Bone targeting therapeutics

Abstract

Provided herein are compositions and methods for targeted regulation of bone resorption. In particular, provided herein are compositions comprising a bone-anchored, activatable agent that provides activated local delivery of therapeutics to mineralized surfaces for localization of therapeutic effects.

Description

[0001] BONE TARGETING THERAPEUTICS STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under DE029359 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING The text of the computer readable sequence listing filed herewith, titled “43834- 601_SEQUENCE_LISTING”, created September 25, 2025, having a file size of 2,751 bytes, is hereby incorporated by reference in its entirety. FIELD Provided herein are compositions and methods for targeted regulation of bone resorption. In particular, provided herein are compositions comprising a bone-anchored, activatable agent that provides activated local delivery of therapeutics to mineralized surfaces for localization of therapeutic effects. BACKGROUND Osteoporosis, a disease of decreased skeletal mass and bone density, affects more than 10 million adults in the US, with direct medical costs estimated at more than $50 billion per year (Compston et al., 2019; Insights, 2019; Tu et al., 2018). Osteoporosis patients experience an increased bone fracture risk. Fractures lead to disability, decreased independence, and death (Black et al., 2020; Rashki Kemmak et al., 2020). Bone loss occurs when the rate of bone degradation exceeds that of bone formation. Traditional anti-resorptive therapies (e.g., bisphosphonates) eliminate bone-degrading cells (osteoclasts) to limit bone loss but inadvertently decrease average bone turnover and new bone formation due to the critical role of osteoclasts in these processes (Tu et al., 2018). Considerable efforts have been made to identify novel anti- resorptive drugs that selectively inhibit bone degradation by osteoclasts without eliminating osteoclasts and negatively affecting bone formation. This includes the development of multiple small molecule inhibitors of the lysosomal protease Cathepsin K (CTSK) (Chen et al., 2016; Dai et al., 2020; Hao et al., 2015) – most prominently, Odanacatib (Merck), which demonstrated strong evidence of efficacy in its phase III clinical trial. Unfortunately, subsequent analysis revealed potential off-target toxicity (i.e., risk of stroke), and the program was discontinued. New solutions are needed. SUMMARY In response to the historic failures and the continued unmet therapeutic need in osteoporosis and other diseases and conditions, the present technology provides efficacious agents, without the unwanted side effects observed with prior therapeutic agents. For example, in some embodiments, provided herein are compositions and methods that locally target resorptive osteoclasts. In some embodiments, provided herein are compositions and methods that use three mechanisms to localize drug activity to the resorptive osteoclast, combining affinity drug targeting to increase accumulation at the bone surface, prodrug formulation to limit off-target toxicity, and activation (e.g., acid activation) to localize drug activity to actively resorbing osteoclasts at sites of active bone turnover. As such, the compositions and methods prevent, slow, stop, or even reverse the progression of osteoporosis and other diseases and conditions. The technology provides a platform for the localization of small molecule and peptide therapeutics to sites of active bone remodeling for treating osteoporosis and other musculoskeletal diseases. Drug development for bone and musculoskeletal diseases has suffered from a lack of specific accumulation of pharmacologic agents at the bone surface, resulting in a need for higher doses and off-target side effects (Wang et al., 2020b). In addition to poor overall biodistribution, drugs may be required at precise times during the bone remodeling cycle and may have adverse effects on bone / cartilage / connective tissue when active at inappropriate stages. A key innovation of the present technology is a combination of physical targeting and prodrug formulation, which allows for specific activity on cells within bone remodeling compartments for disease-specific interventions. This approach allows specific activity on cells within bone remodeling compartments to target the inflammatory osteoclasts responsible for bone erosion. Using this local approach, one can significantly mitigate high therapeutic doses and off-target toxicity concerns, which have hindered the development of recent small molecule candidates in this field. In some embodiments, the compositions and methods utilize three mechanisms to localize drug activity of peptide aldehyde protease inhibitors to resorptive osteoclasts by: 1. affinity targeting to increase local accumulation and retention at the bone surface (BP targeting), 2. prodrug formulation, which limits off-target protease inhibition and toxicity, and 3. acid activation, which releases activated peptide aldehyde protease inhibitors or other compounds within the bone remodeling unit (lacunae). In some embodiments, provided herein are compositions comprising: a) a prodrug of a bone antiresorptive agent; and b) a bone targeting moiety (e.g., wherein the prodrug of the bone antiresorptive agent and the bone targeting moiety are joined by a cleavable linker). In some embodiments, cleavage of the cleavable linker releases and activates the prodrug of the bone antiresorptive agent, thereby producing an active, unbound form of the bone antiresorptive agent. In some embodiments, the bone antiresorptive agent is a peptide aldehyde protease inhibitor. In some embodiments, the bone antiresorptive agent is a CTSK inhibitor. In some embodiments, the CTSK inhibitor is selected from the group consisting of relacatib, balacatinib, odanacatib, and ONO-5334. In some embodiments, the bone antiresorptive agent is a proteasome inhibitor. In some embodiments, the proteasome inhibitor is Mg132. In some embodiments, the bone targeting moiety comprises a phosphonate. In some embodiments, the bone targeting moiety comprises a monophosphonate, a bisphosphonate, or a trisphosphonate. In some embodiments, the bisphosphonate is an alendronate bisphosphonate. In some embodiments, the cleavable linker is an acid-cleavable linker. In some embodiments, the cleavable linker comprises a hydrazone linker. In some embodiments, the prodrug of the bone antiresorptive agent is acid-activated. In some embodiments, the prodrug of the bone antiresorptive agent is acid-activated in response to bone resorption. In some embodiments, the composition is provided as part of a pharmaceutical composition. In some embodiments, the composition is provided on or in an implant. In some embodiments, the pharmaceutical composition comprises: a) a prodrug of a bone antiresorptive agent; b) a bone targeting moiety (e.g., wherein the prodrug of the bone antiresorptive agent and the bone targeting moiety are joined by a cleavable linker), and c) an excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is provided in dosage form (e.g., dosage form of 0.001 mg / kg to 100 mg / kg). Also provided herein are methods of administering a composition to a subject. In some embodiments, the method comprises administering a composition (e.g., a pharmaceutical composition) to subject to treat or prevent a disease or condition. In some embodiments, the subject has osteoporosis, periarticular osteoporosis, osteoarthritis, rheumatoid arthritis, bone fracture healing, periodontal disease, periimplantitis, dental implant osseointegration, alveolar bone regeneration (e.g., ridge augmentation, sinus lift, socket preservation, in combination with bone graft / bone graft substitute / biomaterial or used alone), endodontic applications (e.g., periapical periodontitis, apical abscess, alveolar regeneration after apicoectomy or respective endodontics, pharmacologic agent for use in dental pulp therapy / vital pulp therapy), osteonecrosis of the jaw, osteomyelitis, dental caries arrest / prevention (e.g., anti-caries medicament), dentinal sensitivity, cemental sensitivity, pharmacologic treatment for external or internal root resorption, adjunct to mechanical debridement, scaling, and root planing, or in conjunction with open flap or ostectomy / osteoplasty, heterotopic ossification, primary cancer (e.g., multiple Myeloma, osteosarcoma, chondrosarcoma, chordoma), metastatic cancers or patients at risk for bone metastasis, mantle cell lymphoma, rare bone diseases (e.g., metachondromatosis, pyncodysostosis, Gorham-Stout Disease, Hajdu-Cheney Syndrome, hypophosphatasia), Paget’s Disease, osteolysis, osteopetrosis, hyperparathyroidism, renal osteodystrophy, rickets, osteomalacia, osteogenesis imperfecta, osteonecrosis, osteomyelitis, and / or orthopedic fracture. Further provided herein is the use of any of the above compositions (e.g., pharmaceutical compositions) (e.g., comprising a prodrug of a bone antiresorptive agent and a bone targeting moiety (e.g., wherein the prodrug of the bone antiresorptive agent and the bone targeting moiety are joined by a cleavable linker)). For example, in some embodiments, provided herein is the use of the compositions for the treatment of a bone disease or condition (e.g., osteoarthritis, rheumatoid arthritis, bone fracture healing, periodontal disease, periimplantitis, dental implant osseointegration, alveolar bone regeneration (e.g., ridge augmentation, sinus lift, socket preservation, in combination with bone graft / bone graft substitute / biomaterial or used alone), endodontic applications (e.g., periapical periodontitis, apical abscess, alveolar regeneration after apicoectomy or respective endodontics, pharmacologic agent for use in dental pulp therapy / vital pulp therapy), osteonecrosis of the jaw, osteomyelitis, dental caries arrest / prevention (e.g., anti- caries medicament), dentinal sensitivity, cemental sensitivity, pharmacologic treatment for external or internal root resorption, adjunct to mechanical debridement, scaling, and root planing, or in conjunction with open flap or ostectomy / osteoplasty, heterotopic ossification, primary cancer (e.g., multiple Myeloma, osteosarcoma, chondrosarcoma, chordoma), metastatic cancers or patients at risk for bone metastasis, mantle cell lymphoma, rare bone diseases (e.g., metachondromatosis, pyncodysostosis, Gorham-Stout Disease, Hajdu-Cheney Syndrome, hypophosphatasia), Paget’s Disease, osteolysis, osteopetrosis, hyperparathyroidism, renal osteodystrophy, rickets, osteomalacia, osteogenesis imperfecta, osteonecrosis, osteomyelitis, and / or orthopedic fracture). Definitions Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply. As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a peptide amphiphile” is a reference to one or more peptide amphiphiles and equivalents thereof known to those skilled in the art, and so forth. As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of” and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of” denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of” and / or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language. As used herein, the terms “treat,” “treatment,” and “treating” refer to reducing the amount or severity of a particular condition, disease state, or symptoms thereof, in a subject presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete treatment (e.g., total elimination of the condition, disease, or symptoms thereof). For example, “treating” cancer may refer to reducing the size of a tumor, reducing the number of tumors, eliminating a tumor, reducing the risk of metastasis of a tumor, and the like. As used herein, the terms “prevent,” “prevention,” and preventing” refer to reducing the likelihood of a particular condition or disease state from occurring in a subject not presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete or absolute prevention. The terms “subject” and “patient” are used interchangeably herein and refer to any animal. In some embodiments, the subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). In some aspects, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In some aspects, the mammal is a human. In some aspects, the human is an adult aged 18 years or older. In some aspects, the human has or is suspected of having osteoporosis. The term “bisphosphonate” refers to a class of compounds characterized by two phosphonate groups, and are also referred to as disphosphonates. The structure of bisphosphonates is shown below: The R groups determine the chemical properties of a bisphosphonate and distinguishes individual types of bisphosphonates from one another. Exemplary bisphosphonates include, for example, alendronate, risedronate, etidronate, ibandronate, clodronate, tiludronate, pamidronate, zoledronate, and salts and derivatives thereof. In some embodiments, the bisphosphonate is alendronate (ALD), also referred to as alendronic acid or alendronate sodium. Alendronate has the structure: Description of Figures FIG.1: Sodium alendronate bisphosphonate (ALD), a small molecule with an affinity for hydroxyapatite mineral, is chemically functionalized through additive nucleophilic substitution at its primary amine to yield a hydrazine-modified bisphosphonate (middle). Peptide aldehyde therapeutic payloads are loaded to the hydrazine-modified bisphosphonate vector by hydrazine- aldehyde coupling, yielding a hydrazone-drug adduct. The bisphosphonate moiety provides affinity targeting to the bone surface; the labile hydrazone bond allows for acid-mediated cleavage to release the aldehyde payload (A). Cy5, a far-red fluorescent molecule, is used as a therapeutic payload analog to generate acid-cleavable F1 (ALD-z-Cy5, from Cy5-CHO) and non-cleavable F2 (ALD-Cy5, from Cy5-NHS ester). F1 and F2 are bound to hydroxyapatite beads in vitro and incubated at various pH. Cy5 is detected by spectrophotometer at 640 nm excitation and 670 nm emission (B). F1 models pH-sensitive release at pH below 5.2 but not above, demonstrating its stability at physiologic pH (C). Cathepsin K (CTSK) inhibitor screening assay validates the prodrug formulation and acid-mediated activation of the therapeutic payload (acidified (4)) to inhibit CTSK similar to the free therapeutic warhead (D), while the targeting vector (3), (4) under physiologic conditions, and non-cleavable (6) do not elicit a biologic response. FIG.2: An in vitro bovine bone chip assay was used to evaluate pit and trench formation by osteoclasts stimulated by LPS to induce inflammatory resorption (A). Bone chips were incubated with PBS, Cbz-LLL-CHO, (3) ALD-NHNH2, and (4) GSW001. Pit and trench formation was