclosed herein.

[0164] This specification discloses a method for promoting the repair or regeneration of a tendon attachment during or after orthopedic surgery, comprising administering a molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety, wherein the BP moiety has a PCP structure and the cleavable linker links the bioactive moiety to the BP moiety.

[0165] In some aspects of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the orthopedic surgery is bone cutting, ACL reconstruction, PCL reconstruction, LCL or MCL repair / reconstruction, medial patellofemoral ligament reconstruction, meniscal root repair, meniscal allograft, and / or osteochondral allograft.

[0166] In some embodiments of the methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the molecule is administered immediately before the orthopedic surgery. In some embodiments of the methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the molecule is administered during the orthopedic surgery. In some embodiments of the methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the molecule is administered after the orthopedic surgery.

[0167] In some embodiments of the methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such action, the molecule is administered systemically. In some embodiments, the molecule is administered topically.

[0168] i. Biologically active moiety In some embodiments of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the bioactive moiety is an osteochondral factor, osteogenic factor, tendon morphogenetic factor, or extracellular matrix (ECM) protein. In some embodiments of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the bioactive moiety is an ECM protein, which is collagen, elastin, fibrin, fibronectin, gelatin, laminin, integrin, CCN1, WISP-1, or CTGF.

[0169] In some embodiments of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the bioactive portion is an osteochondral factor, which is NELL-1, TGF-β1, TGF-β2, TGF-β3, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-11, BMP-15, or rhBMP. In some embodiments of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the osteochondral factor is NELL-1 or BMP-2.

[0170] In some embodiments of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the bioactive portion is an osteogenic factor, which is oxy-133 or oxysterol-133.

[0171] In some embodiments of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the bioactive portion is a tendinogenesis factor, which is TGFβ-1, TGFβ-2, TGF-β3, FGF, CTGF, BMP-12, BMP-13, BMP-14, CCN1, or WISP-1. In some embodiments of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the tendinogenesis factor is TGFβ-1 or TGFβ-2.

[0172] In some embodiments of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the bioactive moiety is a bioactive moiety which is parathyroid hormone, parathyroid hormone-related protein, growth differentiation factor 11 (GDF11), activin receptor-like kinase-1-Fc (ALK1-Fc), or activin receptor-like kinase-4-Fc (ALK4-Fc).

[0173] ii. Cuttable linker In some embodiments of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the cleavable linker is a cathepsin K-sensitive peptide linker. In some embodiments of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the cathepsin K-sensitive peptide linker is GHPGGPQG (SEQ ID NO: 1) or GGGMGPSGPWGGK (SEQ ID NO: 2).

[0174] In some embodiments of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the severable linker is an MMP severable linker. In some embodiments of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the MMP severable linker is PLGLAG (SEQ ID NO: 3), or Ac-GCRD-GPQGIWGQ-DRCG (SEQ ID NO: 4) or Ac-GCRD-GPQGIAGQ-DRCG (SEQ ID NO: 5).

[0175] In some embodiments, the cleavable linker is a pH-sensitive linker. A pH-sensitive linker may be one found in US8,063,209, US11,219,697, and US11,840,549, all of which are incorporated herein by reference in their entirety. A pH-sensitive linker may also be found, for example, in US10,383,912 and US10,688,193, both of which are incorporated herein by reference in their entirety.

[0176] iii. Bisphosphonate portion In some embodiments of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such action, the BP portion does not sufficiently or completely inhibit osteoclast function, or inhibits it only minimally. In some embodiments, BP only minimally inhibits farnesyl pyrophosphate synthase (FPSS). In some embodiments of methods disclosed herein for promoting the repair or regeneration of tendon attachments during or after orthopedic surgery in subjects requiring such repair or regeneration, the BP portion is 2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-PyrEBP), 1-hydroxy-2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS), methylene bisphosphonate (MBP), methylene hydroxyl bisphosphonate (MHDP), etidronate (EHDP), methyl hydroxyl diphosphonate, clodronate, isochlordronate, tildronate, 2-hydroxy-2-phosphono-3-(pyridine-3-yl)propanoic acid (3-PEHPC), or 2-hydroxy-3-(imidazo[1,2-c]pyridine-3-yl)-2-phosphonopropanoic acid (3-IP-EHPC).

[0177] 7. Methods for treating osteoporosis, osteoarthritis, and / or cartilage defects. This specification discloses a method for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring such treatment, comprising administering one of the disclosed molecules, compositions, or pharmaceutical compositions to the subject.

[0178] This specification discloses a method for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring treatment, comprising administering a molecule comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety, wherein the BP moiety has a PCP structure and the cleavable linker links the bioactive moiety to the BP moiety.

[0179] In some embodiments of methods disclosed herein for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring such treatment, the sites of osteoporosis, osteoarthritis, and / or cartilage defects are the knee, hip, ankle spine, wrist, shoulder, elbow, hand, fingers, foot, and / or toes.

[0180] In some embodiments of methods disclosed herein for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring treatment, the molecule is administered systemically. In some embodiments of methods disclosed herein for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring treatment, the molecule is administered topically.

[0181] i. Biologically active moiety In some embodiments of methods disclosed herein for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring treatment, the bioactive moiety is an osteochondral factor, osteogenic factor, tendon morphogenetic factor, or extracellular matrix (ECM) protein. In some embodiments of methods disclosed herein for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring treatment, the bioactive moiety is an ECM protein, where the ECM protein is collagen, elastin, fibrin, fibronectin, gelatin, laminin, integrin, CCN1, WISP-1, or CTGF.

[0182] In some embodiments of methods disclosed herein for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring treatment, the bioactive moiety is an osteochondral factor, which is NELL-1, TGF-β1, TGF-β2, TGF-β3, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-11, BMP-15, or rhBMP. In some embodiments of methods disclosed herein for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring treatment, the osteochondral factor is NELL-1 or BMP-2.

[0183] In some embodiments of methods disclosed herein for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring such treatment, the bioactive portion is a bone morphogenetic factor, which is oxy-133 or oxysterol-133.

[0184] In some embodiments of the methods disclosed herein for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring such treatment, the bioactive portion is a tendinogenesis factor, which is TGFβ-1, TGFβ-2, TGFβ3, FGF, CTGF, BMP-12, BMP-13, BMP-14, CCN1, or WISP-1. In some embodiments, the tendinogenesis factor is TGFβ-1 or TGFβ-2.

[0185] In some embodiments of methods disclosed herein for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring treatment, the bioactive moiety is a bioactive moiety which is parathyroid hormone, parathyroid hormone-related protein, growth differentiation factor 11 (GDF11), activin receptor-like kinase-1-Fc (ALK1-Fc), or activin receptor-like kinase-4-Fc (ALK4-Fc).

[0186] ii. Cuttable linker In some embodiments of methods disclosed herein for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring such treatment, the cleavable linker is a cathepsin K-sensitive peptide linker. In some embodiments, the cathepsin K-sensitive peptide linker is GHPGGPQG (SEQ ID NO: 1) or GGGMGPSGPWGGK (SEQ ID NO: 2).

[0187] In some embodiments of methods disclosed herein for treating subjects requiring treatment for osteoporosis, osteoarthritis, and / or cartilage defects, the cleavable linker is an MMP cleavable linker. In some embodiments of methods disclosed herein for treating subjects requiring treatment for osteoporosis, osteoarthritis, and / or cartilage defects, the MMP cleavable linker is PLGLAG (SEQ ID NO: 3), or Ac-GCRD-GPQGIWGQ-DRCG (SEQ ID NO: 4) or Ac-GCRD-GPQGIAGQ-DRCG (SEQ ID NO: 5).

[0188] In some embodiments, the cleavable linker is a pH-sensitive linker. A pH-sensitive linker may be one found in US8,063,209, US11,219,697, and US11,840,549, all of which are incorporated herein by reference in their entirety. A pH-sensitive linker may also be found, for example, in US10,383,912 and US10,688,193, both of which are incorporated herein by reference in their entirety.

