Compositions for the prevention and treatment of joint diseases and methods of making and using same

Abstract

Disclosed herein are particles, pharmaceutical compositions, and methods for the prevention and / or treatment of joint diseases in a subject, and methods of making same.

Description

[0001] DUKE-45610.601

[0002] COMPOSITIONS FOR THE PREVENTION AND TREATMENT OF JOINT DISEASES AND METHODS OF MAKING AND USING SAME

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U. S. Provisional Patent Application No. 63 / 776,365, filed on March 24, 2025, the disclosure of which is incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with Government support under Federal Grant no.

[0006] 1AY2AX000013 awarded by the Advanced Research Projects Agency for Health (ARPA-H). The Federal Government has certain rights to this invention.

[0007] BACKGROUND

[0008] Joint diseases that involve the entire joint, such as osteoarthritis (OA), often require delivery of drugs to chondrocytes residing within the cartilage. Although intra-articular drug delivery has been proposed as a more effective strategy compared to systemic administration, the intra-articular administration of drugs remains a challenge due to their rapid clearance within the joint. While nanocarrier-assisted delivery typically increases the half-life of therapeutic molecules within the joint, the diffusion of nanocarriers into the cartilage tissue is hindered by its dense and negatively charged extracellular matrix (ECM). Drug delivery is further complicated for treatments that target the whole cartilage and / or subchondral bone that require deeper drug penetration. Chondrocytes, situated in the lacunae, are sparsely distributed within the anionic ECM, which comprises high-density collagen fibers intertwined with negatively charged proteoglycans. The challenges associated with delivering drugs to chondrocytes residing within the dense cartilage tissue are considered to be a key bottleneck that contributes to the unsuccessful outcomes of OA treatments.

[0009] Cationic nanocarriers that form electrostatic interactions with the anionic cartilage ECM can be an effective approach to overcome the electrostatic barrier. For an effective therapeutic outcome, the nanocarriers first need to adhere to the cartilage tissue and then penetrate and be retained throughout the full-thickness cartilage. It is therefore important to develop nanomaterials that can rapidly adhere upon intra-articular administration toDUKE-45610.601 prevent clearance from the joint and promote transport across the full-thickness cartilage tissue while maintaining adequate retention within the tissue. Transport of molecules, including nanocarriers, within the cartilage tissue is governed by properties such as size, shape, and surface charge, where the latter strongly influences the cartilage-binding properties of the nanocarriers and their retention within the tissue. An optimal net positive charge that enables weak, reversible interactions with the cartilage tissue is necessary for the nanocarriers to penetrate through the full-thickness cartilage tissue. Additionally, the nanocarriers should be able to support high drug loading such that adequate amounts of drug can be delivered with a lower concentration of cationic materials, which can be cytotoxic. Large amounts of cationic polymers can also alter intra-tissue osmotic pressure which could have detrimental effects to cartilage tissue. Research over the years has led to a number of cationic nanocarriers composed of peptides and proteins, amino acids, liposomes, lipid molecules, or polymers to deliver biomolecules to cartilage tissue and also gene editing, with varying levels of success.

[0010] SUMMARY

[0011] In one aspect, disclosed herein is a particle comprising a polymeric nanocarrier, a branched poly-lysine compound covalently attached to a subset of polymer chains in the polymeric nanocarrier, and one or more pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof, and any combination thereof.

[0012] In some embodiments, the one or more pharmaceutically active compounds are selected from the group consisting of PTH and BMP-2, and a combination thereof. In some embodiments, the particle comprises PTH. In some embodiments, the PTH is teriparatide. In some embodiments, the particle comprises BMP-2. In some embodiments, the BMP-2 is recombinant human BMP-2. In some embodiments, the particle comprises PTH and BMP-2. In some embodiments, the PTH is teriparatide and the BMP-2 is recombinant human BMP-2.

[0013] In another aspect, disclosed herein is a particle comprising a polymeric nanocarrier, a branched poly-lysine compound covalently attached to a subset of polymer chains in the polymeric nanocarrier, and one or more pharmaceutically active compounds selected fromDUKE-45610.601 the group consisting of roflumilast, Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-β (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof, and any combination thereof.