evaluated by light microscopy using toluidine blue staining. Pit and trench formation was quantified by high-throughput image analysis, and the ratio of pits-to-trenches (B) and absolute number (C, D) are quantified as a function of treatment concentration. FIG.3: Bisphosphonate targeting is highly efficient in increasing the binding coefficient of a model small molecule fluorophore (A). When administered intravenously, ALD-pHrodo, a bone-targeted-pH-sensitive fluorescent analog, efficiently detects local osteoclast activity in vitro in a bone chip assay (B). Administered intravenously in mice, bisphosphonate-targeted fluorophores F1 and F2 rapidly circulate in the blood over 60 minutes (C). Their pH-sensitive bisphosphonate targeted analog localizes to the bone surface in vivo (red) and colocalizes with osteoclasts (green, TRAP staining; blue, DAPI) in histologic sections of the femur in both cortical and trabecular bone (D). FIG.4: The therapeutic efficacy of GSW001 was validated in a murine ovariectomy- induced bone loss model in which healthy female mice underwent bilateral ovariectomy at 14 weeks of age. Starting one week after the surgery, mice were given biweekly injections of 1 mg / kg (4), or equimolar equivalent of (3), Cbz-LLL-CHO, (6), or vehicle control. Mice were sacrificed after four weeks, and femurs were harvested for MicroCT evaluation of the trabecular bone (A). Three-dimensional rendering highlighting the region of interest (B). MicroCT parameters for quantitative analysis: BV / TV = bone volume to total volume fraction (C), Conn dens = connectivity density (D), SMI = structural model index (E), Tb. N = trabecular number (F), Tb.Sp = trabecular spacing (G), BMD = bone mineral density (H). N = 8 mice (16 femurs, average of two femurs = 1 mouse) for each condition. One-way ANOVA was employed for statistical analysis. Data presented as mean ± SEM, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Complete statistical analysis and comparisons are shown in Table 1. Representative 3D rendering of the femoral cross-section for each group. FIG.5: MicroCT evaluation of the cortical bone: three-dimensional rendering highlighting the region of interest at the mid-shaft of the femur (A). MicroCT parameters for quantitative analysis: Ct.Ar / Tt.Ar = cortical volume fraction (B), BMD = bone mineral density (C), TMD = total mineral density (D), and Ct.Th = cortical thickness (E). N = 8 mice (16 femurs, average of two femurs = 1 mouse) for each condition. One-way ANOVA was employed for statistical analysis. Data presented as mean ± SEM, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Complete statistical analysis and comparisons are shown in Table 1. Representative 3D rendering of the femoral cross-section for each group. FIG.6: MicroCT evaluation of the L4 vertebrae trabecular bone (A) Three-dimensional rendering highlighting the region of interest at the L4 vertebrae (B). MicroCT parameters for quantitative analysis: BV / TV = bone volume to total volume fraction (C), Conn dens = connectivity density (D), SMI = structural model index (E), Tb. N = trabecular number (F), Tb.Sp = trabecular spacing (G), BMD = bone mineral density (H). N = 8 mice (16 femurs, average of two femurs = 1 mouse) for each condition. One-way ANOVA was employed for statistical analysis. Data presented as mean ± SEM, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Complete statistical analysis and comparisons are shown in Table 1. Representative 3D rendering of the femoral cross-section for each group. FIG.7: Histologic evaluation of femurs collected from mice to evaluate histomorphometry by hematoxylin and eosin staining and osteoclast number by TRAP staining at high and low magnifications. FIG.8: Schematic representation of the bone-anchored far-red cathepsin K (CTSK) fluorescent sensor, utilizing the consensus sequence for CTSK (A). Osteolytic resorption and proteolysis result in CTSK release at the resorption pit. Cleavage of the bone-anchored peptide substrate releases the fluorescent quencher at the distal terminus of the peptide, resulting in fluorescence evolution and emission in the far-red range. Over 90 minutes, both untargeted (8) and bone-targeted (9) detect CTSK activity in vitro with recombinant CTSK, unaffected by the addition of the targeting moiety (B). When anchored to hydroxyapatite beads in vitro, fluorescent signal evolves after 60 minutes of CTSK exposure from (9), but not (8), in the presence of recombinant CTSK, attributed to its affinity anchorage to the hydroxyapatite beads (C). The fluorescent peptide (9) detects dose-dependent inhibition and modulation of CTSK activity in vitro (D). A subcutaneous calvarial injection model was designed for local inflammatory bone resorption (E). The mechanism by which GSW001 exerts its therapeutic effect was assessed in inflammatory (LPS-stimulated) resorption conditions, compared to (3), Cbz-LLL-CHO, and (6), (F). FIG.9: Graphical representation of an exemplary platform. A bone-targeting moiety (blue) is covalently bound to a therapeutic peptide aldehyde payload (green) via hydrazone formation. This acid-sensitive labile bond inactivates the therapeutic effect of the payload by occupying its aldehyde site. In vivo, this delivery vector anchors on the bone surface and is activated at sites of osteoclast resorption, resulting in acid-catalyzed hydrolysis of the hydrazone bond to regenerate the peptide aldehyde payload. DETAILED DESCRIPTION In some embodiments, the present technology provides a composition comprising a targeted, activatable, therapeutic agent. In some embodiments, the composition is provided as a conjugate comprising a prodrug form (e.g. inactive form) of the activatable agent that is released and activated at a site of interest. For example, in some embodiments the composition is provided as a conjugate comprising a prodrug form of the activatable agent and a bone targeting moiety joined by a cleavable linker. Cleavage of the cleavable linker releases and activates the agent at a target site of interest (e.g. the bone). For example, in some embodiments, the composition is an acid-activated compound that converts from the prodrug form of an agent to the active form of the agent in an acidic microenvironment of a bone surface. In some embodiments, the compound preferentially targets bone. In some embodiments, the activatable agent is a bone antiresorptive agent. In some embodiments, the bone antiresorptive agent is a cathepsin K (CTSK) inhibitor. For example, during the development of embodiments of the technology, a small molecule drug delivery system was created for local inhibition of proteases at sites of bone resorption. Cathepsin K is a cysteine protease integral to age-related bone loss (Dai et al., 2020). Ctsk- / - mice demonstrate a radical reduction in inflammation and bone resorption (Hao et al., 2015) through lack of enzyme function and reduced immune cell-mediated activation of OCs (Heo et al., 2021). Ctsk deletion also increases bone formation by preventing the degradation of periostin (Bonnet et al., 2017). In a mouse model of P. gingivalis-induced periodontal disease, AAV-sh-Ctsk silencing therapy offered 80% protection from bone resorption and reduced inflammatory cytokine expression (Chen et al., 2016). CTSK is directly responsible for inflammatory bone resorption and destruction (Hao et al., 2015). Given Odanacatib's clinical efficacy and commercial enthusiasm, CTSK was chosen as a drug target. As shown in the Example section below, a compound, GSW001, was synthesized, which binds to the bone surface in vitro and in vivo and is activated below a critical threshold pH of 5.2. Unlike previous bone-targeted therapeutics, the therapeutic peptide aldehyde payload is kept inactive until exposed to the acidic environment of the osteoclast resorption lacunae, thereby limiting the possibility of off-target effects. The technology successfully demonstrates CTSK inhibition only when acidified by resorbing osteoclasts and shows significant therapeutic efficacy in an animal model of estrogen deficiency-induced bone loss. Peptide aldehyde protease inhibitors represent a compelling class of therapeutic payloads. Peptide aldehydes are an established class of protease inhibitors in several proteolytic enzymes, including aspartic proteases, cysteine proteases, and serine proteases (Damodaran and Harris, 1995; Fehrentz et al., 1995; Moulin et al., 2007). The inhibitory properties result from the tetrahedral hydrates of the C-terminus aldehyde function that mimics the transition state of the substrate during hydrolysis by the enzyme (Moulin et al., 2007). In GSW001 (4), the primary hydrazine (3) reacts with the terminal aldehyde of Cbz-LLL-CHO to form a hydrazone, disrupting the geometry that mimics the substrate transition state, which we have demonstrated computationally using molecular modeling simulations. In other examples of peptide aldehydes, their function is completely lost following the oxidation of the C-terminal aldehyde to a carboxylic acid or reduction to alcohol (Sarubbi et al., 2001). GSW001 (4), has pleiotropic anti- resorptive and anti-osteoclastogenic effects by simultaneously targeting cathepsin K (CTSK) and RANK-mediated proteasomal proteases. In some embodiments, the bone antiresorptive agent is a peptide aldehyde protease. In some embodiments, the peptide aldehyde protease is a proteasome inhibitor. In some embodiments, the peptide aldehyde protease is a cysteine protease inhibitor (e.g., leupeptin, calpain inhibitor I, calpain inhibitor II, Cbz-Phe-Phe-CHO, Cbz-Phe-o-Tyr-CHO (self-masked aldehyde inhibitor)). In some embodiments, the peptide aldehyde protease is a cathepsin inhibitor (e.g., Cbz-Phe-Phe-CHO (for cathepsin L), peptide aldehydes with Leu, Phe, or similar amino acids at P2 position (for cathepsin L and cruzain), peptide aldehydes with optimized sequences for cathepsin K (see e.g., Votta 1997)). In some embodiments, the peptide aldehyde protease is an inflammatory process inhibitor (e.g., Ac-YVAD-CHO (caspase-1 inhibitor)). In some embodiments, the bone antiresorptive agent is ALD-z-DAVY-Ac (from Ac-YVAD-CHO), which is a potent inhibitor of interleukin converting enzyme. In some embodiments, the bone antiresorptive agent is an agent that counteracts a downstream result of CTSK expression or overexpression. For example, in some embodiments, the bone antiresorptive agent is a proteasome inhibitor that rescues the downregulation of SIAH1 expression induced by CTSK overexpression. In some embodiments, the bone antiresorptive agent is a peptide aldehyde. In some embodiments, the bone antiresorptive agent is MG132. Peptide aldehydes are robust protease inhibitors because they form reversible covalent bonds with the active-site nucleophiles of proteases, such as the hydroxyl group of serine or the sulfhydryl group of cysteine (Harer et al., 2012). This interaction typically involves the formation of hemiacetals or hemithioacetals, which are crucial for the inhibition mechanism, allowing peptide aldehydes to effectively block the proteolytic activity of both serine and cysteine proteases (Votta et al., 1997). Cbz-LLL-CHO, also known as MG132, is a peptide aldehyde proteasome that inhibits proteasome activity. MG132 is a reversible inhibitor extensively utilized in various studies to understand its effects on cellular processes, particularly in cancer research (Costanzi et al., 2021; Kim and Kim, 2020; Xu et al., 2022; Zeng et al., 2019). The aldehyde moiety in peptide aldehyde inhibitors like MG132 is critical for their efficacy. This highly electrophilic functional group allows it to form reversible covalent bonds with the active- site nucleophiles of proteases. As such, in designing targeted prodrugs, it is optimal to preserve the aldehyde moiety to maintain the inhibitor's binding affinity and specificity to the target protease. Cbz-LLL-CHO (MG132) is primarily known as a proteasome inhibitor, and its effects on CTSK signaling suggest a broader impact on bone metabolism than previously understood (Kim and Kim, 2020; Xu et al., 2022; Zeng et al., 2019). Modifying CTSK activity indirectly through proteasome inhibition opens new avenues for therapeutic strategies targeting bone diseases when coupled with an appropriate delivery vector that localizes its therapeutic activation specifically to the osteoclast resorption lacunae or disease site. Inhibition of CTSK can lead to reduced bone resorption and potentially increased bone formation, as seen in models where CTSK deletion enhances bone formation by altering the balance of osteoclast and osteoblast activity (Bonnet et al., 2017; Borggaard et al., 2020b; Chen et al., 2016; Dai et al., 2020; Hao et al., 2015; Heo et al., 2021; Votta et al., 1997). Other proteasome inhibitors that find use in the technology include, but are not limited to, MG115 (Cbz-Leu-Leu-norvalinal) and Z-IE(OtBu)AL-H. In some embodiments, the targeting component comprises a phosphonate. In some embodiments, the targeting component comprises a monophosphonate. In some embodiments, the targeting component comprises a bisphosphonate. In some embodiments, the targeting component comprises a trisphosphonate. In some embodiments, the targeting component comprise tetracycline. In some embodiments, the composition comprises a bisphosphonate to provide targeting. Bisphosphonates are a class of drugs developed in the 1970s and continued to be used primarily to treat bone diseases characterized by excessive bone resorption, such as osteoporosis, Paget's disease, and bone metastases from cancer. They function by inhibiting osteoclast-mediated bone resorption by inducing osteoclast apoptosis, attributed to the terminal primary amine (Drake et al., 2008; Fleisch, 1998; Reszka et al., 1999; Rogers et al., 2000). The high affinity of bisphosphonates for hydroxyapatite crystals in bone makes them particularly effective in targeting bone tissue, which has led to their use in affinity-targeting platforms for therapeutic delivery (Farrell et al., 2018). By conjugating bisphosphonates with other therapeutic agents, it is possible to deliver drugs specifically to bone tissue (Sun et al., 2021). In some embodiments, composition comprises the activatable agent (e.g. bone antiresorptive agent) in a prodrug form, bound to a bone targeting moiety (e.g. a bisphosphonate) by a cleavable linker. In the prodrug form, the bone antiresorptive agent is inactive. For example, in some embodiments the bone antiresorptive agent is a peptide aldehyde (e.g. a peptide aldehyde protease inhibitor) bound to the bound targeting moiety by a hydrazone linker which occupies the aldehyde site of the peptide aldehyde protease inhibitor. This occupation of the aldehyde site