[0189] iii. Bisphosphonate portion In some embodiments of methods disclosed herein for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring such treatment, the BP portion does not sufficiently or completely inhibit osteoclast function, or inhibits it only minimally. In some embodiments, BP only minimally inhibits farnesyl pyrophosphate synthase (FPSS). In some embodiments of methods for treating osteoporosis, osteoarthritis, and / or cartilage defects in subjects requiring such treatment, the BP portion is 2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-PyrEBP), 1-hydroxy-2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS), methylene bisphosphonate (MBP), methylene hydroxyl bisphosphonate (MHDP), etidronate (EHDP), methyl hydroxyl diphosphonate, clodronate, isochlordronate, tildronate, 2-hydroxy-2-phosphono-3-(pyridine-3-yl)propanoic acid (3-PEHPC), or 2-hydroxy-3-(imidazo[1,2-c]pyridine-3-yl)-2-phosphonopropanoic acid (3-IP-EHPC).

[0190] 8. Medication regimen A drug regimen is disclosed comprising at least one therapeutic cycle of an effective amount of a disclosed molecule, composition, or pharmaceutical composition.

[0191] This specification discloses drug regimens in which a molecule, composition, or pharmaceutical composition is administered only once.

[0192] This specification discloses drug regimens in which molecules, compositions, or pharmaceutical compositions are administered multiple times over a period of time.

[0193] This specification discloses drug regimens in which the administered molecule, composition, or pharmaceutical composition can be administered at any point between 1 day and 2 weeks post-surgery.

[0194] A treatment cycle may include administration of different dosages of molecules, compositions, or pharmaceutical compositions, and administration at different time points. Molecules, compositions, or pharmaceutical compositions may be administered over a period of time varying in amount for up to 6 months. Molecules, compositions, or pharmaceutical compositions may be administered indefinitely over a period of time varying in amount. In some cases, administration may be carried out for up to 1, 2, 3, 4, 5, or 6 months. For example, a molecule, composition, or pharmaceutical composition may be administered once a week for 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks. In some embodiments, the disclosed molecule, composition, or pharmaceutical composition may be administered approximately every 3 months, approximately every 6 months, or approximately every 12 months.

[0195] The duration of each treatment cycle may vary depending on the amount of molecule, composition, or pharmaceutical composition administered per dose. A treatment cycle may include administration of the molecule, composition, or pharmaceutical composition once, twice, or three times per week. In some embodiments, the molecule, composition, or pharmaceutical composition may be administered daily. In some embodiments, the molecule, composition, or pharmaceutical composition may be administered once every two weeks, or even once a month. In some cases, the molecule, composition, or pharmaceutical composition may be administered every two weeks for 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks. For example, a treatment cycle may include administration of the molecule, composition, or pharmaceutical composition once a week for 4 weeks, or once every two weeks for up to 6 months. Thus, each treatment cycle includes an established duration for administration and an established dosing schedule within that time frame.

[0196] In one embodiment, molecules, compositions, or pharmaceutical compositions may be administered during a therapeutic cycle. Two or more molecules, compositions, or pharmaceutical compositions may be formulated together or in separate compositions. In some cases, one or more molecules, compositions, or pharmaceutical compositions are administered in combination with one or more other therapeutic agents, including but not limited to antibodies, nanobodies, aptamers, liposomes, antioxidants, anti-inflammatory agents, and senolytic agents.

[0197] 9.Dose The dose or dosage of a molecule, composition, or pharmaceutical composition may vary, but is not limited to, depending on many factors such as the patient's age, condition, sex, and severity of the disease, the route of administration, the length of the treatment cycle, or whether other drugs are included in the regimen, and can be determined by a person skilled in the art.

[0198] An effective dosage can be determined empirically, and making such a determination is within the scope of the art of the art. The dosage range for administering the composition is large enough to produce the desired effect in which the disease is treated. For example, the dosage may be an amount that produces or is effective in producing a sustained therapeutic effect even after discontinuation of the treatment (e.g., one or more of the disclosed molecules comprising i) a bioactive moiety, ii) a cleavable linker, and iii) a bisphosphonate moiety, wherein the BP moiety has a PCP structure and the cleavable linker links the bioactive moiety to the BP moiety). The therapeutic effect may be, but is not limited to, improvement of tendon injury, healing of fracture or bone stress injury, faster recovery from orthopedic procedures, improved biomechanical properties such as strength, or improved tissue organization of the repaired tendon attachment compared to an untreated subject. Other biomarkers used to measure the therapeutic effect may be markers of tendon or bone repair. The therapeutic effect can be measured by imaging techniques including MRI, intravascular ultrasound, ultrafast imaging CT scans, B-mode ultrasound, virtual histological intravascular ultrasound, optical coherence tomography, or other known methods.

[0199] The dosage should not be so high as to cause adverse side effects, such as undesirable cross-reactions or anaphylactic reactions. The dosage may be adjusted by the individual physician in the event of any counter-indication. The dosage may vary and may be administered in doses once or more daily for one or several days. Guidance can be found in the literature regarding appropriate dosages for a given class of pharmaceutical products.

[0200] Suitable dosages include, but are not limited to, amounts ranging from 0.01 mg / kg to 20 mg / kg. For example, this specification discloses methods involving the administration of one or more of the disclosed molecules, compositions, or pharmaceutical compositions, where the molecules are administered in amounts ranging from approximately 0.01 mg / kg to approximately 20 mg / kg. For example, the concentration of the molecule may be 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / kg. The dosage will depend on the identity of the bioactive portion. The dose of BP-BMP-2 may be, for example, approximately 10 mg / kg.

[0201] The dosage of the molecule, composition, or pharmaceutical composition may be administered systemically or topically. The dosage of the molecule, composition, or pharmaceutical composition may be administered as a bolus injection or as an infusion over a period of one hour or more.

[0202] 10.Delivery In the methods described herein, the administration or delivery of molecules, compositions, or pharmaceutical compositions may be via a variety of mechanisms. As defined above, this specification discloses methods of treatment, drug regimens, and methods of using those drug regimens for treatment. Drug regimens and methods include compositions containing one or more of the molecules described herein, which may also include carriers such as pharmaceutically acceptable carriers. For example, pharmaceutical compositions are disclosed that include the molecules and compositions disclosed herein, as well as pharmaceutically acceptable carriers.

[0203] The disclosed molecules, compositions, or pharmaceutical compositions may be in solution or suspension (e.g., incorporated into microparticles, liposomes, or cells).

[0204] Any preferred route of administration can be used for the disclosed molecules, compositions, and pharmaceutical compositions. Preferred routes of administration may include, for example, local, enteral, topical, systemic, or parenteral administration. For example, administration may be cutaneous, inhalation, enema, conjunctival, eye drops, ear drops, alveolar, nasal, intranasal, enteral, oral, oral, transoral, intestinal, rectal, intrarectal, transrectal, injection, infusion, intravenous, intraarterial, intramuscular, intracerebral, intraventricular, intraventricular, intracardiac, subcutaneous, intraosseous, intradermal, subarachnoid, intraperitoneal, intravesical, intracavernosal, intramedullary, intraocular, intracranial, percutaneous, transmucosal, transnasal, inhalation, intracisional, epidural, peridural, intravitreal, etc. The disclosed compositions can be used in and in conjunction with any other therapies.

[0205] In another embodiment, one or more components of the solution may be provided as a “concentrate,” for example, in a storage container (e.g., a pre-measured volume) that is ready for dilution, or in a soluble capsule that is ready for addition to a certain volume of water.

[0206] The formulations and methods of administration described herein are illustrative and not limiting. It should be understood that other suitable formulations and modes of administration can be readily devised using the teachings provided herein.