[0014] In some embodiments, the one or more pharmaceutically active compounds are selected from the group consisting of roflumilast, PTH, and LDN-214117, and any combination thereof. In some embodiments, the particle comprises roflumilast. In some embodiments, the particle comprises PTH. In some embodiments, the PTH is teriparatide. In some embodiments, the particle comprises LDN-214117. In some embodiments, the particle comprises roflumilast, PTH, and LDN-214117. In some embodiments, the PTH is teriparatide.

[0015] In some embodiments, the branched poly-lysine compound comprises a branched poly(L-lysine) compound. In some embodiments, the branched poly-lysine compound is covalently attached to the polymeric nanocarrier via a direct bond. In some embodiments, the branched poly-lysine compound is covalently attached to the polymeric nanocarrier via a linker.

[0016] In some embodiments, the polymeric nanocarrier comprises a polyester. In some embodiments, the polyester is poly(lactic-co-glycolic) acid (PLGA). In some embodiments, the PLGA comprises about 70 mol% to about 80 mol% lactic acid and about 20 mol% to about 30 mol% glycolic acid. In some embodiments, the PLGA comprises about 75 mol% lactic acid and about 25 mol% glycolic acid. In some embodiments, the PLGA comprises about 60 mol% to about 70 mol% lactic acid and about 30 mol% to about 40 mol% glycolic acid. In some embodiments, the PLGA comprises about 65 mol% lactic acid and about 35 mol% glycolic acid. In some embodiments, the PLGA comprises about 45 mol% to about 55 mol% lactic acid and about 45 mol% to about 55 mol% glycolic acid. In some embodiments, the PLGA comprises about 50 mol% lactic acid and about 50 mol% glycolic acid.

[0017] In some embodiments, a second subset of polymer chains within the polymeric nanocarrier are functionalized with a polyalkylene oxide. In some embodiments, the polyalkylene oxide is polyethylene glycol).DUKE-45610.601 In some embodiments, a third subset of polymer chains within the polymeric nanocarrier are functionalized with a polyalkylene oxide and a compound that binds to bone. In some embodiments, the poly alkylene oxide is poly (ethylene glycol) and the compound that binds to bone is alendronate.

[0018] In some embodiments, the one or more pharmaceutically active compounds is encapsulated within the polymeric nanocarrier. In some embodiments, at least one pharmaceutically active compound is encapsulated in a sugar glass micelle, and the sugar glass micelle is encapsulated within the polymeric nanocarrier. In some embodiments, the sugar glass micelle comprises a sugar selected from trehalose and sucrose, and a surfactant selected from dioctyl sulfosuccinate, hexadecyltrimethylammonium bromide, lecithin, imidazolium-based surfactants, polyoxyethylene ethers, sodium di-(n-octyl)phosphinate, and 3,3-dimethyl-1-butysulfosuccinate sodium salt.

[0019] In some embodiments, the particle comprises the one or more pharmaceutically active compounds in an amount of about 0.01 pg / mg to about 5.0 pg / mg.

[0020] In another aspect, disclosed herein is a pharmaceutical composition comprising a plurality of any of the particles disclosed herein, and a pharmaceutically acceptable carrier.

[0021] In some embodiments, each particle in the plurality of particles comprises a single pharmaceutically active compound. In some embodiments, the single pharmaceutically active compound is BMP-2. In some embodiments, the single pharmaceutically active compound is PTH. In some embodiments, the single pharmaceutically active compound is roflumilast. or a pharmaceutically acceptable salt thereof. In some embodiments, the single pharmaceutically active compound is LDN-214117, or a pharmaceutically acceptable salt thereof.

[0022] In some embodiments, each particle in the plurality of particles comprises two pharmaceutically active compounds. In some embodiments, the two pharmaceutically active compounds are BMP-2 and PTH.

[0023] In some embodiments, each particle in the plurality of particles comprises three pharmaceutically active compounds. In some embodiments, the three pharmaceutically active compounds are roflumilast, PTH, and LDN-214117, or pharmaceutically acceptable salts thereof.DUKE-45610.601 In some embodiments, the plurality of particles comprises a first subset of particles comprising a first pharmaceutically active compound, and a second subset of particles comprising a second pharmaceutically active compound. In some embodiments, the first pharmaceutically active compound is BMP-2, and the second pharmaceutically active compound is PTH.