inactivates the peptide aldehyde protease inhibitor, thus in bound state the peptide aldehyde protease inhibitor is inactive, e.g. a prodrug form. In some embodiments, cleavage of the cleavable linker releases and activates the prodrug of the bone antiresorptive agent, thereby producing an active, unbound form of the bone antiresorptive agent. For example, cleavage of the hydrazone bond releases the occupation of the aldehyde site of the peptide aldehyde protease inhibitor, thereby activating the agent and producing an unbound, active form. The use of activatable prodrug compositions of the present technology provides efficacious localization of active agent activity, without potentially harmful activity in regions of the tissue (e.g., bone) that might otherwise be targeted by a targeting agent. Targeting the resorption process, or subtypes of inflammatory or aggressive resorption, involves drug delivery vectors with distinctive chemical functionalities to facilitate their precise activation and localization. Indiscriminate targeting of the bone surface, like that accomplished with bisphosphonate affinity ligands, does not account for the specific cellular environments or disease states within the bone, which can lead to suboptimal therapeutic outcomes. For instance, bisphosphonates may not effectively target specific diseased areas or cells, potentially leading to toxicity in healthy bone cells or physiologic resorption sites, thus limiting the efficacy of the drug delivery system. In some embodiments, the cleavable linker is acid-cleavable. In some embodiments, the prodrug of the bone antiresorptive agent is acid activated. As such, in some embodiments in an appropriately acidic environment (e.g. the acidic environment of the osteoclast resorption lacunae) the acid-cleavable linker is cleaved, thus releasing the prodrug from the bone targeting moiety, and the prodrug is activated, thereby producing the bone antiresorptive agent in its unbound, active form. In some embodiments, the cleavable linker comprises a hydrazone bond. Hydrazones are organic compounds formed through the reaction of hydrazines with aldehydes or ketones, resulting in a hydrazone linkage characterized by a C=N-NH bond. This reaction involves the nucleophilic attack of the hydrazine nitrogen on the carbonyl carbon of the aldehyde, leading to the formation of an intermediate that subsequently loses water to form the stable hydrazone compound. Hydrazones exhibit notable acid sensitivity, which allows their use in drug delivery systems that exploit pH changes (Sonawane et al., 2017; Verma et al., 2014). Under acidic conditions, such as those found in specific biological environments, hydrazone linkages can undergo hydrolysis, leading to the release of the drug (Yildiz, 2016). Hydrazone-based drug delivery systems have been explored for their ability to release drugs in response to pH changes. For instance, hydrazone linkages have been utilized in polymeric micelles to achieve controlled, pH-dependent release of paclitaxel, where the drug is released more rapidly in acidic environments such as those found in tumors (Liu et al., 2023). Another example is the use of hydrazone-containing triblock copolymeric micelles, which facilitate the release of doxorubicin in acidic subcellular compartments like endosomes and lysosomes, enhancing the drug's efficacy against cancer cells (Qi et al., 2018). In some embodiments, the acid-sensitive release of compositions of the present technology find use in targeting osteoclast resorption activity, where the acidic microenvironment generated during bone resorption triggers the release of drugs from a hydrazone-based delivery system. In designing osteoclast-modulating therapeutics, it is important to consider the intricate coupling between osteoclasts and osteoblasts to avoid indiscriminately targeting or eliminating all osteoclasts. Osteoclasts are responsible for bone resorption, while osteoblasts are involved in bone formation, and their activities are tightly coupled to ensure that bone resorption by osteoclasts is followed by bone formation by osteoblasts, a process essential for healthy bone remodeling. Disrupting this balance, such as through excessive inhibition of osteoclasts can impair the release of osteoclast-derived coupling factors that are vital for osteoblast differentiation and function (Chen et al., 2018; Kim et al., 2020; Zhu et al., Int. J. Biol. Sci.2022, Lotinun et al., J. Clin. Invest., 2013). Therefore, therapeutics like GSW001 should aim to modulate osteoclast activity without completely abolishing it, preserving the necessary signaling for osteoblast activation and maintaining the balance of bone remodeling. This approach can help prevent the adverse effects associated with traditional antiresorptive therapies, such as reduced bone formation and compromised bone strength, ultimately leading to more effective and sustainable treatment outcomes for osteoporosis (Sims and Martin, 2015; Takegahara et al., 2024). The compositions find use to treat and / or prevent a number of different diseases and conditions. For example, in some embodiments, the compositions find use in the treatment or prevention of osteoporosis, periarticular osteoporosis, osteoarthritis, rheumatoid arthritis, bone fracture healing, periodontal disease, periimplantitis, dental implant osseointegration, alveolar bone regeneration (e.g., ridge augmentation, sinus lift, socket preservation, in combination with bone graft / bone graft substitute / biomaterial or used alone), endodontic applications (e.g., periapical periodontitis, apical abscess, alveolar regeneration after apicoectomy or respective endodontics, pharmacologic agent for use in dental pulp therapy / vital pulp therapy), osteonecrosis of the jaw, osteomyelitis, dental caries arrest / prevention (e.g., anti-caries medicament), dentinal sensitivity, cemental sensitivity, pharmacologic treatment for external or internal root resorption, adjunct to mechanical debridement, scaling, and root planing, or in conjunction with open flap or ostectomy / osteoplasty, heterotopic ossification, primary cancer (e.g., multiple Myeloma, osteosarcoma, chondrosarcoma, chordoma), metastatic cancers or patients at risk for bone metastasis, mantle cell lymphoma, rare bone diseases (e.g., metachondromatosis, pyncodysostosis, Gorham-Stout Disease, Hajdu-Cheney Syndrome, hypophosphatasia), Paget’s Disease, osteolysis, osteopetrosis, hyperparathyroidism, renal osteodystrophy, rickets, osteomalacia, osteogenesis imperfecta, osteonecrosis, osteomyelitis, and / or orthopedic fracture. In some embodiments, the compositions are used prophylactically to prevent or delay onset of disease. In some embodiments, the compositions are used therapeutically to halt the progression of disease. In some embodiments, the compositions are used regeneratively, to reverse the progression of disease. Drug compositions may be formulated in any of a variety of suitable ways. For example, in some embodiments, the compositions are adsorbed to particulate bone graft material or bone graft substitute, such as particulate hydroxyapatite or a mineralized biomaterial and the graft material is administered to a subject. In some embodiments, the compositions are adsorbed or administered with a dental or orthopedic implant or irrigated at the osteotomy site. In some embodiments, the compositions are combined with a biomaterial delivery vector (e.g., nanoparticles, liposomes, etc.). In some embodiments, the compositions are combined with a biomaterial scaffold or synthetic grafting substitute (e.g., hydrogel, microspheres, polymer scaffolds, 3D printed scaffolds, etc.). In some embodiments, the compositions are formulated with a gastric coating for oral administration. In some embodiments, the compositions are formulated as a tablet, capsule, solution, suspension, or emulsion. In some embodiments, the compositions are formulated in a controlled-release formulation, extended-release formulation, delayed-release formulation, or pulsatile-release formulation. In some embodiments, the compositions are administered to subjects in combination with one or more additional agents or therapeutic or diagnostic approaches. The agents may be formulated together, administered together, or administered separately, but sequentially. Agents or therapeutic or diagnostic approaches for use in combination include, but are not limited to, neutralizing monoclonal antibodies (e.g., anti-RANKL, anti-Sclerostin, anti-TNFα), a bisphosphonate, a chemotherapeutic agent, cell transplantation, surgical resection, prosthesis implantation, an implantable device (e.g., a prosthetic implant), a biomaterial scaffold, distraction, osteogenesis, and / or mechanical debridement or repositioning. In some embodiments, the present invention provides pharmaceutical compositions comprising any of the compositions described herein. Such pharmaceutical compositions may optionally comprise one or more additional therapeutically-active substances. In accordance with some embodiments, a method of administering a pharmaceutical composition comprising inventive compositions to a subject in need thereof is provided. In some embodiments, inventive compositions are administered to humans. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and / or dogs; and / or birds, including commercially relevant birds such as chickens, ducks, geese, and / or turkeys. The pharmaceutical compositions may be administered by any route. In some embodiments, the pharmaceutical compositions of the present invention are administered by a variety of routes, including oral, implant, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and / or drops), transdermal, mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with one or more excipients and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit. A pharmaceutical composition of the invention may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The relative amounts of the active ingredient, the pharmaceutically acceptable excipient(s), and / or any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient. Pharmaceutical formulations of the present invention may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21St Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Md., 2006) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents. In certain embodiments, the compositions of the invention may be administered at therapeutic agent in amounts ranging from about 0.001 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 20 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, or from about 1 mg / kg to about 25 mg / kg, of subject body weight. The compositions may be administered at any suitable dosing frequency. For example, the compositions may be administered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks, every two months, every three months, every four months, every six months, every seven months, every eight months, every nine months, every ten months, every 11 months, yearly, or less than one time per year. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). In some embodiments, provided herein are kits comprising one or more of the compositions. For example, in some embodiments, a kit comprises a drug composition and instructions for use. A kit may comprise any of a number of additional components or reagents in any combination. All of the various combinations are not set forth explicitly but each combination is included in the scope of the invention. Kits will generally include one or more vessels or containers so that some or all of the individual components and reagents may be separately housed. Kits may also include a components for enclosing individual containers in relatively close confinement for commercial sale, e.g., a plastic box, in which instructions, packaging materials such as styrofoam, etc., may be enclosed. An identifier, e.g., a bar code, radio frequency identification (ID) tag, etc., may be present in or on the kit or in or one or more of the vessels or containers included in the kit. An identifier can be used, e.g., to uniquely identify the kit for purposes of quality control, inventory control, tracking, movement between workstations, etc. EXAMPLES To enhance local accumulation and safety, we incorporated a small-molecule CTSK inhibitor into a drug delivery system that combines affinity targeting to the bone surface, prodrug formulation to prevent off-target activity, and OC-mediated activation. GSW001 was synthesized from a bisphosphonate derivative and peptide aldehyde inhibitor of CTSK (CTSKi). We characterized targeting to bone and pH-sensitive release using fluorescent analogs. A commercial assay assessed the inhibition of recombinant CTSK, and activation by OC was confirmed on bone chips. We tested efficacy in 14-week-old female mice undergoing bilateral ovariectomy, comparing GSW001 to free CTSKi, functionalized bisphosphonate alone, a non-cleavable form of the conjugate (GSW001NC), and vehicle controls.1 mg / kg doses or equimolar equivalents were given biweekly and outcomes were assessed via microcomputed tomography (uCT), histomorphometry, and TRAP staining.1-way ANOVA with the Turkey test was used to determine significance. At 24 hours post-injection, a fluorescent analog of GSW001 demonstrated retention at the bone surface (Kd = 25 uM). GSW001, but not GSW001NC, demonstrates pH-responsive release below a critical threshold of pH 5.2, above which minimal release is measured. At physiologic pH, GSW001 demonstrated enzyme inhibition only at the highest concentration (100 nM) and was less effective than a 10-fold lower free drug concentration. Acidified GSW001 is a potent inhibitor of CTSK as low as 0.1 nM. In vitro, GSW001 selectively inhibits LPS-induced OCs to favor pit resorption over trench resorption. In ovariectomized mice, biweekly injection of GSW001 (0.2956±0.01) produced significant increases in the femur and L4 vertebral BV / TV, as compared to CTSKi (0.181±0.02, p<0.0001), GSW001NC (0.177±0.01, p<0.0001) and vehicle control (0.181±0.0.01, p<0.0001). Other indices demonstrated a similar pattern between groups, with significantly increased connective tissue density (p<0.0001) and decreased SMI in GSW001-treated mice (p<0.0001). GSW001 shows no toxicity toward reducing TRAP+ OC number. Materials & Methods Materials: Unless otherwise specified, all materials are purchased from Sigma Aldrich and used as received. Synthesis of GSW000 delivery platform (3): Scheme 1: Synthesis of (1) to prepare the hydrazine-containing moiety for conjugation to sodium alendronate bisphosphonate. The tetrafluorophenol group provides a favorable leaving group for subsequent nucleophilic substitution, as shown in Scheme 2.