[0207] 11. Combination therapy In one embodiment of the disclosed method, a molecule, composition, or pharmaceutical composition may be administered alone or in combination with one or more additional therapeutic agents. The additional therapeutic agents are selected based on the disease or condition being treated. A description of suitable pharmacological agents and drugs of various classes can be found in Goodman and Gilman, The Pharmacological Basis of Therapeutics, (11th Ed., McGraw-Hill Publishing Co.) (2005). For example, a pharmaceutical composition containing a molecule may be administered in combination with one or more known therapeutic agents for the treatment of atherosclerosis.

[0208] Examples of therapeutic agents for treating tendon injuries include, but are not limited to, anti-inflammatory agents, analgesics, anti-rheumatic drugs, and immunomodulators, biophysical agents (e.g., shock wave therapy, ultrasound, magnetic fields, joint and tissue passive movement devices), dry needling, thermotherapy (e.g., ice, heat), and therapeutic exercises (e.g., physical therapy, eccentric strengthening).

[0209] The molecule, composition, or pharmaceutical composition may be administered in conjunction with or after any of the additional therapeutic agents disclosed.

[0210] Combination therapy may include administering molecules, compositions, or pharmaceutical compositions, and additional therapeutic agents during the treatment cycle of a medication regimen. [Examples]

[0211] It should be understood that the methods and compositions disclosed are not limited to the specific methodologies, protocols, and reagents described, as they may vary. It should also be understood that the terms used herein are solely for the purpose of describing specific embodiments and are not intended to limit the scope of the invention, which would be limited only by the appended claims.

[0212] Those skilled in the art will be able to recognize or confirm, by routine experimentation alone, many equivalents to specific embodiments of the methods and compositions described herein. Such equivalents are intended to be covered by the following claims.

[0213] Example 1: Biologically linked bisphosphonate chaperones effectively deliver molecules to the site of soft tissue-bone healing. Tendon injuries are common and often treated surgically, but current tendon repair healing results in poorly organized fibrous tissue. Certain growth factors have been reported to improve both the strength and organization of repaired tendon attachments, but their clinical applicability is severely limited due to the lack of appropriate delivery strategies. In this study, we evaluated Osteoadsorptive Fluorogenic Sentinel-3 (OFS-3), a recently developed fluorescent probe consisting of a bone-targeting bisphosphonate moiety linked to a fluorescent dye and a quencher molecule linked via a cathepsin K-sensitive peptide sequence. Using a mouse Achilles tendon-to-bone repair model, the bisphosphonate-based and / or Ctsk-linked imaging probe was applied either topically or systemically. Fluorescence imaging was used to quantify the signal obtained in vivo. After tendon-bone repair, animals that received either topical or systemic administration of the imaging probe showed significantly higher fluorescence signals at the repair site compared to the sham surgery group at all time points (p<0.001), with the signal peaking at 7–10 days post-surgery. This finding demonstrates the feasibility of using novel bisphosphonate-based targeted and Ctsk-activated molecular delivery to tendon-to-bone repair sites, laying the foundation for further development of this platform as an effective strategy for delivering bioactive molecules to musculoskeletal injury sites.

[0214] Bisphosphonates (BPs) are molecules that have a specific affinity for hydroxyapatite (HAP) calcium-phosphate minerals exposed in the bone's extracellular matrix (ECM) and preferentially bind to newly reabsorbed bone surfaces. HAP minerals can be exposed in bone through mechanical destruction, such as cortical detachment during tendon repair, or by physiologically activated osteoclasts during increased bone metabolic activity. During tendon-to-bone surgical repair, mechanically destroyed local bone in the tendon footprint is exposed to HAP, creating an environment in which local bone-derived progenitor cells communicate directly with the repaired tendon. In addition to mesenchymal progenitor cells, osteoclasts are also known to be active at the site of tendon repair and are associated with bone loss related to tendon-to-bone repair. 16~19 Given that activated osteoclasts secrete degrading bone enzymes such as cathepsin K (Ctsk) during the repair process, this well-characterized biology can be leveraged to facilitate the targeted delivery of molecules to the site of tendon-bone repair.

[0215] method Mouse model of tendon-bone repair Twelve-week-old male C57BL / 6 mice underwent right hindlimb surgery for either Achilles tendon-to-bone repair or sham surgery. Achilles tendon-to-bone repair consisted of a transtendinous 5-0 nylon suture positioned perpendicular to the long axis of the Achilles tendon, and a transosseous suture positioned laterally to medially and placed in the posterior calcaneus. After proper suture capture, the Achilles tendon was sharply excised from its attachment in the calcaneus, followed by posterior superior cortical detachment of the calcaneus with a dental bur. The Achilles tendon was then repaired to bone by directly attaching the proximal end of the excised tendon to the cortical detachment area of ​​the calcaneus using a pre-positioned 5-0 nylon suture (Figure 1). The surgical hindlimb was not immobilized, and the animals were given supportive weight to an acceptable extent immediately after the procedure. At necropsy, rupture of the repair structure was rare, affecting less than 5% of the study animals.

[0216] In vivo imaging We obtained AF647-ZOL (Alexa Fluor 647), a commercially available imaging probe consisting of BP conjugated with a fluorescent dye, from BioVinc, LLC (Pasadena, CA). OFS-3 was synthesized as previously described. 20 Simply put, OFS-3 consists of a BP molecule conjugated to the fluorescent dye sulfocyanine 5 and the Ctsk-sensitive peptide sequence GHPGGPQG. The other end of this peptide sequence is conjugated to BlackBerry Quencher 650 (BBQ-650), a dye that internally absorbs the emission spectrum from the sulfocyanine 5 fluorescent dye when properly excited via a fluorescence resonance energy transfer (FRET) mechanism. Cat K 680 FAST (Perkin-Elmer, Inc.) is a commercially available in vivo imaging agent that does not have a BP component but consists of a similar fluorescent dye-quencher dye pair connected via a Ctsk-sensitive peptide. When activated by Ctsk, the Cat K 680 FAST probe also produces fluorescence when exposed to light energy in the appropriate excitation spectrum, although it does not specifically bind to bone. Each study drug was resuspended in sterile phosphate-buffered saline (PBS) at pH 7.4.

[0217] Animals in the topical treatment group (n=7 per group) received a single dose of 5 microliters (μL) of a 1000 nM solution of the study drug (AF647-ZOL or OFS-3) via a micropipette, administered manually and directly to the repaired tendon-bone junction before skin closure, following Achilles tendon-bone repair. The topical treatment group did not include Cat K 680 FAST, as this product does not contain a bone-localized bisphosphonate component. Similarly, topical OSF-3 was not included in the placebo group because these animals only had skin injuries, and they had no evidence of mechanically exposed bone matrix containing hydroxyapatite minerals and / or activation of locally present osteoclasts. Therefore, it is not expected that OSF-3 would show significant differential targeting between placebo animals.

[0218] Animals in the systemic treatment group (n=7 per group for OFS-3 and AF647, n=3 per group for Cat K 680 FAST) received a single dose via subcutaneous injection immediately after surgery, either 100 μL of 50 nM research drug (AF647-ZOL or OFS-3) or 2 nmol in 100 μL of Cat K 680 FAST, following Achilles tendon-bone repair. In each experiment, the sham surgery group (n=7 per group) received a skin incision and suture closure without destruction of the Achilles tendon or calcaneus, followed by a systemic injection of either 100 μL of 50 nM research drug (AF647-ZOL or OFS-3) or 2 nmol in 100 μL of Cat K 680 FAST.

[0219] The treatment groups were designed to compare the current gold standard clinical approach (topical application) with the potentially most challenging scenario and a future alternative approach (systemic administration). Currently, the only growth factor (BMP-2) clinically available for the treatment of orthopedic injuries is applied topically to the repair site during surgery. Delivery of the study compounds is likely to be via transdermal injection to the repair site. In rodents, a portion of any transdermal injection of any of the investigational compounds is likely to be delivered to areas adjacent to the anatomical target, in addition to the anatomical target itself. Consequently, evaluating the localization of imaging compounds in the most challenging administration scenario (i.e., far from the Achilles tendon-calcaneal repair site) will provide data on the targeting capabilities of the investigational compounds.