[0024] In some embodiments, the plurality of particles comprises a first subset of particles comprising a first pharmaceutically active compound, a second subset of particles comprising a second pharmaceutically active compound, and a third subset of particles comprising a third pharmaceutically active compound. In some embodiments, the first pharmaceutically active compound is roflumilast or a pharmaceutically acceptable salt thereof, the second pharmaceutically active compound is PTH, and the third pharmaceutically active compound is LDN-214117 or a pharmaceutically acceptable salt thereof.

[0025] In some embodiments, the pharmaceutical composition further comprises a delivery vehicle. In some embodiments, the delivery vehicle comprises hyaluronic acid. In some embodiments, the delivery vehicle comprises a functionalized hyaluronic acid compound, wherein the functionalized hyaluronic acid compound comprises a hyaluronic acid backbone with one or more side chains attached thereto, wherein at least one side chain comprises a ureidopyrimidinone moiety.

[0026] In another aspect, disclosed herein is a method of treating a joint disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein.

[0027] In another aspect, disclosed herein is a method of treating pain associated with a joint disease or joint tissue damage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein.

[0028] In another aspect, disclosed herein is a method of delivering a pharmaceutically active compound to cartilage and / or subchondral bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein. In some embodiments, the subject is suffering from a joint disease.DUKE-45610.601 In another aspect, disclosed herein is a method of treating an injury to a joint or cartilage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein. In some embodiments, the joint disease comprises arthritis. In some embodiments, the joint disease comprises osteoarthritis. In some embodiments, the joint disease comprises post-traumatic osteoarthritis. In some embodiments, the joint disease comprises joint cartilage or bone damage.

[0029] In some embodiments, the pharmaceutical composition is administered via intraarticular injection.

[0030] In another aspect, disclosed herein is a kit comprising any of the pharmaceutical compositions disclosed herein.

[0031] In some embodiments, the kit further comprises a syringe. In some embodiments, the kit further comprises instructions for using the pharmaceutical composition to treat a joint disease, to treat pain associated with a joint disease or joint tissue damage, to deliver a pharmaceutically active compound to cartilage and / or subchondral bone, or to treat an injury to a joint or cartilage in a subject.

[0032] In another aspect, disclosed herein is a pharmaceutical composition as disclosed herein, for use as a medicament.

[0033] In another aspect, disclosed herein is a pharmaceutical composition as disclosed herein, for use in treating a joint disease, treating pain associated with a joint disease or joint tissue damage, delivering a pharmaceutically active compound to cartilage and / or subchondral bone, and / or treating an injury to a joint or cartilage. In some embodiments, the joint disease comprises arthritis. In some embodiments, the joint disease comprises osteoarthritis. In some embodiments, the joint disease comprises post-traumatic osteoarthritis. In some embodiments, the joint disease comprises joint cartilage or bone damage.

[0034] In another aspect, disclosed herein is a pharmaceutical composition comprising at least two pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc. zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof, and aDUKE-45610.601 pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises PTH and BMP-2.

[0035] In another aspect, disclosed herein is a pharmaceutical composition comprising at least two pharmaceutically active compounds selected from the group consisting of roflumilast. Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-β (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises roflumilast, PTH, and LDN-214117.

[0036] In some embodiments, the pharmaceutical composition is formulated for intraarticular injection.

[0037] In another aspect, disclosed herein is a method of treating a joint disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof.

[0038] In another aspect, disclosed herein is a method of treating pain associated with a joint disease or joint tissue damage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof.

[0039] In another aspect, disclosed herein is a method of delivering a pharmaceutically active compound to cartilage and / or subchondral bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof. In some embodiments, the subject is suffering from a joint disease.DUKE-45610.601 In another aspect, disclosed herein is a method of treating an injury to a joint or cartilage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH). bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of PTH and BMP-2.

[0040] In another aspect, disclosed herein is a method of treating a joint disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of roflumilast, Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-β (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof.

[0041] In another aspect, disclosed herein is a method of treating pain associated with a joint disease or joint tissue damage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of roflumilast, Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-β (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof.

[0042] In another aspect, disclosed herein is a method of delivering a pharmaceutically active compound to cartilage and / or subchondral bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of roflumilast, Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-β (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof. In some embodiments, the subject is suffering from a joint disease.DUKE-45610.601 In another aspect, disclosed herein is a method of treating an injury to a joint or cartilage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of roflumilast, Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-β (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of roflumilast, PTH, and LDN-214117.