[0002] Synthesis of (1), Scheme 1: Tri-boc-hydrazine acetate (90 mg, 0.23 mmol) and 2,3,4,5- tetrafluorophenol (1.0 mol eq., 38.38 mg, 0.23 mmol) are dissolved in 5.00 mL chloroform at room temperature. N’-dicyclohexylcarboiimide (DCC, 1.10 mol eq, 52.32 mg, 0.254 mmol) is dissolved in 5.00 mL chloroform at room temperature. The solution containing DCC is added dropwise to the first solution over five minutes. Substantial dicyclohexylurea precipitate is observed after thirty minutes. Thin layer chromatography using normal phase silica is used to assess reaction progress (85% hexane, 15% acetonitrile mobile phase, F254 normal phase silica), assessed by UV+ species. The solvent is evaporated by rotary evaporation. The product is redissolved in hexane and filtered by gravity filtration. The solvent is removed by rotary evaporation, and the product is separated on a neutral silica column using 85% hexane and 15% acetone mobile phase, yielding (1). Rf(product) = 0.55-0.5; Rf(TFP) = 0.25. The isolated product (1) is immediately used in the subsequent step. ESI-MS: [M+Na]+ = 561.1838 (calc = 561.183), [M+K]+ = 577.156 (calc = 577.157).1H NMR (CDCl3): 1.43-1.62 ppm (mm, Boc CH3, 27H), 4.46-4.51 ppm (dd, CH2, 2H), 7.26-7.27 ppm (s, CH, 1H). Scheme 2: The activated boc-protected hydrazine (1) is reacted with sodium alendronate by nucleophilic substitution, catalyzed by triethylamine base, yielding (2), a boc-protected hydrazine bisphosphonate—deprotection with HCl results in hydrazine bisphosphonate (3). The crude product (2) is extracted against hexane three times to remove residual triethylamine and (1). Then, it is extracted against ether three times to remove residual tetrafluorophenol. The product is diluted with deionized water and lyophilized to recover a solid, which is partially pure, still containing excess alendronate. The compound is separated on a neutral silica column with a 5:1 ratio of acetonitrile to water.1H NMR confirms the removal of triethylamine (absence of 0.970 ppm, 2.425 ppm peaks);19F NMR confirms the removal of tetrafluorophenol (-144 ppm; expect no peaks in a pure compound). Rf(2) = 0.80, Rf(organic SM) = 1.0, Rf(ALD SM) = 0.0. Unreacted alendronate remains on the column. The purity and identity of products (2) are confirmed by TLC to identify fractions for subsequent use and lyophilization. ESI-MS: [M-H]- = 620.199 (calc = 620.197), [M-Boc]- = 520.15 (calc = 520.145), [M-2Boc]- = 420.17 (calc = 420.165).1H NMR (CDCl3): 1.22-1.42 ppm (mm, Boc CH3, 27H), 1.62-1.64 ppm (m, CH2, 2H), 2.00-2.03 (m, CH2, 2H), 3.64-3.70 (m, CH2, 2H), 4.03-4.07 (m, CH2, 4H), 6.0-6.5 ppm (m, OH, 5H).31P NMR (CDCl3): 18.14-18.25 (t). Synthesis of (3), Scheme 2: Immediately before use in subsequent synthesis, (2) is deprotected. Treating solid (2) with 1M hydrochloric acid, diluted in methanol, for three hours at room temperature. HCl is removed, and the product is diluted with deionized water, which is recovered by lyophilization. ESI-MS and 1H NMR monitor deprotection. The dry powder (3) is washed thrice with ethanol to remove residual organics. The product remains solid and is separated by centrifugation at 10,000 rpm for 5 minutes for each of the three washes, and ethanol is discarded. The compound is dried under vacuum or lyophilization and reconstituted at 5 mg / mL in deionized water. ESI-MS: [M+H]+ 322.05 (calc = 322.055).1H NMR (D2O): 1.17- 1.21 (m, CH2, 2H), 1.74-1.77 (m, CH2, 2H), 3.12 (m, CH2, 2H), 3.20 (m, CH2, 2H), 5.55-3.58 (s, NH2, 2H), 3.70 (s, NH, 2H), 4.67-4.85 (m, broad, OH, 5H).31P NMR (D2O): 18.72 ppm (s). Scheme 3: Hydrazine bisphosphonate (3) is conjugated to the peptide aldehyde cathepsin K protease inhibitor Cbz-LLL-CHO (Mg132) by hydrazine-aldehyde coupling in situ, yielding (4), GSW001. Synthesis of GSW001 (4), Scheme 4: 7.0 mg (1.0 mol. eq.) of compound (3) is constituted in a minimum volume of deionized water to dissolve the substrate, yielding a clear, colorless solution. Separately, 11.39 mg (1.1 mol. eq.) Cbz-LLL-CHO is constituted in a minimum volume of 200 proof EtOH, roughly at 20 mg / mL, yielding a clear colorless solution, considered 1.0 vol. eq., with an equal volume of DMSO. Compound (3) is diluted to 1.5 vol. eq. total volume in ddH2O.0.02 vol. eq.1M HCl(EtOH) is added to the peptide solution in ethanol. The solution of (3) is added dropwise to the peptide solution over 5 minutes. The resulting solution is reacted at room temperature for 12 hours; ESI-MS and TLC monitor the reaction progress (reverse phase C18 silica, 5% acetonitrile, 95% ddH2O mobile phase). The resulting product is isolated by column chromatography using a C18 reverse phase silica SepPak cartridge and isocratic 5% acetonitrile; 95% deionized water gradient. Fractions are collected and checked by ESI-MS and TLC under the same conditions. The fractions containing the product were combined, and the product was diluted in deionized water and isolated by lyophilization. The product is stored lyophilized at -80C for further use and reconstituted in PBS or 0.9% NaCl saline solution for future use. ESI-MS: [M+H]+ = 779.373 (calc = 779.35).1H NMR (D2O): 0.81-0.85 ppm (mm, Leu CH3, 18H), 1.38-1.40 (mm, CH2, 6H), 1.47 (m, BP CH2, 2H), 1.77 (m, Leu CH2, 6H), 1.90-1.94 (m, BP CH2, 2H), 2.97 (m, BP CH2, 2H), 3.13.3-21 (m, BP CH2, 2H), 3.71-3.73 (m, Leu CH, 2H), 4.70-4.95 (s, OH, 5H), 5.02 (m, Cbz CH2, 2H), 7.33-7.35 (mm, Cbz C=CH, 5H), 7.37 (m, HC=N, 1H). While Cbz was used as the protecting group at the terminus, a number of alternative protecting groups could be used including, but not limited to, Boc, Fmoc, Ac, Troc, Teoc, PMB, Trityl, Bz, Piv, MOM, MEM, various aliphatic ethers (benzyl ether, methyl ether, ethyl ether, tBut ether), Ts / Ms, methoxyphenyl (PMP), and hydrazone derivatives. Scheme 4: A non-cleavable analog was synthesized to evaluate the hypothesis that acid activation is important to the therapeutic efficiency of GSW001. In Step 1, Cbz-LLL-CHO, the therapeutic peptide aldehyde was oxidized by oxone in DMF, yielding Cbz-LLL-COOH, (5). In Step 2, (5) is coupled NHS-ester and conjugated to sodium alendronate (bisphosphonate) by EDC-coupling, yielding (6) the non-cleavable bisphosphonate-targeted Cbz-LLL molecule. Synthesis of Cbz-LLL-COOH (5), Scheme 5: 5.0 mg solid Cbz-LLL-CHO is constituted in DMF at 20 mg / mL and subject to ozone-mediated oxidation as first reported by Travis et al., Organic Letters 2003.1 mol. eq Oxone reagent (6.25 mg) was added to the DMF solution at once and then reacted at room temperature for three hours. After three hours, the resulting product was treated with 1M HCl (aq), and the oxidized peptide was extracted into ethyl acetate, then washed with brine and dried with Na2SO4. Care was taken to avoid any alcohol-containing species reported to lead to ester product formation. After removing the solvent, a white solid was recovered and reconstituted in DMF for subsequent ESI-MS analysis. ESI-MS: [M+H]+ = 506.33 (506.327 calc), [M+NH4]+ = 523.46 (523.35 calc). The product was immediately used for subsequent steps. Synthesis of GSW001NC (6), Scheme 4: Solid Cbz-LLL-COOH is recovered by recrystallization.0.93 mg (2 mol. eq.) N-hydroxuccinimide (NHS) and 1.26 mg (92 mol. eq.) 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were dissolved in 50 uL dimethylformamide (DMF). This 50 uL DMF solution was added to a vial containing 2.0 mg Cbz-LLL-COOH. Separately, 2.02 mg (2 mol. eq.) sodium alendronate (ALD) trihydrate was constituted in 100 uL ddH2O containing 3.0 uL triethylamine (5 mol. eq.) and agitated until a clear colorless solution as formed. The ALD solution was slowly added dropwise to the peptide solution in DMF and reacted at room temperature. ESI-MS assessed reaction progress. The resulting product was separated on a normal phase silica column using a 65% acetonitrile and 35% deionized water solution; fractions were collected and assayed for the product. Rf product = 0.80. The product fractions were combined and diluted with deionized water, lyophilized, and stored at -80C. ESI-MS: [M+H]+ = 736.32 (calc = 736.317).