[0220] Fluorescence signals (p / s) were acquired using the IVIS Lumina II imaging system (Perkin-Elmer Inc., Shelton, CT) while the animals were under isoflurane anesthesia on postoperative day 1 (POD), and then every 72 hours for either 3 weeks (AF647-ZOL and OFS-3 treatment groups) or 11 days (Cat K 680 FAST treatment group). Fluorescence signals from target areas in the hind feet of the surgical limbs (pseudo and repair groups) and the contralateral non-surgical hind limbs were quantified using IVIS Living Image® advanced in vivo imaging software (Perkin-Elmer Inc., Shelton, CT). The hind feet of the surgical hind limbs were designated as "on-target" among animals that underwent tendon repair, as they exhibited the highest fluorescence signal throughout the entire course of the experiment. The forefoot of the non-surgical hind limbs was designated as "off-target" among all animals, as this region exhibited the second highest fluorescence signal throughout the entire course of the experiment. In the animals in the sham treatment group, the forefoot of the hind limb was designated as the "on-target" because this region is closest to the surgical site, and it was noted that the forefoot had a higher fluorescence signal than the hindfoot throughout the entire experiment.

[0221] Histology and immunohistochemistry Four days after Achilles tendon excision and repair, mouse hind limb specimens were collected and subsequently fixed in 4% paraformaldehyde for three days. The specimens were then decalcified with formic acid (Statlab, Cat: 1414-32) for two days and embedded in paraffin. Sagittal sections 5 micrometers thick were obtained using a microtome and stained with hematoxylin (Fisher brand, Cat: 245-656) and eosin (Sigma-Aldrich, Cat: 17372-87-1) (H&E) using a previously described standard histological protocol, and counterstained with safranin-O (Sigma-Aldrich, Cat: S8884-25G) and Fast Green (Sigma-Aldrich, Cat: F7258-25G) (SOFG). 21The evaluation of activated osteoclasts was performed by immunohistochemical staining of sections for cathepsin K (Abcam, Cat: ab19027) at a dilution of 1:100 using the standard protocol used as previously described. 22 Images were taken with an Olympus BX60 bright-field microscope using 4x and 10x objective lenses. Representative sections of the entire area of the posterior calcaneal tendon attachment were selected for histological analysis.

[0222] Statistical analysis The mean fluorescence signal was compared across groups and between "on" and "off-target" using repeated measures (mixed) analysis of variance models. A repeated measures model was required because the same animals were measured over time, and these observations were not independent. Examination of the residual error normal quantile plot confirmed that the data had a normal distribution on the log scale, enabling the use of this parametric model.

[0223] Results In vivo imaging with AF647-ZOL Animals in the repair cohort that received either local or systemic AF647-ZOL showed significantly higher on-target fluorescence signals compared to the sham group at all time points except POD21 (p = 0.032) (p < 0.002), and the signal reached a peak at 7 - 10 days post-surgery (Figure 3). Representative images from each treatment group at POD10 are included in Figure 4. There was no significant difference in the on-target signal between the local and systemic treatment groups at all time points studied. Fluorescence (on-target signal) at the site of repair in the repaired (both locally and systemically treated) cohort was significantly higher than off-target fluorescence in the sham and repaired cohorts, except at POD1 in the systemic AF647-ZOL treatment group (p = 0.31) and at POD17 and 21 in both the systemic and local treatment groups (p < 0.001). This likely reflects a signal level that returns to baseline at approximately POD17 or about 2 weeks post-surgery.

[0224] In vivo imaging with Cat K 680 FAST Animals in the repair group treated with Cat K 680 FAST showed significantly higher on-target fluorescence signals compared to the sham group at all time points except POD0 (p=0.0065, Figure 5) (p<0.0001). Fluorescence signals in the repair cohort peaked approximately 4 days post-surgery. Off-target fluorescence signals in the repair group compared to the sham group were significantly different at POD0 (p=0.048), but not significantly different at the remaining time points studied. Repair group animals showed significantly less on-target fluorescence signals than off-target signals at POD0 (p=0.033), but significantly greater on-target fluorescence signals than off-target signals at POD4 (p=0.022). However, sham group animals showed significantly increased off-target signals compared to on-target signals at all time points (p<0.014). Representative images from each treatment group at POD4 are included in Figure 6.

[0225] In vivo imaging with OFS-3 Animals in the repair cohort that received either local or systemic OFS-3 showed significantly higher on-target fluorescence signals compared to the sham group at all time points, except for repair in the local OFS-3 treatment group at POD1 (P=0.66, Figure 7) (p<0.001). Signaling in the repair cohort peaked at 7–10 days post-surgery. Notably, there was a significant difference in on-target signaling between the local and systemic treatment groups at POD1 (p=0.0001). However, after POD1, there was no significant difference in on-target signaling between the local and systemic treatment groups. On-target fluorescence signaling in the repair cohort was significantly higher than off-target fluorescence in either the sham or repaired cohorts at POD4–14 (p<0.001), but there was no significant difference in repair in the systemic OFS-3 treatment group at POD1 (p<0.14, Figure 8). Following POD14, there was no significant difference in on-target signals compared to off-target signals in the repair cohort (Figure 8). Similar to the results for AF647-ZOL, this likely reflects the level of signaling returning to baseline approximately two weeks post-surgery.

[0226] Histology and immunohistochemistry Uninjured contralateral hind limbs (Figure 9A, B) showed normal bone, tendon, and tendon attachment architecture on H&E and SOFG staining, with a marked absence of cathepsin K-positive cells in the posterior superior calcaneus. Sections were decalcified to allow sectioning through bone. This prevented direct localization of OSF-3 or AF647, as divalent calcium cations that interact within the hydroxyapatite mineral to which bisphosphonate molecules (BPs) bind must be available for association with the bone matrix. However, Ctsk activity was examined. Animals treated with Achilles tendon-to-bone repair (Figure 9C and Figure 9D) showed cortical detachment areas of the posterior superior calcaneus with marked Ctsk-positive staining in immunohistochemistry in this region of cortical detachment bone (black brackets, Figure 9D). Additional areas of Ctsk-positive cells were present with less staining in adjacent skin tissue and the remaining distal Achilles tendon stump (black arrows, Figure 9D).

[0227] Consideration Currently, targeting of bioactive molecules like BMP-2 to tendon-bone repair sites in clinical settings is limited to macroscopic tissue levels and to hyperphysiological doses administered only during surgical procedures. 23 BP-based compounds can effectively target tendon-bone repair sites in vivo. Furthermore, bio-linked Ctsk-dependent molecular delivery strategies can effectively target tendon-bone repair sites, whether delivered locally or systemically.

[0228] This study evaluated the BP portion not as a therapeutic drug, but as a means of targeting active bone remodeling in the context of Achilles tendon repair. The use of the BP molecule to enhance soft tissue-to-bone healing has been previously investigated. However, this was mainly in the context of animal models investigating the effects of BP on osteoclast inhibition, and the results of this approach are debatable. Xu et al. reported that risedronate was beneficial for rotator cuff healing in osteoporotic rats. 24 Liu et al. noted enhanced tendon graft-to-bone healing in a rat model of ACL reconstruction. 25 In contrast, Hjorthaug et al. pointed out adverse effects on tendon-to-bone healing in their studies of rotator cuff repair in rats with zolendronate. 26 In their model of tendon-to-bone healing in dogs, Thhomopoulos et al. showed that after treatment with alendronate, load-bearing to final rupture improved and the incidence of sutures pulling on the tendon was lower. 27 Notably, all of these studies have very limited relevance to current trials because the BPs included in these studies inhibit osteoclasts and are not biologically linked to other molecules as in the case of OFS-3. Sung et al. showed that low-dose alendronate had little effect in vitro on the viability, proliferation, and wound healing capacity of human rotator cuff fibroblasts.28 Finally, while there is some potential risk of increased tendon injury reported to be associated with the clinical use of BP, it is difficult to attribute this risk solely to the use of BP. 29 In addition, bisphosphonates are used in the treatment of (e.g., osteoporosis) 30 and diagnostically (e.g., bone scintigraphy) 31 Given the widespread use of both, transient use of these drugs appears safe. Furthermore, as in the case of OFS-3, it is possible to manipulate bisphosphonate molecules that bind to HAP without significantly impairing local osteoclast activity. 32;33 .