[0043] In some embodiments, the joint disease comprises arthritis. In some embodiments, the joint disease comprises osteoarthritis. In some embodiments, the joint disease comprises post-traumatic osteoarthritis. In some embodiments, the joint disease comprises joint cartilage or bone damage.

[0044] In some embodiments, the at least two pharmaceutically active compounds are administered via intra- articular injection. In some embodiments, the at least two pharmaceutically active compounds are administered simultaneously. In some embodiments, the at least two pharmaceutically active compounds are administered sequentially.

[0045] In another aspect, disclosed herein is a kit comprising at least two pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof. In some embodiments, the kit comprises PTH and BMP- 2.

[0046] In another aspect, disclosed herein is a kit comprising at least two pharmaceutically active compounds selected from the group consisting of roflumilast, Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-P (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof. In some embodiments, the kit comprises roflumilast, PTH, and LDN-214117.

[0047] In some embodiments, the kit further comprises a syringe. In some embodiments, the kit further comprises instructions for using the pharmaceutically active compounds toDUKE-45610.601 treat a joint disease, to treat pain associated with a joint disease or joint tissue damage, to deliver a pharmaceutically active compound to cartilage and / or subchondral bone, or to treat an injury to a joint or cartilage in a subject.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a reaction scheme with the synthesis of BPL functionalized with the disulfide group. Cbz-Lys: Ne-benzyloxycarbonyl-L-lysine; Cbz-Lys-NCA: Ne-benzyloxycarbonyl-L-lysine N-carboxyanhydride; Fmoc-Lys(Fmoc)-OH: Na, Ne-Di(9-fluorenylmethoxycarbonyl-L-lysine).

[0049] FIG. 2 shows a reaction scheme with the synthesis of BPL-azide. Cbz-Lys: Ne-benzyloxycarbonyl-L-lysine; Cbz-Lys-NCA: Ne-benzyloxycarbonyl-L-lysine N-carboxyanhydride; Fmoc-Lys(Fmoc)-OH: Na, Ne-Di(9-fluorenylmethoxycarbonyl-L-lysine).

[0050] FIG. 3 shows a reaction scheme for the synthesis of PLGA-PEG, via chemical conjugation of PEG to PLGA.

[0051] FIG. 4 shows a reaction scheme for the synthesis of PLGA-DBCO, via chemical conjugation of DBCO to PLGA.

[0052] FIG. 5 shows a reaction scheme for the synthesis of PLGA-BPL, via chemical conjugation of BPL to PLGA.

[0053] FIG. 6 shows a reaction scheme for the synthesis of PLGA- Ain, via the chemical conjugation of alendronate (azido-PEG-Aln) to PLGA-DBCO.

[0054] FIG. 7 is a schematic showing the assembly of nanocarriers for pharmaceutically active compounds provided herein in accordance with one embodiment of the present disclosure.

[0055] FIG. 8 is a schematic showing the entrapment of protein-based pharmaceutically active compounds (e.g., PTH and BMP-2) into sugar glass micelles in accordance with one embodiment of the present disclosure.

[0056] FIG. 9 shows bone measures on human osteochondral explants for alternative agents at 14-days in culture. The graphs quantify PSR+ bone perimeter / total bone perimeter for control and treated human osteochondral explants from at least 3 patients. Each icon is one measured section. Sections were measured by two individual blinded reviewers. N=3DUKE-45610.601 replicates per sample. The percentages indicate average percent improvement over pairwise untreated controls.

[0057] FIG. 10 shows cartilage measures on human osteochondral explants for alternative agents. Graph quantifying SafO+ area / total cartilage area for control and human osteochondral explants from 3-5 patients. Sections were measured by 3 individual blinded reviewers. Scores are normalized to control. Mean + SEM is indicated. The percentages indicate average percent improvement over pairwise untreated controls.

[0058] FIG. 11 shows a schematic for studies whereby human osteochondral explants were evaluated pairwise for anabolic cartilage or bone activity using SafO or PSR measurements as the primary outcome for TAI or TA2 respectively. The outcomes of alternative agents were compared to those from the proposed lead compounds.