[0003] Scheme 5: (3) is coupled to Cy5-CH0, a fluorescent aldehyde-containing molecule analogous to an aldehyde-based therapeutic payload.

[0004]

[0005] Synthesis of ALD-z-Cy5 (Fl), Scheme 5: Cy5-CHO was purchased from AAT Bioquest and used as received. 2.0 mg Cy5- CHO was withdrawn from a 10 mg / mL stock solution of Cy5-CHO in DMSO. Separately, compound (3) was constituted in a deionized water stock solution, and 3.77 mg (5 mol. eq.) was added slowly to the Cy5-CHO solution and reacted at room temperature for 12 hours. The identity of the product was confirmed by ESI-MS: [M+3H]3+ = 378.785 (calc = 378.787), [M+2H]2+ = 567.679 (calc = 567.677).

[0006] Scheme 6: Sodium alendronate bisphosphonate is coupled to Cy5-NHS ester, a fluorescent molecule, yielding a non-cleavable bisphosphonate-targeted fluorophore.

[0007] Synthesis of ALD-Cy5 (F2), Scheme 6: Cy5-NHS ester was purchased from Lumiprobe and used as received. (F2) was synthesized by combining 1.0 mol. eq Cy5-NHS-ester with 5 mol. eq. sodium alendronate trihydrate, dissolved in deionized water and activated with triethyl amine, reacted at room temperature for 12 hours. ESI-MS confirmed the product's identity: [M+H]+ = 888.235 (calc = 888.237). Acid-sensitive release kinetics: To ensure pH stability, the fluorescence of F1 and F2 incubated at various pH at 37C over time was assessed, and standard curves were generated using a fluorescence plate reader. Hydroxyapatite beads were prepared and functionalized with the colorimetric reagents ALD-z-Cy5 and ALD-Cy5. The study was conducted over 100 hours, with the pH of the solution ranging from 4 to 7.4. The pH was adjusted using a phosphate- buffered solution, and the test compounds' stability was monitored. The pH of the solution was controlled using a CO2 / HCO3− buffer system, which is physiologically relevant and commonly used in such studies. Aliquots were taken every 4 hours to determine the release of the colorimetric reagents over time. The fluorometric response was analyzed using a plate-reader platform. Samples were digested with 14% EDTA at the endpoint to determine the total amount of compound remaining on the beads after release. CTSK inhibition assay: A commercially available Cathepsin K Inhibitor Screening Kit (Fluorometric) was purchased and used according to the manufacturer’s directions (Abcam, ab185439) to determine the ability of (4) to inhibit CTSK activity in vitro. Stock solutions of Cbz-LLL-CHO, (3), (4), and (6) were prepared. Separately, an aliquot of (4) was acidified at pH 4 for one hour, neutralized, and diluted in PBS to the same concentrations as the stock solution prepared and not subjected to acidification. The assay was repeated in triplicate with three independent biologic replicates. Osteoclast fate modulation: Long bones were dissected from 4–6-month C57Bl / 6 female mice. The epiphyses were removed from both femurs and tibias, and the bone marrow was flushed with 1X PBS. Bone marrow was treated with Red Cell Lysis Buffer (eBioscience, Inc., San Diego, CA, USA). Cells were plated in 10 cm dishes (Corning, Corning, NY, USA) with α-MEM supplemented with 10% (vol / vol) fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT, USA) and 25 ng / mL macrophage colony-stimulating factor (M-CSF) for 24 hours. Cells were then plated into 24-well plates at 4e5 / well with osteoclast differentiation media (100 ng / mL RANKL and 25 ng / mL macrophage colony-stimulating factor (M-CSF)) for 72 hours. Cells were lifted using 100uL / well of 0.02% EDTA (Bio-rad Laboratories) and P100 pipette tips to scrape the bottom of each well. Bovine femur bone slices (Fisher (immunodiagnostic systems) were incubated for 60 minutes at room temperature with compounds and washed three times with DPBS. After 72 hours of differentiation, osteoclasts were plated on the coated bone slices at 1x105 per bone slice. After 24 hours, OCs were lysed, and bone slices were polished in ddH2O with a cotton swab, washed, dried, and stained with toluidine blue (1% toluidine blue (w / v) in 1% sodium borate (w / v)). To determine proteolytic phenotype, bone chips were first treated with NHS-ester-AF647. This fluorescent molecule conjugates to free primary amine proteins on the bone chip's surface and then incubates with the test compounds described above. Quantification of Bone Resorption: Bone slices were analyzed by light microscopic examination, and quantifications were performed blinded to trace the resorption pit borders across the slices. The quantifications were performed using Fiji. Determination of Kd: ALD-FITC was synthesized as a model drug for a bisphosphate- targeted fluorophore. Fluorescein isothiocyanate is dissolved in DMSO at 5 mg / mL. Separately, sodium alendronate trihydrate is dissolved in a 0.1 M carbonate buffer adjusted to pH 9.4 at 2 mg / mL.2.38 mL of 5 mg / mL FITC-ISO was slowly added to 5 mL of BP solution in water and reacted in the dark for 30 minutes. The product was diluted and lyophilized to recover the product.31P NMR was used to determine unreacted BP.31P NMR (D2O) = 17.94 ppm. The bone-binding properties were confirmed by briefly dissolving the product, ALD-FITC, in PBS at 0.5 mg / mL and incubating with a bovine bone chip at 37C on a shaker plate, compared to PBS and untargeted fluorescein for 60 minutes. The bone chips were washed 5x with PBS; then, the chips were imaged by confocal laser microscopy using a 488 nm laser to assess fluorescence. The binding affinity (Kd) of the bisphosphonate-targeted fluorophore ALD-FITC to hydroxyapatite was determined using a fluorescence-based assay. Hydroxyapatite beads (50 mg) were incubated with increasing concentrations of ALD-FITC (0.1 nM to 100 μM) in 1 mL of phosphate-buffered saline (PBS, pH 7.4) for 2 hours at room temperature with gentle shaking. The beads were then washed three times with PBS to remove unbound fluorophore. The fluorescence intensity of the bound ALD-FITC was measured using a plate reader (excitation 485 nm, emission 528 nm). A standard curve of ALD-FITC fluorescence intensity versus concentration was used to determine the amount of bound fluorophore. The data were fitted to a one-site binding model using nonlinear regression to calculate the Kd. The same procedure was performed as a control using non-targeted fluorescein at equivalent concentrations. The specific binding of ALD-FITC was calculated by subtracting the nonspecific binding of fluorescein from the total binding of ALD-FITC at each concentration. The Kd was determined from the concentration of ALD-FITC at which half-maximal specific binding was observed. In vivo pharmacokinetic assay: pH-sensitive probe assay: Commercially available pHrodo STP-ester (Thermo Fisher, pHrodoTM iFL STP ester) was conjugated to sodium alendronate to confer bone affinity (termed ALD-pHrodo), separated by column chromatography, and used to identify sites of active bone resorption in vitro and in vivo. In vitro, the probe was incubated with a bovine bone chip before inoculation with primary osteoclasts, as described above. In vivo, 1 mg / kg ALD-pHrodo was administered intravenously to 4-month-old C57BL / 6 female mice. After four hours, the mice were sacrificed, and femurs were collected. All animal procedures followed a protocol approved by the University of Michigan Institutional Animal Care and Use Committee (IACUC). Femurs were collected, and soft tissue was removed in 4% paraformaldehyde before fixation for 72 hours. Femurs were placed into a 12% sucrose solution for 72 hours. Femurs were washed in PBS once and embedded in OCT (Optimal Cutting Temperature Embedding Medium) (Fisher) in plastic cassettes. Samples were sectioned at 8 um thickness from frozen tissue blocks. ELF™ 97 Phosphatase Substrate (ELF™ 97 Phosphate), 0.2 µm Filtered (Invitrogen) (Catalog number: E6588) was applied to vertebral sections in conjunction with the Acid Phosphatase Leukocyte (TRAP) Kit (Sigma-Aldrich) (Catalog number: 387A) to determine osteoclast number, and mounted for microscopy using ProLongTM Gold Antifade mountant with DAPI. In vivo osteoclast toxicity: C57BL / 64-month-old female mice were injected intravenously with either GSW001 or equimolar sodium alendronate. Vehicle mice were injected with an equal volume of 1X DPBS saline solution. Mice were euthanized 24 hours post- injection, and long bones and vertebrates were collected. All animal procedures followed a protocol approved by the University of Michigan Institutional Animal Care and Use Committee (IACUC, PRO00009377). L4 vertebrates were collected, and soft tissue was removed before fixation in 4% paraformaldehyde for 72 hours. Vertebrates were placed into a 12% sucrose solution for 72 hours. Bones were washed in PBS once and embedded in OCT (Optimal Cutting Temperature Embedding Medium) (Fisher) in plastic cassettes. Samples were sectioned at 8 um thickness from frozen tissue blocks. ELF™ 97 Phosphatase Substrate (ELF™ 97 Phosphate), 0.2 µm Filtered (Invitrogen) (Catalog number: E6588) was applied to vertebral sections in conjunction with the Acid Phosphatase Leukocyte (TRAP) Kit (Sigma-Aldrich) (Catalog number: 387A) to determine osteoclast number, and mounted for microscopy using ProLongTM Gold Antifade mountant with DAPI. Surgical ovariectomy model: 13-week-old healthy female C57BL / 6 mice were received from Jackson Laboratories (000664). Surgical ovariectomy (OVX) was performed as previously described (Peng et al., 2014; Sims et al., 1996; Zhao et al., 2012). Briefly, mice were acclimated for one week and then anesthetized with a constant plane of isoflurane. Pre-operative carprofen (5 mg / kg) was given via subcutaneous injection. Hair was removed from the dorsal midline, and the surgical site was scrubbed with alternating betadine and saline three times. A 1 cm skin incision was made along the dorsal midline to expose the dorsolateral abdominal muscles in the lower back just below the rib cage. A 0.25 cm incision was made bilaterally to locate the ovaries. Holding the edge of the incision, the fat pad attached to the ovary was retracted from the abdominal cavity to expose the oviduct. In “sham control” mice, the ovary and fat pad were immediately returned to the peritoneal cavity. In “OVX” mice that received the entire surgery, the exposed ovary and the oviduct were carefully removed using sterile scissors, and hemostasis was achieved. The uterus and remaining oviduct were placed back into the abdominal cavity. The incision in the abdominal wall was closed with a resorbable suture (5-0 vicryl). The remaining skin incision was closed with 4-0 Ethilon monofilament sutures or suture clips. Post- operative carprofen was administered at 24 hours (5 mg / kg subcutaneous injection), and animals were monitored daily for the first ten days and then twice weekly until euthanasia. Bone-targeted and untargeted sclerostin antibodies and targeted control (non-functional) antibodies (5mg / kg) were administered weekly via retroorbital injection, starting seven days post-surgery. After four weeks, mice were euthanized by CO2asphyxiation and bilateral pneumothorax. Long bones and vertebrae were harvested at the endpoint, fixed with 4% paraformaldehyde at 4°C for 48 hours, and then kept in PBS at 4°C for subsequent analysis. All animal procedures were performed according to guidelines by the IACUC (PRO00009377) Microcomputed Tomography Analysis: Micro-CT analysis followed published guidelines (Bouxstein et al., JBMR 2010). Briefly, samples were placed in a 19 mm diameter specimen holder and scanned over the entire length of the tibia using a micro-CT system (µCT100 Scanco Medical, Bassersdorf, Switzerland) with voxel size 10 μm, 70 kVp, 114 μA, 0.5 mm AL filter, and integration time 500 ms. For femur analysis: A 1.0 mm region of the trabecular compartment was analyzed immediately below the growth plate using a fixed global threshold of 18%, and a 0.3 mm region of the cortical compartment at the midpoint was analyzed using a fixed global threshold of 28% (280 on a grayscale of 0–1,000). For vertebral analysis, the L4 vertebrae were identified; the trabecular compartment was analyzed with a fixed global threshold of 18%. Trabecular bone volume fraction (BV / TV), connectivity density (Conn dens), structure model index (SMI), trabecular thickness (Tb. Th), trabecular number (Tb. N), trabecular separation (Tb. Sp), cortical bone volume fraction (BV / TV), cortical porosity, cortical thickness, tissue mineral density (TMD), sub-periosteal area and sub-endosteal area were analyzed using an evaluation software from the manufacture. Three-dimensional renderings were generated using Scanco software to evaluate the histomorphometric characteristics of the bones in cross-section and gross morphology. Representative images are shown. Histologic Preparation: Femurs from ovariectomized mice were dissected, debrided from soft tissue and muscle, fixed in 4% paraformaldehyde for 72 hours, then exchanged for PBS and dehydrated with an ethanol gradient. The fixed femurs were decalcified in 14% ethylenediaminetetraacetic acid (EDTA) solution (pH 7.4) for 14 days at 4°C. The EDTA solution was changed every 2-3 days to ensure efficient decalcification. Decalcification was confirmed by physical assessment using a fine needle. Following decalcification, the femurs were processed through a series of graded ethanol solutions (70%, 80%, 95%, and 100%) for dehydration, cleared in xylene, and embedded in paraffin wax. The embedded samples were oriented to allow for longitudinal sectioning. Paraffin blocks containing the femur samples were sectioned at 5 μm thickness and were mounted on positively charged glass slides. For H&E staining, sections were deparaffinized in xylene and rehydrated to distilled water through a graded ethanol series. Slides were stained with hematoxylin and eosin according to standard protocols and mounted with an xylene-based mounting medium. For TRAP staining, sections were deparaffinized and rehydrated as described above. According to the manufacturer's instructions, TRAP staining was performed using a commercial kit (Sigma-Aldrich, 387A-1KT). Briefly, sections were incubated in the TRAP staining solution (naphthol AS-BI phosphate and fast garnet GBC) at 37°C for 1 hour in the dark. After incubation, the slides were rinsed in distilled water and counterstained with hematoxylin for 2 minutes. Sections were then air-dried and mounted using an aqueous mounting medium. Slides were imaged within 24 hours of staining to ensure preservation of the TRAP signal. Stained sections were examined using a light microscope (Olympus BX53) with a digital camera. Fluorescent Detection of Cathepsin K Activity, Scheme 7: Scheme 7: DBCO-sulfo-STP-ester is covalently conjugated to sodium alendronate bisphosphonate by triethylamine catalyzed nucleophilic substitution, yielding a bisphosphonate- targeted DBCO moiety (7) capable of downstream Click chemistry coupling. Subsequently, (7) is reacted with a custom-synthesized peptide (8) containing a terminal azido-lysine residue capable of DBCO-N3 Click coupling, yielding (9).