[0229] Bone loss at the site of tendon repair is a well-documented phenomenon, and it has been suggested that this bone loss is associated with the presence of activated osteoclasts. 16~19 This finding is supported by histological findings, as evidenced by the marked cathepsin K positivity in immunohistochemical staining of the repaired specimens. Interestingly, in addition to more prominent Ctsk-positive staining at the site of tendon repair to bone, there was some Ctsk positivity at the excised tendon ends. The presence of Ctsk on POD4 and POD7 immunohistochemical stained sections correlates well with in vivo imaging data from POD4 and POD7. The biology of locally activated osteoclasts provides the predictable biological distribution observed in this study.

[0230] Following treatment with the study drugs at POD0, AF647-ZOL and OFS-3-treated animals were observed to have peak fluorescence signal intensity 7–10 days post-surgery, while Cat K 680 FAST-treated animals were observed to have peak signal intensity at approximately POD4. The finding that these three different compounds exhibit signal intensity between POD4 and POD10 may suggest that this timeframe corresponds to peak osteoclast activity at the site of tendon-bone repair in this model, which could have implications for future studies evaluating the timing of administration of bioactive compounds using this model. However, the peak signal intensity observed between POD4 and POD10 may also reflect overlap in the pharmacokinetic properties of these compounds with BP-containing compounds that have more similar pharmacokinetic profiles. Inferences related to the timeline of osteoclast activity at the repair site can also be made from results using Ctsk-sensitive imaging agents (i.e., Cat K 680 FAST and OFS-3). Animals in the repair group treated with Cat K 680 FAST (via systemic administration) showed significantly higher on-target fluorescence signals compared to the sham group at all time points except POD0. Similarly, in the repair cohort treated with OFS-3 (local and systemic), on-target fluorescence signals were significantly higher than off-target fluorescence at POD4-POD10 (p<0.0015) and POD14 (p<0.049). However, at POD1 in the repaired systemic OFS-3 treatment group, there was no significant difference between on-target and off-target signals (p=0.14).

[0231] The data support the use of biologically linked bisphosphonate targeting to the site of tendon-to-bone repair, with minimal differences in outcomes regardless of whether the BP-containing compound was administered topically or systemically. Animals treated with AF647-ZOL showed no significant difference in on-target signaling when comparing topical and systemic treatment groups at all time points studied. On-target fluorescence signaling in the repaired cohort (topical and systemic) treated with AF674-ZOL was also significantly higher than off-target signaling in both the sham and repaired cohorts until the signal returned to baseline at POD17, except in the systemic treatment group at POD1 (p=0.31) (p<0.001). Similarly, animals in the repaired group treated with Cat K 680 FAST showed significantly higher on-target fluorescence signaling compared to the sham group at all time points except POD0. Animals in the repair cohort that received either local or systemic OFS-3 showed significantly higher on-target fluorescence signals compared to the sham group at all time points, except for the local OFS-3 treatment group at POD1 (p=0.66) (p<0.001).

[0232] Following an injury to any of the limbs, animals typically alter their normal weight bearing capacity on those injured limbs. The injured limb is subjected to an increased proportion of the animal's weight-bearing load on the forefoot compared to baseline forefoot weight-bearing capacity. This is also likely to avoid pain during hindfoot incision, while reducing the weight-bearing load on the injured hindfoot. The other limbs are also subjected to a greater load than their normal weight-bearing capacity. This likely leads to a hidden bone stress response in the forelimbs of both the forelimbs and hindlimbs across all study animals, which may lead to the detection of signals after administration of the imaging probes used in this study.

[0233] Animals in the repair group treated with OFS-3 showed a significant reduction in on-target signals in the topical treatment group compared to the systemic treatment group at POD1, but there was no significant difference after POD1. This observation of early detection of signal reduction in the topical application group may be related to the administration technique, which may allow the study drug to leak from the wound before skin closure, potentially leading to a lower overall initial dose than the parenteral administration route.

[0234] Animals in the Cat K 680 FAST treatment group received 400 times the dose of animals in the AF647-ZOL or OFS-3 treatment groups, setting a very high upper limit threshold for background activity from Cat K 680 FAST. Despite this large dose difference, similar signal levels were achieved across these groups. However, it is also noteworthy that both peak signaling and signal retention in the Cat K 680 FAST treatment group were of shorter duration, despite the much larger dose administered.

[0235] Sham animals treated with Cat K 680 FAST showed significantly increased off-target signals compared to on-target signals at all time points (p<0.014). This was influenced by at least two factors: 1) as mentioned above, Cat K 680 FAST was administered at a dose 400 times greater than that of BP-containing compounds, according to the manufacturer's recommendations, making it likely that off-target sites were saturated; and 2) sham animals also likely showed reduced weight support in the surgical limbs, while lacking the physiological stress on the mechanically fractured calcaneus (i.e., no application of bars during surgery and subsequent activated osteoclast infiltration).

[0236] The detection of increased fluorescence signals in the repair groups using each of these imaging probes at early time points is promising, but the lack of significant differences in on-target signals compared to off-target signals at later time points (POD14 and POD17) in the OFS-3 and AF647-ZOL treated repair cohorts is also promising. As discussed herein, this return to baseline fluorescence signals may reflect either local osteoclast activity or the pharmacokinetic properties of these compounds, each of which is significant for future studies on the timing of administration of the study drugs. Targeting bioactive molecules to the site of tendon-bone repair beyond the time of surgery is a clear advantage of the BP-targeted Ctsk-linked delivery strategy. This can enable optimized delivery of progenitor cells to the repair site, followed by targeted delivery of growth factors that signal proliferation and / or differentiation, which can significantly improve local tissue regeneration. In humans, transdermal delivery of these specialized agents makes this alternative approach very attractive.

[0237] Off-target signaling is a definite concern in any drug discovery investigation. One explanation for the observation of signaling in the uninjured limb between animals in this study may be a physiological response to increased load exposure in the uninjured limb after unilateral Achilles tendon to bone repair. Increased weight-bearing in the uninjured limb to reduce the load on the injured limb may result in increased bone turnover. This phenomenon correlates well with clinical observations of athletes exposed to increased repetitive weight-bearing that leads to bone stress fracture injuries. In addition, if the pathogenesis of this signaling is increased weight-bearing resulting in mild increased bone resorption, the sensitivity of these probes described here as off-target signaling may have clinical significance as an assessment of increased bone turnover among patients with musculoskeletal injuries or further animals (e.g., racehorses) and athletes at risk of bone stress injuries.

[0238] While this initial study noted undesirable off-target signaling, this finding has room for improvement and can be addressed in dose optimization studies. Off-target delivery also shows a tendency for reduced off-target signaling with local administration compared to systemic administration of OFS-3, with significantly less signaling observed in POD1 (p=0.0004) and POD7 (p=0.0496), and nearly significant in POD10 (p=0.058), suggesting that it can be further optimized by administration technique. This observed difference in off-target signaling between the local and systemic treatment groups may be attributed to improved access via direct application of BP to mechanically fractured HAP minerals in the calcaneus during repair. Furthermore, when bone-inducing agents such as BMP-2, TGFβ, or NELL-1 are delivered using this BP-targeted Ctsk activation strategy, the less transiently delivered bone-forming signals are less likely to be associated with significant sequelae.

[0239] Appropriate biological cues to guide tissue regeneration are recognized as a critical, unmet need in the environment of tendon repair. 8 However, hyperphysiological drug administration during surgery is known to be associated with several well-documented complications, including ectopic bone, excessive inflammatory responses leading to airway obstruction, and seroma formation, which places patients at high risk of wound infection. In addition, as discussed herein, intraoperative delivery of bioactive molecules may miss a critical time to induce biological signaling after sufficient proliferation of the transfer and / or local progenitor cell population. The results obtained from low-dose administration of the compound in this study suggest that a BP-targeted Ctsk-linked delivery approach may allow for a significant reduction in the dose administered if it can deliver physiologically relevant levels of bioactive molecules at the cellular level rather than the macroscopic tissue level.

[0240] conclusion The data presented herein demonstrate the feasibility of using novel bisphosphonate-based targeted and cathepsin K-linked delivery of molecules to sites of tendon-to-bone repair. Through guided biological distribution and temporal optimization of bioactive molecule delivery at the cellular level, this approach has great potential for improving soft tissue-to-bone healing and other sites of musculoskeletal injury. However, further investigation is certainly needed.

[0241] Example 2: Manufacturing As in the case of BP-FQ, NELL-1 and BMP-2 were covalently bonded to the BP molecule via a peptide sequence that is a cleavable substrate for the cat-K enzyme using click chemistry. Click chemistry is a type of simple atomic economy reaction commonly used to join two selected molecular entities. Siverino et al. J Vis Exp. 2018 Mar 29;(133):56616.

[0242] Example 3: In vitro BMP receptor activation In vitro BMP receptor activation was evaluated using a dual luciferase assay in which Rinella plasmids and BMP response element (BRE) plasmids were transfected into ATDC5 cells known to express the BMP receptor. These transgenic ATDC5 cells were then subjected to one of five treatments: 1) commercial rhBMP-2, 2) variant BMP-2, 3) Ctsk digested BP-vBMP-2, 4) undigested BP-vBMP-2, or 5) phosphate-buffered saline. Luciferase 1 expression from the BRE plasmid was detected and normalized to control Rinella plasmid luciferase 2 expression (Figure 11). These results indicate that Ctsk digested BP-BMP-2 can activate the BMP receptor in a similar manner to commercial rhBMP-2, and that undigested BP-BMP-2 does not activate the BMP receptor above control levels.

[0243] Example 4: In vitro study Considering the presence of human mesenchymal stem cells (MSCs) in the human tendon repair site, human MSCs were purchased from Lonza and maintained for fewer than five passages in a culture medium consisting of RPMI 1640 medium with 10% FBS. The same number of cells were plated into each culture. After 48 hours, the culture medium was then changed to a serum-free medium containing one of the following: no additives (group 1), bisphosphonate alone (group 2), BMP-2 protein (group 3), BP-BMP-2 alone (group 4), or BP-BMP-2 with cathepsin K enzyme (group 5) (see Table 4). The dose of BMP-2 was set at 100 ng / ml based on previously published in vitro data (Pang S et al., Stem Cells. 2015;33(3):904-915), and this value was used to calculate the molar equivalent of BP-BMP-2 used in groups 4 and 5, with or without cathepsin K. The culture medium is changed every three days. On days 3 and 10 after transfer to these experimental medium solutions, proliferation assays, cell viability assays, and RT-PCR evaluations of gene expression for osteogenic, chondrogenic, and adipogenic gene transcripts are performed. The use of early and late time points provides insights into potentially accelerated differentiation and longer-term cell viability. Cell viability is examined using a live / dead assay kit (Thermofisher, Inc.), and cell proliferation assays are performed using a CyQUANT assay (Thermofisher, Inc.) according to the manufacturer's protocol. All treatment groups, assays, and time points are performed in triplicate. Total RNA is isolated using TRIzol reagent (Invitrogen, Inc.) and stored at -80°C. Differences in gene expression are assessed via TaqMan real-time PCR assay (Thermofisher, Inc.) according to the manufacturer's protocol, including genes related to osteogenic differentiation (osteopontin, osteocalcin, alkaline phosphatase), chondrogenic differentiation (Sox9, Col2A), and adipogenic differentiation (PPARγ). [Table 4]

[0244] As described above, the same methodology is used to evaluate BP, NELL-1 protein, and BP-NELL-1 molecule, except that the positive control group (group 6) uses a NELL-1 dose of 800 ng / ml based on previously published in vitro data (Table 2) (Pang S et al., Stem Cells. 2015;33(3):904-915). Again, based on this standard dose of NELL-1, the molar equivalents of BP-NELL-1, with or without cathepsin K, are used in groups 7 and 8 for comparison.

[0245] Statistical methods: Statistical analysis of the obtained data will utilize one-way analysis of variance models (one set for the BMP-2 experiment and one set for the NELL-1 experiment). For each set of models, the gene expression of various genes will be the result. These models include terms for processing. If the distribution of gene expression values ​​is abnormal, transformations will be considered, and pairwise group tests will be performed using Tukey's post-hoc test.

[0246] Example 5: In vivo study A dose of 1.25 mg / kg of recombinant human NELL-1 (rhNELL-1) administered intravenously has been shown to induce osteogenesis in a mouse model of osteoporosis (James AW et al. Nat Commun. 2015;6:7362). In addition, they describe the pharmacokinetics of systemically administered NELL-1-PEG, a chemically modified version of the NELL-1 protein using polyethylene glycol (also known as PEGylation), administered systemically once weekly at a dose of 1.25 mg / kg. This treatment has been shown to enhance fracture healing in the absence of abnormalities detected in toxicological studies and no evidence of ectopic osteogenesis on radiography or organ recovery analysis (Kwak JH et al. Biomaterials. 2015;57:73-83; Tanjaya J et al. Am J Pathol. 2018;188(3):715-727). To further improve pharmacokinetics, BP-NELL-1-PEG is created by covalently bonding bone NELL-1-PEG to the BP molecule. BP-NELL-1-PEG at a dose of 10 mg / kg once per week has shown the ability to preserve bone volume among mice in a space environment (confidential data). These results guide the initial dosing scheme for BP-NELL-1. Systemic administration of rhBMP-2 at doses of 0.5 mg, 1 mg, and 5 mg per mouse per day is associated with a statistically significant increase in total bone volume among osteopenic mice (Turgeman G et al. J Cell Biochem. 2002;86(3):461-474). Given that mice are approximately 10% of rat body weight, there is data supporting systemic administration of 5 mg–10 mg of rhBMP-2 in rats. These data guide the initial dosing scheme for BP-BMP-2. Initial drug-dosing pilot experiments using the Achilles tendon-bone repair model also include postmortem IHC staining for NELL-1 and BMP-2 proteins at the site of tendon repair relative to the contralateral, uninjured tendon attachment, 3 days after injection of BP-NELL-1 and BP-BMP-2.Pharmacokinetic evaluation will be performed by labeling BP-NELL-1 and BP-BMP-2 with amine-reactive near-infrared fluorescent dyes (VivoTag 680XL, Perkin Elmer) and then imaging them daily using the IVIS Lumina II imaging system until no further light signals are detected. Dose escalation studies will be conducted as needed, based on initial results from pilot studies. Regarding timing, single doses administered immediately after surgical procedures will be investigated initially, based on improved pharmacokinetics and localization associated with BP chaperones (e.g., BP-NELL-1-PEG data). If the initial single-dose systemic dosing protocol is ineffective, topical application, multiple systemic doses, and / or delayed systemic dosing regimens of these compounds will be investigated.