[0008] Fluorescent Peptide (8): (4) was custom-synthesized by CPC Scientific (Sunnyvale, CA; Product: 943164; Lot: CU-02-01531) from the sequence: QSY21-Gly-His-Pro-Gly-Gly-Pro-Gln- Lys(Cy5)-Lys(N3) (MW = 2234.6 Da). QSY21 is a quencher with a broad range of 590-720 nm. A primary amine-modified oligonucleotide is covalently attached with QSY21 via NHS conjugation chemistry. Cy5 is a bright, far-red-fluorescent molecule with excitation designed for use with the 633 nm or 647 nm laser lines and reacts at the primary amine functional group of lysine, yielding Lys(Cy5). Based on RP-HPLC analysis, the purity of the trifluoroacetic peptide is 97.6% and is subsequently used as received at 1 mg / mL in DMSO. ESI-MS independently confirms its identity. ESI-MS(+): 745.4 (m / z)3+, 1117.4 (m / z)2+. Synthesis of (7): 10.0 mg (0.0331 mmol, 4.0 mol. eq.) alendronate sodium trihydrate consisted of 500 uL MilliQ deionized water with 8.00 uL (0.0580 mmol, 7.0 mol. eq.) triethylamine and agitated until the solution becomes clear. Separately, 5.00 mg (0.00827 mmol, 1.0 mol. eq.) Sulfo DBCO-2,3,5,6 tetrafluorophenol Ester was constituted in 250 uL acetonitrile to form a colorless solution. The ALD solution is slowly added dropwise to DBCO-TFP (MeCN) and reacted at room temperature for 12 hours. Reaction progress is assessed by thin-layer chromatography using normal-phase silica and a 3:1 MeCN / ddH2O. Analytes are identified by UV absorption at 254 nm (DBCO-TFP, (3)) and potassium permanganate stain. Rf(DBCO-TFP) ~0.95-1.0, Rf(3) = 0.65, Rf(ALD) = 0. The product is isolated by rotary evaporation and is redissolved in a minimum volume of 3:1 MeCN / ddH2O. (3) is isolated by column chromatography with normal phase silica and a 3:1 MeCN / ddH2O mobile phase. ESI-MS and 31P NMR confirm the product. The isolated product is stored and lyophilized at -20 °C for subsequent use. ESI-MS(+): m / z 687.37, [M+2K-H]+ 764.57, [M+3H]3+ 230.25.31P NMR: 18.25 ppm. Synthesis of (9): (4) is diluted to 0.31 mM in ddH2O, with 10% v / v DMSO (500 uL volume). Slowly, 285 uL of 3.5 mg / mL ALD-DBCO (10.0 mol. eq.) is added to the solution containing (4). The reaction mixture is protected from light and reacted at room temperature, in the dark, for 12 hours. Reaction efficiency is assessed by normal phase TLC using a 10% MeCN + 90% ddH2O mobile phase. Analytes are identified by UV absorption at 254 nm. Rf(5) = 0.80, Rf(4) = 0. (5) is isolated by column chromatography with normal phase silica and a 65% MeCN + 35% ddH2O mobile phase; fractions are spotted to TLC plates. Rf(5) = 0.75-0.80, Rf(3) = 0.55-0.60, Rf(DBCO-TFP) ~0.95-1.0, Rf(4) = 0. The isolated product is stored and lyophilized at -20 °C for subsequent use. Nanodrop assesses reconstituted products at A640 to determine concentration. In Vitro Activation and Sensitivity by Recombinant CTSK: Cathepsin K (CTSK) was purchased from Abcam (ab185439) and stored at -80°C, protected from light, until used, diluted in 100 uL of CTSK Reaction Buffer (Abcam, ab185439). The CTSK activity assay was performed according to manufacturer instructions, using (5) instead of a commercially available substrate (Ac-LR-AFC).1 uL of CTSK and 1 uL CTSK Reagent (Abcam, ab185439) were diluted to 50 uL with sterile PBS for each well of a 96-well plate needed and aliquoted at 50 uL / well. When an inhibitor was used, either FF-FMK (1 mM) or Mg132 (variable concentration, final concentration = 10-100 uM) was added in a 10 uL aliquot. If no inhibitor was used, 10 uL PBS was added. Separately, 5 uM (5) was prepared in PBS, and 40 uL was added to each well and mixed well in each well. If another protease (Trypsin, Collagenase) was used, it was used at the same concentration. Fluorescence at Ex / Em = 640 / 670 nm was measured using a microplate reader in kinetic mode for up to 60 minutes. Relative inhibition is calculated by comparing each test sample to an enzyme (uninhibited) control. FF-FMK is an irreversible inhibitor of Cathepsin K activity with a slope of 0, making its relative inhibition 100%. All measurements were replicated > n=3 and averaged. Hydroxyapatite-bound Detection of Recombinant CTSK: (4) and (5) were diluted to 11 uM in PBS. (4) or (5) was added to 25 uL of hydroxyapatite beads (25 mg / mL stock solution) and diluted to a final volume of 75 uL in an Eppendorf tube. Samples were kept at 37°C for 1 hour; then, the HA beads were isolated by centrifugation at 5,000 rpm for 3 minutes. HA beads were reconstituted in fresh PBS for five washes.1 uL CTSK Reagent and 1 uL CTSK were diluted to 70 uL PBS and added to each well; no-enzyme controls received 1 uL CTSK reagent in 70 uL PBS. Samples were incubated at 37C for one hour. Fluorescence was measured by microplate reader assay (Ex / Em = 640 / 670 nm) and immediately imaged by confocal laser microscopy. Calvarial Subcutaneous Resorption Assay: To determine baseline binding of ALD- Cy5 or (5) to calvarial bone by localized injection, mice are anesthetized by isoflurane general anesthesia, the site is sterilized with povidone / saline alternating washes, and 100 uL of either compound (1 uM or 10 uM) or vehicle (PBS) control is administered subcutaneously with a 28G needle to the left of the midline, aiming for the center of the left parietal bone. After 4, 24, or 72 hours, mice are euthanized by CO2 asphyxiation, and calvariae are harvested. Calvariae are fixed with 4% paraformaldehyde at 4 °C for 48 hours and then kept in PBS at four °C, protected from light. To induce local resorption, 20 mg / kg of 0111:B4 strain (E. coli) LPS was injected subcutaneously in the calvariae following safety protocols, including a respirator mask and safety glasses. 100 uL (20 mg / kg) of LPS was administered at t=0h, and animals recovered from general anesthesia normally. Four hours later, 100 uL of a therapeutic inhibitor (reversible: Mg132; irreversible: FF-FMK) was dosed. At t=24h, 100 uL of 10 uM (5) was dosed, and animals were sacrificed four hours later (t=28h). Mice are euthanized by CO2 asphyxiation, and calvariae are harvested. Calvariae are fixed with 4% paraformaldehyde at 4 °C for 48 hours and then kept in PBS at 4 °C, protected from light. Mass spectroscopy: An Agilent 6230 TOF instrument recorded all electrospray ionization spectra. Nuclear magnetic resonance spectroscopy: All compounds were characterized by nuclear magnetic resonance (NMR) spectroscopy to confirm their molecular identity.1H spectra were recorded with a Bruker 600 MHz NEO600 spectrometer operating at room temperature. Spectra were observed from compounds dissolved in deuterated chloroform (CDCl3) and conformed to our previously reported spectroscopy. Spectral analysis is carried out in ICONNMR (Bruker) and Mestre Nova (Version 12.0.0-2000080, Metrelab Research). Absorbance Spectroscopy: Compounds were diluted from stock solutions in a quartz cuvette. Spectra were recorded from 200 to 700 nm using a Hitachi U-2910 spectrophotometer. Fluorescence Spectroscopy: Excitation spectra were observed to confirm maximum excitation wavelengths relevant to future applications with confocal laser microscopy. Spectra were recorded from quartz cuvettes on a Horiba Quanta Master spectrometer with a xenon arc lamp and PMT detector. A plate reader was used for assays with emission and excitation wavelengths predetermined from optimization with the Horiba instrument. Statistical Methods: All data are reported as mean ± standard deviation and represent a minimum sample size of n>3. Statistical analysis was carried out in GraphPad Prism v9. Student’s t-test was used to determine the statistical significance of observed values between experimental groups where p < 0.05 was considered significant. N>4 biologic replicates for all groups / time points. All graphics note significance as: * p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001. Preparation of a bone-targeted, acid-activatable delivery vector Given the importance of the terminal aldehyde moiety in the therapeutic functionality of peptide aldehyde protease inhibitors, we hypothesized that its involvement would inactivate the peptide as a result of hydrazone bond formation, effectively masking its active site until its release under resorptive conditions at sites of bone turnover. Based on the general principle shown in FIG.9, whereby sodium alendronate (ALD), a bisphosphonate (BP) with affinity to hydroxyapatite and mineralized bone, is covalently bound to an inactive peptide aldehyde protease inhibitor through a hydrazone bond, enabled by end group modification of the terminal primary amine (FIG.1A). Hydrazine-functionalized ALD was synthesized in a two-step process. First, Boc-protected hydrazine was covalently modified to have a favorable yet unstable tetrafluorophenol leaving group, (1), (Scheme 1) for immediate subsequent nucleophilic substitution by triethylamine activated sodium alendronate, yielding (2) (Scheme 2). The bisphosphonate-functionalized Boc-hydrazine (2) is deprotected, yielding alendronate-hydrazine (ALD-NHNH2, “GSW000”) (3). ALD-NHNH2 (3) is readily conjugated to aldehyde-containing payloads through terminal hydrazine-aldehyde coupling under mildly acidic conditions via a hemiaminal intermediate and dehydration reaction. First, we synthesized ALD-z-Cy5 (F1) as shown in Scheme 5, using Cy5 as a fluorescent model payload to determine the acid-sensitive release. As a control, we also synthesized a non-cleavable ALD-Cy5 (F2), as shown in Scheme 6, by NHS / EDC coupling to the terminal primary amine of sodium alendronate. F1 and F2 are bound to hydroxyapatite beads in vitro and incubated in a physiologic buffer at various pHs. Cy5 is released from F1 at acidic pH but not F2, validating the acid-sensitivity of F1 and stability of F2 FIG.1B). F1 is similarly stable at physiologic pH and pH as low as 6. Below pH 5.2, Cy5 is released from F1 over 100 hours (FIG.1C). EDTA was used to elute residual bound fluorophore from the bone surface. Percent release was calculated as the amount of fluorophore released into the supernatant (fsup) / (fsup + fbound) x 100. A design feature of the GSW platform is prodrug formulation, such that therapeutic warheads are meant to be inactive until released by acid-mediated hydrolysis. Based on this proof of principle, we optimized the synthesis of (4), ALD-z-LLLY-Cbz, from Cbz-LLL-CHO, a potent peptide aldehyde protease inhibitor, and ALD-NHNH2 (3), as shown in Scheme 3, coined “GSW001”. As a control, we oxidized Cbz-LLL-CHO to its carboxylic acid form and directly coupled to ALD via EDC / NHS coupling to form a non-cleavable analog of GSW001, called “GSW001NC” (5) as shown in Scheme 4. We measured the potential of (4) to inhibit recombinant CTSK activity in vitro, compared to free Cbz-LLL-CHO, ALD-NHNH2 (3), acidified (4), and GSW001NC (6). As shown in FIG.1D, the test compound, GSW001 (4), demonstrated enzyme inhibition only at the highest concentration (30 uM) and was less effective than a 10-fold lower free drug concentration. The small amount of enzymatic inhibition seen with GSW001 (4) was ultimately attributed to a slow rate of hydrolysis and release of free drug in the proprietary buffer (pH 6) used in the commercial CTSK inhibitor assay (Abcam, pH confirmed by the manufacturer). Similarly, ALD-NHNH2 (3), the delivery vector without a therapeutic payload, shows no significant inhibition of CTSK. We subjected GSW001 (4) to acidic conditions to mimic its local activation by osteoclast resorption and assessed the acidified drug’s potency to inhibit CTSK. As intended, acidified GSW001 (4), but not GSW001 (4) at physiologic pH, is a potent inhibitor of CTSK. The non-cleavable version, GSW001NC (6), did not appreciably inhibit CTSK activity at any of the concentrations tested. GSW001 Modulates the Activity of Resorbing Osteoclasts without Toxicity Concerns We assessed the anti-resorptive activity of GSW001 by culturing resorbing (LPS- stimulated) osteoclasts on bone chips pre-treated with the drug, using ALD-NHNH2 (3) and the untargeted CTSK inhibitor Cbz-LLL-CHO as controls, compared to vehicle treatment (FIG.2A). We observed a dose-dependent reduction in overall bone resorption area and an apparent dose- dependent skewing towards pitting rather than trench formation due to the drug in LPS-treated osteoclasts but a less significant effect in PBS-treated or physiologically remodeling osteoclasts (FIG.2B). Pit formation remains relatively consistent between PBS-treated and LPS-treated OCs under each treatment condition, while trench formation is significantly attenuated by GSW001 (4) (FIG.2C, D). Based on this, GSW001 (4) selectively inhibits LPS-induced aggressive resorbing osteoclasts to favor physiologic “pit” resorption over inflammatory “trench” resorption (Borggaard et al., 2020b; Merrild et al., 2015). The Kd for a model bisphosphonate-small molecule conjugate binding to hydroxyapatite is approximately 25 uM (FIG.3A), confirming its affinity for hydroxyapatite compared to an untargeted fluorophore, which shows negligible affinity. We conjugated a commercially available pH-sensitive fluorophore, pHrodo, to ALD, yielding ALD-pHrodo, to demonstrate local activation at the site of osteoclast resorption. In vitro, the fluorescence signal is observed from ALD-pHrodo-treated bone chips plated with resorbing osteoclasts but not from pHrodo alone. The shape of the lacunae is well visualized by confocal laser microscopy, indicating osteoclasts’ acidic resorption of the hydroxyapatite surface (white arrows, FIG.3B). In vivo, fluorescent model compounds F1 and F2 determine pharmacokinetics in healthy adult female mice. We demonstrate rapid circulation and clearance of F1, F2, and untargeted Cy5 fluorophore within 60 minutes following intravenous administration. Despite the rapid circulation, we demonstrate bone surface localization and activation of ALD-pHrodo (red) to identify sites of active bone resorption in vivo (FIG.3D). Acidification of resorption lacunae is a key contributor to osteoclast-mediated bone resorption. We note pH-sensitive fluorescence at the bone surface directly colocalized with actively resorbing osteoclasts (TRAP+; green) following in vivo administration. Sodium alendronate (Fosamax) exerts its clinical effect by indiscriminately killing osteoclasts to reduce resorption. Although effective for short-term reduction in bone resorption, alendronate and other primary amine bisphosphonates significantly reduce bone turnover, a process critical for maintaining bone health. It is critical that new osteoporosis therapeutics need anti-osteoclast toxicity to preserve the natural bone turnover process, ensuring that bone formation and resorption remain balanced, thereby maintaining bone strength and reducing the risk of atypical fractures. We administered intravenous alendronate or GSW001 (4) to healthy adult female mice in a single dose and assayed osteoclast toxicity by Elf97 (TRAP+) staining after 24 hours, compared to vehicle control. As little as 1 mg / kg ALD is sufficient to induce a significant decrease in TRAP+ area and reduced osteoclast number, with its effect increasing with dose. On the other hand, no significant toxicity is observed at equimolar doses of GSW001 (4) within the doses tested, up to 10 times the therapeutic dose used in subsequent experiments, compared to vehicle control. Therapeutic Efficacy of Bone-Targeted Acid-Activated GSW001 (4) in a Pre-Clinical Trial Using a Mouse Model of Osteoporosis We sought to compare the therapeutic efficacy of GSW001 (4), Cbz-LLL-CHO, ALD- NHNH2 (3), and GSW001NC (6) in a murine model of ovariectomy-induced bone loss (OVX). Before testing therapeutics, a pilot study of the OVX model was performed to guide sample size calculation and ensure adequate statistical power.14-week-old female C57BL / 6J wild-type mice were subjected to a bilateral ovariectomy (Souza et al., 2019). Beginning one week after surgery, biweekly 1 mg / kg doses of ALD-NHNH2 (3), GSW001 (4), Cbz-LLL-CHO, GSW001NC (6), or vehicle control. Dosing was based on a pilot study performed by our group. As shown in FIG.4, volumetric analysis of the femur trabecular compartment in the defined region of interest (FIG.4A) demonstrated a significant therapeutic effect for GSW001 (4) compared to untargeted Cbz-LLL-CHO, ALD-NHNH2 (3), GSW001NC (6) or vehicle- treated OVX mice, reflected in the three-dimensional reconstructions of the micro CT scans (FIG.4B). Mice treated with GSW001 (4) had significantly greater BV / TV (14.11% ± 0.9%) than those in the other groups (4.66% ± 1.5% vs.5.94% ± 1.3% vs.5.46% ± 1.7% for GSW000, Cbz-LLL-CHO, GSW000NC, and vehicle, respectively, p < 0.0001) (FIG.4C). GSW001 treatment was also associated with significant changes in connectivity density (109.5 ± 19.5 vs. 16.79 ± 11.3 vs.28.75 ± 16.47 vs.22.13 ± 14.27 vs.19.63 ± 9.054, p < 0.0001) and SMI (2.140 ± 0.13 vs.3.298 ± 0.33 vs.3.102 ± 0.15 vs.3.43 ± 0.64 vs.3.391 ± 0.63, p < 0.0001) (FIG.4D, E). The data shows that OVX mice treated with GSW001 (4) exhibited values comparable to those of sham (healthy, age-matched) controls. This indicates that the targeted treatment effectively maintains well-organized and morphometrically normal bone mass while also protecting against estrogen-induced bone loss. Effects on trabecular morphology (Tb.N and Tb.Sp) and bone mineral density (BMD) are shown in FIG.4F, G, and H, respectively. The impact of OVX and various treatments on midshaft femoral cortical bone are shown in FIG.5. OVX had no significant effect on cortical bone metrics compared to sham surgery. Only GSW001, not Cbz-LLL-CHO or GSW001NC, proved effective in preserving cortical bone mass, mineral density, and thickness. FIG.6 shows a micro CT analysis of the lumbar vertebrae trabecular bone. As with the femur, three-dimensional reconstructions of L4 vertebrae matched the quantitative findings from micro CT analysis (FIG.6A). Consistent with the effects seen on femoral bone, GSW001 (4) significantly improved metrics of trabecular bone mass, morphology, and structure in L4 vertebrae of OVX mice after four weeks. GSW001 treatment significantly maintained BV / TV (29.56% ± 1.9%) compared to GSW000 (3), Cbz-LLL-CHO, GSW001NC (6) or vehicle control (18.30% ± 1.4% vs.18.10% ± 2.51% vs.17.68% ± 1.47% vs.18.18% ± 1.56%, p < 0.001, FIG. 6C). Similarly, only GSW001 effectively maintained connectivity density and SMI (FIG.6D, E). Effects on other metrics are shown in FIG.6F-H. Histological evaluation using Hematoxylin and Eosin (H&E) staining at 4x and 20x magnifications revealed distinct differences among the groups (FIG.7, top). The sham-operated mice, serving as healthy controls, exhibited standard bone architecture, dense trabecular structures, and normal marrow morphology. In contrast, the OVX + Veh group showed significant bone loss, reduced trabecular density, increased marrow space, and marrow adiposity. Treatment with ALD-NHNH2 (3) did not prevent bone loss, as the bone structure appeared similar to the vehicle group. Notably, mice treated with GSW001 (4) displayed improved bone morphology, increased trabecular density, reduced marrow space, and somewhat reduced adiposity, indicating a protective effect against orthopedic degeneration. Cbz-LLL-CHO treatment resulted in bone architecture similar to the vehicle group, suggesting minimal efficacy; similarly, the inactive form of GSW001, OVX + (6) GSW001NC, did not improve bone morphology. TRAP staining at 20x and 40x magnifications was used to assess osteoclast activity (FIG. 7). The sham group showed few TRAP-positive osteoclasts, consistent with normal bone remodeling. In contrast, the OVX + Veh group exhibited an increase in TRAP-positive osteoclasts, indicating enhanced osteoclastic activity, as expected. ALD-NHNH2 (3) treatment did not reduce osteoclast numbers, similar to the vehicle group, indicating no overt osteoclast toxicity of the delivery vector. GSW001 (4) treatment reduced TRAP-positive osteoclast number but did not cause significant osteoclast depletion, suggesting effective inhibition of bone resorption without osteoclast toxicity. Reducing aggressive resorption without significantly reducing osteoclast number is essential for maintaining osteoblast-osteoclast coupling. In the Cbz-LLL-CHO group, osteoclast numbers remained high, indicating little impact on osteoclast activity. The GSW001NC (6) group also showed high osteoclast numbers, comparable to the vehicle group. GSW001 Reduces CTSK Activity In Vivo Cathepsin K (CTSK) is a lysosomal cysteine protease secreted by osteoclasts (OC), which plays a crucial role in extracellular matrix proteolysis during bone remodeling. We sought to assess the mechanism by which Cbz-LLL-CHO, a proteasome inhibitor, exerts its protective therapeutic effects against excessive bone resorption. This hypothesis required the development of a CTSK activity probe to detect changes in CTSK activity at the bone surface, both in vitro and in vivo. Previously reported CTSK activity probes have focused on detecting pathologic enzymatic activity in the setting of cancer-related osteolysis, and in vitro assay probes require an acidic non-physiologic medium (Lemke et al., 2021; Song et al., 2023). It is unclear if these probes have the sensitivity or specificity to detect regular enzymatic activity, supranormal proteolysis in the setting of bone diseases, or pharmacologic inhibition of CTSK in vivo. We aimed to develop a low molecular weight probe that localizes to the bone surface and reports CTSK activity using a fluorophore-quencher pair compatible with the autofluorescence of normal bone tissue, shown schematically in FIG.8A. We synthesized a nine-residue peptide based on the consensus sequence of CTSK, modified with a far-red fluorescent residue adjacent to its C-terminus (Cy5) and broad-spectrum quencher (QSY21) at its N-terminus: QSY21-GHPGGPQ-K(Cy5)-K(N3) (SEQ ID NO:1), “CTSKr” (8). The C-terminal azidolysine was orthogonally conjugated to a bisphosphonate dibencocyclooctyne (ALD-DBCO, (7), Scheme 7) to yield ALD-CTSKr (9). We demonstrated the ability of this ALD-CTSKr (10 uM) to detect CTSK protease activity in a fluorometric assay, comparable to a commercially available 7-amino-4-trifluoromethylcoumarin inhibitor screening assay (Abcam) at a physiologically relevant wavelength (640 nm excitation, 670 nm emission). We demonstrated no abberant effect of the bisphosphonate targeting on detection of fluorescent signal evolution in (9), compared to (8), in a fluorescence assay with recombinant CTSK (FIG. 8B). ALD-CTSKr efficiently localizes to the surface of hydroxyapatite nanoparticles (HA-NP) in solution. When treated with recombinant CTSK, the fluorescent signal of (9) from the surface of HA-NP at 670 nm is detected by fluorimeter and confocal at a high quantum yield, but not (8) (FIG.8C). Subsequently, when recombinant CTSK was inhibited by FF-FMK (1 mM), an irreversible inhibitor, or Cbz-LLL-CHO, the fluorescence signal of (9) was abolished, demonstrating its ability to detect therapeutic and dose-dependent inhibition of CTSK within a physiologically relevant dynamic range (FIG.8D). We developed a subcutaneous calvarial injection assay where lipopolysaccharide (LPS) is administered to induce rapid bone resorption in mice. Four hours post-LPS injection, the treatment is administered subcutaneously, and 24 hours later, the fluorescent sensor (9) is administered to measure cathepsin K (CTSK) activity, indicating the level of local bone resorption by confocal laser microscopy at endpoint. The experimental design is shown schematically in FIG.8E. LPS treatment significantly increased CTSK activity, as indicated by the heightened fluorescence intensity compared to the PBS control. Notably, the administration of GSW001 (4) markedly reduced CTSK-mediated bone resorption, similar to equimolar Cbz- LLL-CHO, resulting in a substantial decrease in fluorescence intensity compared to the LPS + Veh group. Neither ALD-NHNH3 (3) nor GSW001NC (6) showed appreciable decrease in fluorescent signal.

[0009] (2) tri-tert-butyl 2-(2-oxo-2-(2,3,5,6-tetrafluorophenoxy)ethyl)hydrazine-1,1,2- tricarboxylate Chemical Formula: C23H30F4N2O8 Exact Mass: 538.19 Molecular Weight: 538.49 ESI-MS: [M+Na]+ calc = 561.183 [M+Na]+ obs = 561.184 [M+K]+ calc = 577.157 [M+K]+ obs = 577.156

[0010] (4) (1-hydroxy-4-(2-(1,2,2-tris(tert-butoxycarbonyl)hydrazineyl)acetamido)butane-1,1- diyl)bis(phosphonic acid) Chemical Formula: C21H41N3O14P2 Exact Mass: 621.21 Molecular Weight: 621.51 ESI-MS: [M-Boc]- calc = 520.15 [M-Boc]- obs = 520.145 [M-H]- calc = 620.199 [M-H]- obs = 520.197

[0011] (6) (4-(2-hydrazineylacetamido)-1-hydroxybutane-1,1-diyl)bis(phosphonic acid) Chemical Formula: C6H17N3O8P2 Exact Mass: 321.05 Molecular Weight: 321.16 ESI-MS: [M+H]+ calc = 322.05 [M+H]+ obs = 322.055

[0012] (8) ((5S,8S,11S,Z)-21-hydroxy-5,8,11-triisobutyl-3,6,9,16-tetraoxo-1-phenyl-2-oxa- 4,7,10,13,14,17-hexaazahenicos-12-ene-21,21-diyl)bis(phosphonic acid) Chemical Formula: C32H56N6O12P2 Exact Mass: 778.34 Molecular Weight: 778.78 ESI-MS: [M+H]+ calc = 779.35 [M+H]+ obs = 779.373

[0013] (10) (5S,8R,11S)-5,8,11-triisobutyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10- triazatridecan-13-oic acid Chemical Formula: C27H43N3O6 Exact Mass: 505.32 Molecular Weight: 505.66 ESI-MS: [M+H]+ calc = 506.327 [M+H]+ obs = 506.33 [M+NH4]+ calc = 523.35 calc [M+NH4]+ obs = 523.46

[0014] (12) ((5S,8R,11S)-18-hydroxy-5,8,11-triisobutyl-3,6,9,13-tetraoxo-1-phenyl-18- phosphono-2-oxa-4,7,10,14-tetraazaoctadecan-18-yl)phosphonate Chemical Formula: C31H53N4O12P2-Exact Mass: 735.31 Molecular Weight: 735.73 ESI-MS: [M+H]+ calc = 736.317 [M+H]+ obs = 736.32