[0247] Based on dosing data from the pilot experiment, characterize the effectiveness of BP-NELL-1 and BP-BMP-2 in an Achilles tendon-to-bone repair model. Previous rat tendon repair studies have shown that initial healing was completed approximately 2 weeks after surgical repair and that most of the healing occurred by 6 weeks (Galatz LM et al. J Orthop Res. 2006;24(3):541-550; Kremen et al. Am J Sports Med. 2019:47(11):2737-2744; Zhao S et al. J Surg Res. 2015;193(1):33-42). Twelve groups of rats (n = 15 animals / group) undergo acute Achilles tendon-to-bone repair as described in Example 1. Groups 1A and 1B receive repair alone without augmentation. Groups 2A and 2B receive systemically delivered intravenous (IV) BP alone. Groups 3A and 3B receive an rhBMP-2 protein solution delivered at the repair site via an implanted type I bovine collagen sponge (DuraGen, Integra LifeSciences). Groups 4A and 4B receive systemic BP-BMP-2. Groups 5A and 5B receive rhNELL-1 protein in normal saline delivered via an implanted type I bovine collagen sponge. Groups 6A and 6B receive systemic BP-NELL-1. Groups 1A, 2A, 3A, 4A, 5A, and 6A are euthanized at 2 weeks postoperatively. Groups 1B, 2B, 3B, 4B, 5B, and 6B are euthanized at 6 weeks postoperatively (see Table 5). Evaluating early and late time points provides evidence of any accelerated healing. After euthanasia, the operative extremities are harvested. Twelve animals from each group are dedicated to testing of cyclic load and load to failure using an Instron materials testing machine as previously described (Galatz LM et al. J Orthop Res. 2006;24(3):541-550). Three animals from each group are fixed in 4% formalin, decalcified using 0.5 M EDTA, and subjected to hematoxylin and eosin staining, collagen birefringence microscopy, and safranin O staining to evaluate the presence of fibrocartilage.

Table 5

[0248] Statistical method: The statistical analysis of the obtained data utilizes a two-way analysis of variance model. The results evaluated in these models are the cyclic load and the load until fracture. For each model, the terms in the model include treatment (see Table 5), sacrifice time (2 weeks, 6 weeks), and the interaction effect of treatment over time. Terms for the main effect of gender and the interaction effect with gender (e.g., gender by treatment interaction) in these models are added. When the distribution of biomechanical values is not normal, transformations are considered and Tukey's post hoc test is used to perform pairwise group tests. With a sample size of 12 per group (6 male rats and 6 female rats) in each cell of a 2×6 factorial experiment, assuming ANOVA analysis and a two-sided 0.05 significance level, there is 80% power to detect an effect size of at least 1.2 for the time effect within treatment and an effect size of 0.83 between pairs of treatments.

[0249] Example 6: PTH and PTHrP Both PTH and PTHrP are proteins that can be synthesized using techniques similar to those for the approach to modified BMP-2 synthesis (described by Silverino C, et al JOVE 2018, PMID: 29658921). This enables the incorporation of unnatural amino acids compatible with click chemistry, which allows these bioactive moieties to be conjugated to our BP-cathepsin K-linked molecular cargo delivery approach. Subsequently, the effects of the BP-(Ctsk-sensitive peptide)-PTH or BP-(Ctsk-sensitive peptide)-PTHrP compounds are evaluated in vivo using an animal model of fracture healing, the effects on subchondral bone and cartilage tissue using an osteoarthritis (OA) model, healing from soft tissue to bone, and implant osseointegration using mechanical tests as described by Morinaga, et al., Biomaterials vol. 192 (2019): 62 - 74.

[0250] It will be apparent to those skilled in the art that various modifications and variations are possible in the present invention without departing from the scope or spirit of the invention. Other embodiments of the present invention will be apparent to those skilled in the art from consideration of the specification and practice of the present invention disclosed herein. This specification and examples are intended to be considered illustrative only, and the true scope and spirit of the invention are given by the following claims.

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Claims

1. It is a molecule, i) The bioactive portion, ii) A linker that can be cut, iii) including a bisphosphonate (BP) moiety, The aforementioned BP portion has a P-C-P structure, A molecule in which the cleavable linker connects the bioactive portion to the BP portion.

2. The molecule according to claim 1, wherein the bioactive portion is an osteochondral factor, an osteogenic factor, a tendon morphogenetic factor, or an extracellular matrix (ECM) protein.

3. The molecule according to claim 2, wherein the osteochondral factor is NELL-1, TGF-β1, TGF-β2, TGF-β3, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-11, BMP-15, or rhBMP.

4. The molecule according to claim 3, wherein the osteochondral factor is NELL-1 or BMP-2.

5. The molecule according to claim 2, wherein the tendon formation factor is TGFβ-1, TGFβ-2, TGFβ3, FGF, CTGF, BMP-12, BMP-13, BMP-14, CCN1, or WISP-1.

6. The molecule according to claim 5, wherein the tendon formation factor is TGFβ-1 or TGFβ-2.

7. The molecule according to claim 2, wherein the ECM protein is collagen, elastin, fibrin, fibronectin, gelatin, laminin, integrin, CCN1, WISP-1, or CTGF.

8. The molecule according to claim 1, wherein the biologically active portion is parathyroid hormone, parathyroid hormone-related protein, growth differentiation factor 11 (GDF11), activin receptor-like kinase-1-Fc (ALK1-Fc), or activin receptor-like kinase-4-Fc (ALK4-Fc).

9. The molecule according to any one of claims 1 to 8, wherein the cleavable linker is a cathepsin K-sensitive peptide linker.

10. The molecule according to claim 9, wherein the cathepsin K-sensitive peptide linker is GHPGGPQG (SEQ ID NO: 1) or GGGMGPPSGPWGGK (SEQ ID NO: 2).

11. The molecule according to any one of claims 1 to 8, wherein the linkable linker is an MMP-cleavable linker.

12. The molecule according to claim 11, wherein the MMP-cleavable linker is PLGLAG (SEQ ID NO: 3), or Ac-GCRD-GPQGIWGQ-DRCG (SEQ ID NO: 4), or Ac-GCRD-GPQGIAGQ-DRCG (SEQ ID NO: 5).

13. The molecule according to any one of claims 1 to 8, wherein the cleavable linker is a pH-sensitive linker.

14. The molecule according to any one of claims 1 to 13, wherein the BP portion does not inhibit osteoclast function and / or only minimally inhibits farnesyl pyrophosphate synthase (FPSS).

15. The molecule according to any one of claims 1 to 14, wherein the BP portion is 2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-PyrEBP), 1-hydroxy-2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS), methylene bisphosphonate (MBP), methylene hydroxyl bisphosphonate (MHDP), etidronate (EHDP), methyl hydroxyl diphosphonate, clodronate, isochlorodronate, tildronate, 2-hydroxy-2-phosphono-3-(pyridine-3-yl)propanoic acid (3-PEHPC), or 2-hydroxy-3-(imidazo[1,2-c]pyridine-3-yl)-2-phosphonopropanoic acid (3-IP-EHPC).

16. A composition comprising the molecule described in any one of claims 1 to 15.

17. A pharmaceutical composition comprising a molecule according to any one of claims 1 to 15 or a composition according to claim 16.

18. A method for performing molecular targeting on the site of injury, i) The bioactive portion, ii) A linker that can be cut, iii) including a bisphosphonate (BP) moiety, The aforementioned BP portion has a P-C-P structure, A method comprising administering a molecule in which the cleavable linker links the bioactive portion to the BP portion.

19. The method according to claim 18, wherein the site of injury is tendon attachment tissue.

20. The method according to claim 18 or 19, wherein the site of injury is the rotator cuff, distal biceps tendon, pectoralis major muscle, patellar tendon, quadriceps tendon, or triceps tendon.

21. The method according to claim 18 or 19, wherein the site of injury is also the site of surgery, and the surgery is anterior cruciate ligament (ACL) reconstruction, posterior cruciate ligament (PCL) reconstruction, lateral collateral ligament (LCL) or medial collateral ligament (MCL) repair / reconstruction, medial patellofemoral ligament reconstruction, meniscal root repair, meniscal allograft, or osteochondral allograft.

22. The method according to any one of claims 18 to 21, wherein the site of injury is located at the site of bone integration of an implantable prosthesis or at the site of bone integration of allogeneic tissue.