[0015] (F1): 1-ethyl-2-((1E,3E)-5-((E)-1-(6-((2-(4-((Z)-(2-(2-((4-hydroxy-4,4- diphosphonobutyl)amino)-2- oxoethyl)hydrazineylidene)methyl)benzamide)ethyl)amino)-6-oxohexyl)-3,3- dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium- 5-sulfonate Chemical Formula: C49H65N7O16P2S2 Exact Mass: 1133.34 Molecular Weight: 1134.16 ESI-MS: [M+3H]3+ calc = 378.787 [M+3H]+ obs = 378.785 [M+2H]2+ calc = calc = 567.677 [M+2H]+ obs = 567.679 (F2): 1-ethyl-2-((1E,3E)-5-((E)-1-(6-((4-hydroxy-4,4-diphosphonobutyl)amino)-6- oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl- 3H-indol-1-ium-5-sulfonate Chemical Formula: C37H51N3O14P2S2 Exact Mass: 887.23 Molecular Weight: 887.89 ESI-MS: [M+H]+ calc = 888.237 [M+H]+ obs = 888.235 References Black, D.M., Geiger, E.J., Eastell, R., Vittinghoff, E., Li, B.H., Ryan, D.S., Dell, R.M., and Adams, A.L. (2020). Atypical Femur Fracture Risk versus Fragility Fracture Prevention with Bisphosphonates. N Engl J Med 383, 743-753. Bonnet, N., Brun, J., Rousseau, J.-C., Duong, L.T., and Ferrari, S.L. (2017). Cathepsin K Controls Cortical Bone Formation by Degrading Periostin. Journal of Bone and Mineral Research 32, 1432-1441. Borggaard, X.G., Pirapaharan, D.C., Delaissé, J.-M., and Søe, K. (2020a). Osteoclasts’ Ability to Generate Trenches Rather Than Pits Depends on High Levels of Active Cathepsin K and Efficient Clearance of Resorption Products. International Journal of Molecular Sciences 21. Borggaard, X.G., Pirapaharan, D.C., Delaisse, J.M., and Soe, K. (2020b). Osteoclasts' Ability to Generate Trenches Rather Than Pits Depends on High Levels of Active Cathepsin K and Efficient Clearance of Resorption Products. Int J Mol Sci 21. Chen, W., Gao, B., Hao, L., Zhu, G., Jules, J., MacDougall, M.J., Wang, J., Han, X., Zhou, X., and Li, Y.P. (2016). The silencing of cathepsin K used in gene therapy for periodontal disease reveals the role of cathepsin K in chronic infection and inflammation. J Periodontal Res 51, 647- 660. Chen, X., Wang, Z., Duan, N., Zhu, G., Schwarz, E.M., and Xie, C. (2018). Osteoblast-osteoclast interactions. Connect Tissue Res 59, 99-107. Compston, J.E., McClung, M.R., and Leslie, W.D. (2019). Osteoporosis. The Lancet 393, 364- 376. Costanzi, E., Kuzikov, M., Esposito, F., Albani, S., Demitri, N., Giabbai, B., Camasta, M., Tramontano, E., Rossetti, G., Zaliani, A., et al. (2021). Structural and Biochemical Analysis of the Dual Inhibition of MG-132 against SARS-CoV-2 Main Protease (Mpro / 3CLpro) and Human Cathepsin-L. Int J Mol Sci 22. Dai, R., Wu, Z., Chu, H.Y., Lu, J., Lyu, A., Liu, J., and Zhang, G. (2020). Cathepsin K: The Action in and Beyond Bone. Frontiers in Cell and Developmental Biology 8. Damodaran, A., and Harris, R.B. (1995). Preparations of Ψ-peptide bond and peptide-aldehyde inhibitors of atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor. Journal of Protein Chemistry 14, 431-440. Drake, M.T., Clarke, B.L., and Khosla, S. (2008). Bisphosphonates: mechanism of action and role in clinical practice. Mayo Clin Proc 83, 1032-1045. Farrell, K.B., Karpeisky, A., Thamm, D.H., and Zinnen, S. (2018). Bisphosphonate conjugation for bone specific drug targeting. Bone Rep 9, 47-60. Fehrentz, J.-A., Paris, M., Heitz, A., Velek, J., Liu, C.-F., Winternitz, F., and Martinez, J. (1995). Improved solid phase synthesis of C-terminal peptide aldehydes. Tetrahedron Letters 36, 7871- 7874. Fleisch, H. (1998). Bisphosphonates: Mechanisms of Action. Endocrine Reviews 19, 80-100. Hao, L., Zhu, G., Lu, Y., Wang, M., Jules, J., Zhou, X., and Chen, W. (2015). Deficiency of cathepsin K prevents inflammation and bone erosion in rheumatoid arthritis and periodontitis and reveals its shared osteoimmune role. FEBS Lett 589, 1331-1339. Harer, S.L., Bhatia, M.S., and Bhatia, N.M. (2012). Proteasome inhibitors mechanism; source for design of newer therapeutic agents. The Journal of Antibiotics 65, 279-288. Heo, S.C., Kim, Y.N., Choi, Y., Joo, J.Y., Hwang, J.J., Bae, M.K., and Kim, H.J. (2021). Elevated Expression of Cathepsin K in Periodontal Ligament Fibroblast by Inflammatory Cytokines Accelerates Osteoclastogenesis via Paracrine Mechanism in Periodontal Disease. Int J Mol Sci 22. Insights, F.B. (2019). Osteoporosis treatment market size, share & industry analysis, by drug class, route of administration, distribution channel, and regional forecast 2019-2026. Kim, J.M., Lin, C., Stavre, Z., Greenblatt, M.B., and Shim, J.H. (2020). Osteoblast-Osteoclast Communication and Bone Homeostasis. Cells 9. Kim, Y.M., and Kim, H.J. (2020). Proteasome Inhibitor MG132 is Toxic and Inhibits the Proliferation of Rat Neural Stem Cells but Increases BDNF Expression to Protect Neurons. Biomolecules 10. Lemke, C., Benýšek, J., Brajtenbach, D., Breuer, C., Jílková, A., Horn, M., Buša, M., Ulrychová, L., Illies, A., Kubatzky, K.F., et al. (2021). An Activity-Based Probe for Cathepsin K Imaging with Excellent Potency and Selectivity. Journal of Medicinal Chemistry 64, 13793-13806. Liu, Z., Chen, X., Jin, Q., Li, M., Zhu, S., Zhang, Y., Zhi, D., Zhao, Y., Li, L., and Zhang, S. (2023). Dual functionalized hyaluronic acid micelles loading paclitaxel for the therapy of breast cancer. Front Bioeng Biotechnol 11, 1230585. Merrild, D.M.H., Pirapaharan, D.C., Andreasen, C.M., Kjærsgaard-Andersen, P., Møller, A.M.J., Ding, M., Delaissé, J.-M., and Søe, K. (2015). Pit- and trench-forming osteoclasts: a distinction that matters. Bone Research 3. Moulin, A., Martinez, J., and Fehrentz, J.A. (2007). Synthesis of peptide aldehydes. J Pept Sci 13, 1-15. Peng, J., Lai, Z.G., Fang, Z.L., Xing, S., Hui, K., Hao, C., Jin, Q., Qi, Z., Shen, W.J., Dong, Q.N., et al. (2014). Dimethyloxalylglycine prevents bone loss in ovariectomized C57BL / 6J mice through enhanced angiogenesis and osteogenesis. PLoS One 9, e112744. Qi, P., Wu, X., Liu, L., Yu, H., and Song, S. (2018). Hydrazone-Containing Triblock Copolymeric Micelles for pH-Controlled Drug Delivery. Frontiers in Pharmacology 9. Rashki Kemmak, A., Rezapour, A., Jahangiri, R., Nikjoo, S., Farabi, H., and Soleimanpour, S. (2020). Economic burden of osteoporosis in the world: A systematic review. Med J Islam Repub Iran 34, 154. Reszka, A.A., Halasy-Nagy, J.M., Masarachia, P.J., and Rodan, G.A. (1999). Bisphosphonates act directly on the osteoclast to induce caspase cleavage of mst1 kinase during apoptosis. A link between inhibition of the mevalonate pathway and regulation of an apoptosis-promoting kinase. J Biol Chem 274, 34967-34973. Rogers, M.J., Gordon, S., Benford, H.L., Coxon, F.P., Luckman, S.P., Monkkonen, J., and Frith, J.C. (2000). Cellular and molecular mechanisms of action of bisphosphonates. Cancer 88, 2961- 2978. Sarubbi, E., Seneci, P.F., Angelastro, M.R., Peet, N.P., Denaro, M., and Islam, K. (2001). Peptide aldehydes as inhibitors of HIV protease. FEBS Letters 319, 253-256. Sims, N.A., and Martin, T.J. (2015). Coupling Signals between the Osteoclast and Osteoblast: How are Messages Transmitted between These Temporary Visitors to the Bone Surface? Front Endocrinol (Lausanne) 6, 41. Sims, N.A., Morris, H.A., Moore, R.J., and Durbridge, T.C. (1996). Increased bone resorption precedes increased bone formation in the ovariectomized rat. Calcif Tissue Int 59, 121-127. Sonawane, S.J., Kalhapure, R.S., and Govender, T. (2017). Hydrazone linkages in pH responsive drug delivery systems. Eur J Pharm Sci 99, 45-65. Song, Z., Miao, J., Miao, M., Cheng, B., Li, S., Liu, Y., Miao, Q., Li, Q., and Gao, M. (2023). Cathepsin K‐Activated Probe for Fluoro‐Photoacoustic Imaging of Early Osteolytic Metastasis. Advanced Science 10. Souza, V.R., Mendes, E., Casaro, M., Antiorio, A., Oliveira, F.A., and Ferreira, C.M. (2019). Description of Ovariectomy Protocol in Mice. Methods Mol Biol 1916, 303-309. Sun, S., Tao, J., Sedghizadeh, P.P., Cherian, P., Junka, A.F., Sodagar, E., Xing, L., Boeckman, R.K., Jr., Srinivasan, V., Yao, Z., et al. (2021). Bisphosphonates for delivering drugs to bone. Br J Pharmacol 178, 2008-2025. Swanson, W.B., Durdan, M., Eberle, M., Woodbury, S., Mauser, A., Gregory, J., Zhang, B., Niemann, D., Herremans, J., Ma, P.X., et al. (2022). A library of Rhodamine6G-based pH- sensitive fluorescent probes with versatile in vivo and in vitro applications. RSC Chemical Biology 3, 748-764. Takegahara, N., Kim, H., and Choi, Y. (2024). Unraveling the intricacies of osteoclast differentiation and maturation: insight into novel therapeutic strategies for bone-destructive diseases. Experimental & Molecular Medicine 56, 264-272. Tu, K.N., Lie, J.D., Wan, C.K.V., Cameron, M., Austel, A.G., Nguyen, J.K., Van, K., and Hyun, D. (2018). Osteoporosis: A Review of Treatment Options. P T 43, 92-104. Verma, G., Marella, A., Shaquiquzzaman, M., Akhtar, M., Ali, M.R., and Alam, M.M. (2014). A review exploring biological activities of hydrazones. J Pharm Bioallied Sci 6, 69-80. Votta, B.J., Levy, M.A., Badger, A., Bradbeer, J., Dodds, R.A., James, I.E., Thompson, S., Bossard, M.J., Carr, T., Connor, J.R., et al. (1997). Peptide aldehyde inhibitors of cathepsin K inhibit bone resorption both in vitro and in vivo. J Bone Miner Res 12, 1396-1406. Wang, H., Yang, G., Xiao, Y., Luo, G., Li, G., and Li, Z. (2020a). Friend or Foe? Essential Roles of Osteoclast in Maintaining Skeletal Health. Biomed Res Int 2020, 4791786. Wang, H., Zhang, H., Srinivasan, V., Tao, J., Sun, W., Lin, X., Wu, T., Boyce, B.F., Ebetino, F.H., Boeckman, R.K., Jr., et al. (2020b). Targeting Bortezomib to Bone Increases Its Bone Anabolic Activity and Reduces Systemic Adverse Effects in Mice. J Bone Miner Res 35, 343- 356. Xu, J., Wu, H., Sun, J., Gong, Z., Lu, X., Yang, E., Chen, Z., Huang, S., Nong, X., and Zhang, D. (2022). The anti-tumor effect of proteasome inhibitor MG132 for human adenoid cystic carcinoma: correlate with the emerging role of Nrf2 / Keap1 signaling pathway. Head & Face Medicine 18. Yildiz, I. (2016). A DFT Approach to the Mechanistic Study of Hydrozone Hydrolysis. J Phys Chem A 120, 3683-3692. Zeng, W., Qi, W., Mu, J., Wei, Y., Yang, L.-L., Zhang, Q., Wu, Q., Tang, J.-Y., and Feng, B. (2019). MG132 protects against renal dysfunction by regulating Akt-mediated inflammation in diabetic nephropathy. Scientific Reports 9. Zhao, Q., Shen, X., Zhang, W., Zhu, G., Qi, J., and Deng, L. (2012). Mice with increased angiogenesis and osteogenesis due to conditional activation of HIF pathway in osteoblasts are protected from ovariectomy induced bone loss. Bone 50, 763-770.

Claims

CLAIMS We claim:

1. A composition comprising: a) a prodrug of a bone antiresorptive agent; and b) a bone targeting moiety; wherein the prodrug of the bone antiresorptive agent and the bone targeting moiety are joined by a cleavable linker.

2. The composition of claim 1, wherein cleavage of the cleavable linker releases and activates the prodrug of the bone antiresorptive agent, thereby producing an active, unbound form of the bone antiresorptive agent.

3. The composition of claim 1, wherein the bone antiresorptive agent is a peptide aldehyde protease inhibitor.

4. The composition of claim 1, wherein the bone antiresorptive agent is a CTSK inhibitor.

5. The composition of claim 4, wherein said CTSK inhibitor is selected from the group consisting of relacatib, balacatinib, odanacatib, and ONO-5334.

6. The composition of claim 1, wherein the bone antiresorptive agent is a proteasome inhibitor.

7. The composition of claim 6, wherein the proteasome inhibitor is Mg132.

8. The composition of claim 1, wherein the bone targeting moiety comprises a bisphosphonate.

9. The composition of claim 7, wherein the bisphosphonate is an alendronate bisphosphonate.

10. The composition of claim 1, wherein the cleavable linker is an acid-cleavable linker.

11. The composition of claim 10, wherein the cleavable linker comprises a hydrazone linker.

12. The composition of claim 1, wherein the prodrug of the bone antiresorptive agent is acid-activated.

13. The composition of claim 12, wherein the prodrug of the bone antiresorptive agent is acid-activated in response to bone resorption.

14. A pharmaceutical composition comprising the composition of any of claims 1 through 13 and an excipient.

15. The pharmaceutical composition of claim 14 in dosage form.

16. The pharmaceutical composition of claim 15 in dosage form of 0.001 mg / kg to 100 mg / kg.

17. An implant comprising a composition of any of claims 1 through 13.

18. A method comprising: administering a composition of any of claims 1 through 13, the pharmaceutical composition of any of claims 14-16, or the implant of claim 17 to a subject.

19. The method of claim 18, wherein the subject has osteoporosis.

20. The method of claim 18, wherein the subject has periarticular osteoporosis, osteoarthritis, rheumatoid arthritis, bone fracture healing, periodontal disease, periimplantitis, dental implant osseointegration, alveolar bone regeneration (e.g., ridge augmentation, sinus lift, socket preservation, in combination with bone graft / bone graft substitute / biomaterial or used alone), endodontic applications (e.g., periapical periodontitis, apical abscess, alveolar regeneration after apicoectomy or respective endodontics, pharmacologic agent for use in dental pulp therapy / vital pulp therapy), osteonecrosis of the jaw, osteomyelitis, dental caries arrest / prevention (e.g., anti-caries medicament), dentinal sensitivity, cemental sensitivity, pharmacologic treatment for external or internal root resorption, adjunct to mechanical debridement, scaling, and root planing, or in conjunction with open flap or ostectomy / osteoplasty, heterotopic ossification, primary cancer (e.g., multiple Myeloma, osteosarcoma, chondrosarcoma, chordoma), metastatic cancers or patients at risk for bone metastasis, mantle cell lymphoma, rare bone diseases (e.g., metachondromatosis, pyncodysostosis, Gorham-Stout Disease, Hajdu-Cheney Syndrome, hypophosphatasia), Paget’s Disease, osteolysis, osteopetrosis, hyperparathyroidism, renal osteodystrophy, rickets, osteomalacia, osteogenesis imperfecta, osteonecrosis, osteomyelitis, and / or orthopedic fracture.

21. Use of a composition of any of claims 1 through 13 or a pharmaceutical composition of any of claims 14 through 16.

22. Use of a composition of any of claims 1 through 13 or a pharmaceutical composition of any of claims 14 through 16 for the treatment of a bone disease or condition.

23. The use of claim 22, wherein the bone disease or condition is osteoporosis, osteoarthritis, rheumatoid arthritis, bone fracture healing, periodontal disease, periimplantitis, dental implant osseointegration, alveolar bone regeneration (e.g., ridge augmentation, sinus lift, socket preservation, in combination with bone graft / bone graft substitute / biomaterial or used alone), endodontic applications (e.g., periapical periodontitis, apical abscess, alveolar regeneration after apicoectomy or respective endodontics, pharmacologic agent for use in dental pulp therapy / vital pulp therapy), osteonecrosis of the jaw, osteomyelitis, dental caries arrest / prevention (e.g., anti-caries medicament), dentinal sensitivity, cemental sensitivity, pharmacologic treatment for external or internal root resorption, adjunct to mechanical debridement, scaling, and root planing, or in conjunction with open flap or ostectomy / osteoplasty, heterotopic ossification, primary cancer (e.g., multiple Myeloma, osteosarcoma, chondrosarcoma, chordoma), metastatic cancers or patients at risk for bone metastasis, mantle cell lymphoma, rare bone diseases (e.g., metachondromatosis, pyncodysostosis, Gorham-Stout Disease, Hajdu-Cheney Syndrome, hypophosphatasia), Paget’s Disease, osteolysis, osteopetrosis, hyperparathyroidism, renal osteodystrophy, rickets, osteomalacia, osteogenesis imperfecta, osteonecrosis, osteomyelitis, and / or orthopedic fracture.

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