23. The method according to any one of claims 18 to 22, wherein the molecule is administered systemically.

24. The method according to any one of claims 18 to 22, wherein the molecule is administered locally.

25. The method according to any one of claims 18 to 24, wherein the bioactive portion is a bone chondrocyte factor, a bone morphogenetic factor, a tendon morphogenetic factor, or an extracellular matrix (ECM) protein.

26. The method according to claim 25, wherein the osteochondral factor is NELL-1, TGF-β1, TGF-β2, TGF-β3, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-11, BMP-15, or rhBMP.

27. The method according to claim 26, wherein the osteochondral factor is NELL-1 or BMP-2.

28. The method according to claim 25, wherein the tendon formation factor is TGFβ-1, TGFβ-2, TGFβ3, FGF, CTGF, BMP-12, BMP-13, BMP-14, CCN1, or WISP-1.

29. The method according to claim 28, wherein the tendon formation factor is TGFβ-1 or TGFβ-2.

30. The method according to claim 25, wherein the ECM protein is collagen, elastin, fibrin, fibronectin, gelatin, laminin, integrin, CCN1, WISP-1, or CTGF.

31. The method according to any one of claims 18 to 24, wherein the bioactive portion is parathyroid hormone, parathyroid hormone-related protein, growth differentiation factor 11 (GDF11), activin receptor-like kinase-1-Fc (ALK1-Fc), or activin receptor-like kinase-4-Fc (ALK4-Fc).

32. The method according to any one of claims 18 to 31, wherein the cleavable linker is a cathepsin K-sensitive peptide linker.

33. The method according to claim 32, wherein the cathepsin K-sensitive peptide linker is GHPGGPQG (SEQ ID NO: 1) or GGGMGPPSGPWGGK (SEQ ID NO: 2).

34. The method according to any one of claims 18 to 31, wherein the linkable linker is an MMP-cuttable linker.

35. The method according to claim 34, wherein the linker capable of MMP cleavage is PLGLAG (SEQ ID NO: 3), or Ac-GCRD-GPQGIWGQ-DRCG (SEQ ID NO: 4), or Ac-GCRD-GPQGIAGQ-DRCG (SEQ ID NO: 5).

36. The method according to any one of claims 18 to 31, wherein the cleavable linker is a pH-sensitive linker.

37. The method according to any one of claims 18 to 36, wherein the BP portion does not inhibit osteoclast function and / or only minimally inhibits farnesyl pyrophosphate synthase (FPSS).

38. The method according to any one of claims 18 to 37, wherein the BP portion is 2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-PyrEBP), 1-hydroxy-2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS), methylene bisphosphonate (MBP), methylene hydroxyl bisphosphonate (MHDP), etidronate (EHDP), methyl hydroxyl diphosphonate, clodronate, isochlorodronate, tildronate, 2-hydroxy-2-phosphono-3-(pyridine-3-yl)propanoic acid (3-PEHPC), or 2-hydroxy-3-(imidazo[1,2-c]pyridine-3-yl)-2-phosphonopropanoic acid (3-IP-EHPC).

39. A method for treating tendon injuries, fractures, and / or bone stress injuries, and / or promoting the healing of soft tissues to bone, i) The bioactive portion, ii) A linker that can be cut, iii) including a bisphosphonate (BP) moiety, The aforementioned BP portion has a PCP structure, A method comprising administering a molecule in which the cleavable linker links the bioactive portion to the BP portion.

40. The method according to claim 39, wherein the tendon injury is the rotator cuff, distal biceps tendon, pectoralis major, patellar tendon, quadriceps tendon, and / or triceps tendon.

41. A method for performing this procedure on subjects who require enhancement of allograft tissue integration or promotion of tendon attachment repair during or after orthopedic surgery, i) The bioactive portion, ii) A linker that can be cut, iii) including a bisphosphonate (BP) moiety, The aforementioned BP portion has a PCP structure, A method comprising administering a molecule in which the cleavable linker links the bioactive portion to the BP portion.

42. The method according to claim 41, wherein the orthopedic surgery is bone cutting, ACL reconstruction, PCL reconstruction, LCL or MCL repair / reconstruction, medial patellofemoral ligament reconstruction, meniscal root repair, meniscal allograft, and / or osteochondral allograft.

43. The method according to claim 41 or 42, wherein the molecule is administered immediately before the orthopedic surgery.

44. The method according to claim 41 or 42, wherein the molecule is administered during the orthopedic surgery.

45. The method according to claim 41 or 42, wherein the molecule is administered after the orthopedic surgery.

46. A method for performing treatment in subjects requiring treatment for osteoporosis, osteoarthritis, and / or cartilage defects, i) The bioactive portion, ii) A linker that can be cut, iii) including a bisphosphonate (BP) moiety, The aforementioned BP portion has a PCP structure, A method comprising administering a molecule in which the cleavable linker links the bioactive portion to the BP portion.

47. The method according to any one of claims 39 to 46, wherein the molecule is administered systemically.

48. The method according to any one of claims 39 to 46, wherein the molecule is administered locally to the site of the tendon injury or surgical procedure.

49. The method according to any one of claims 39 to 48, wherein the bioactive portion is a bone chondrocyte factor, a bone morphogenetic factor, a tendon morphogenetic factor, or an extracellular matrix (ECM) protein.

50. The method according to claim 49, wherein the osteochondral factor is NELL-1, TGF-β1, TGF-β2, TGF-β3, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-11, BMP-15, or rhBMP.

51. The method according to claim 50, wherein the osteochondral factor is NELL-1 or BMP-2.

52. The method according to claim 49, wherein the tendon formation factor is TGFβ-1, TGFβ-2, TGFβ3, FGF, CTGF, BMP-12, BMP-13, BMP-14, CCN1, or WISP-1.

53. The method according to claim 52, wherein the tendon formation factor is TGFβ-1 or TGFβ-2.

54. The method according to claim 49, wherein the ECM protein is collagen, elastin, fibrin, fibronectin, gelatin, laminin, integrin, CCN1, WISP-1, or CTGF.

55. The method according to any one of claims 39 to 48, wherein the bioactive agent is parathyroid hormone, parathyroid hormone-related protein, growth differentiation factor 11 (GDF11), activin receptor-like kinase-1-Fc (ALK1-Fc), or activin receptor-like kinase-4-Fc (ALK4-Fc).

56. The method according to any one of claims 39 to 55, wherein the cleavable linker is a cathepsin K-sensitive peptide linker.

57. The method according to claim 56, wherein the cathepsin K-sensitive peptide linker is GHPGGPQG (SEQ ID NO: 1) or GGGMGPPSGPWGGK (SEQ ID NO: 2).

58. The method according to any one of claims 39 to 55, wherein the linkable linker is an MMP linkable linker.

59. The method according to claim 58, wherein the linker capable of MMP cleavage is PLGLAG (SEQ ID NO: 3), or Ac-GCRD-GPQGIWGQ-DRCG (SEQ ID NO: 4), or Ac-GCRD-GPQGIAGQ-DRCG (SEQ ID NO: 5).

60. The method according to any one of claims 39 to 55, wherein the cleavable linker is a pH-sensitive linker.

61. The method according to any one of claims 39 to 60, wherein the BP portion does not inhibit osteoclast function and / or only minimally inhibits farnesyl pyrophosphate synthase (FPSS).

62. The method according to any one of claims 39 to 61, wherein the BP portion is 2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-PyrEBP), 1-hydroxy-2-(pyridine-4-yl)ethane-1,1-diylbisphosphonic acid (p-RIS), methylene bisphosphonate (MBP), methylene hydroxyl bisphosphonate (MHDP), etidronate (EHDP), methyl hydroxyl diphosphonate, clodronate, isochlorodronate, tildronate, 2-hydroxy-2-phosphono-3-(pyridine-3-yl)propanoic acid (3-PEHPC), or 2-hydroxy-3-(imidazo[1,2-c]pyridine-3-yl)-2-phosphonopropanoic acid (3-IP-EHPC).

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