[0059] FIGS. 12A-12C show primary bone measures for human osteochondral explants treated with PTH (100 nM) for 12 days, 7 days, and 1 day in culture. The graphs quantify PSR+ bone perimeter / total bone perimeter for control and PTH treatments (100 nM) of human osteochondral explants at: (A) 14 days, (B) 7 days, and (C) 1 day. Sections were measured by blinded reviewers. 14 day culture results in (A) are representative of the majority of samples that show results greater than 10% of the mean of the control. This was not achievable at 7 days or 1 day in culture. N=3 replicates per sample.

[0060] FIGS. 13A-13B show bone and cartilage measures on human osteochondral explants for TAI agents alone and in combinations following 14-days in culture. The graphs quantify the relative increase or decrease in PSR+ perimeter / total bone perimeter (A) and SafO+ area / total cartilage area (B) for control and TA1 drug treated human osteochondral explants. Sections were measured by at least two individual blinded reviewers.

[0061] FIGS. 14A-14B show bone and cartilage staining of human osteochondral explants following 14-days in culture with or without PTH+BMP2. Representative images for PSR staining (A) and SafO staining (B) for control and PTH+BMP2 drug treated human osteochondral explants. Control and treated samples are derived from single patients.

[0062] FIGS. 15A-15B show results from additional experiments with sequential treatments for the lead TAI formulation, in human osteochondral explants, as described in Example 2.DUKE-45610.601 FIG. 16 shows a schematic summarizing the results of alternative agent testing in vitro and in ex vivo osteochondral explant tissues.

[0063] FIG. 17 shows primary cartilage measures on human osteochondral explants for TA2 agent combinations that included alternative agents following 14 days in culture. The graphs quantify the relative increase or decrease in SafO+ area / total cartilage for control and TA2 drug treated human osteochondral explants. Mean + SEM is indicated. Sections were measured by at least two individual blinded reviewers. The percentages indicate average percent improvement over pairwise untreated controls.

[0064] FIGS. 18A-18B show SafO cartilage staining of human osteochondral explants following 14-days in culture with or without GDFll+roflumilast+EDN (GRE) and roflumilast+EDN+PTH (REP). Representative images for SafO staining for control and GRL treated (A) and control and RLP treated (B) human osteochondral explants. Control and treated samples are derived from single patients.

[0065] FIGS. 19A-19B show results from additional experiments with sequential treatments for TA2 formulations, in human osteochondral explants, as described in Example 2. The lead formulation / sequence (roflumilast + PTH + LDN) outperformed other tested formulations.

[0066] FIG. 20 shows MicroCT assessments of Connectivity Densities for the subchondral plate of normal and MMNX induced OA joints with or without TAI treatments individually or in combination. PTH= PTH 1-34, BMP=rBMP2, ALK=ALK4-Fc.

[0067] FIG. 21 shows representative 2D microCT images in the frontal plane at 3 levels (Anterior, Middle, and Posterior) of the tibia medial subchondral plate / bone compartment. A comparison of subchondral plate / bone changes between 4-wpi MMNX injured OA, uninjured no OA contralateral controls, PTH+BMP2 treated MMNX injured OA samples at 8-wpi, and PTH treated MMNX injured OA samples at 8-wpi. Red, yellow, and green colored lines indicate the location of 2D slices within the rat knee joint.

[0068] FIGS. 22A-22B show secondary cartilage measures on uninjured control and MMNX injured rat knees with and without TAI agent treatments. The graphs quantify the OARSI scores (A) and SafO+ area / total cartilage are measurements (B) for control and TA1 drug treated rats. Sections were measured by at least two individual blinded reviewers. The cartoon (red dashed lines) shows the 3 levels in the sagittal plane collected for cartilageDUKE-45610.601 measures. One-way ANOVA with multiple comparisons. p<0.05. Each data point represents a mean OARSI score for an individual rat.

[0069] FIG. 23 shows representative SafO / Fast Green stained images of uninjured control and MMNX injured rat knees with and without PTH+BMP2 TAI agent treatments. The cartoon (red dashed lines) shows the 3 levels in the sagittal plane (Outer, Middle, Central) collected for cartilage measures at Levels 1, 2, and 3. Level 2 images are shown at higher magnification.

[0070] FIGS. 24A-24B show: (A) Von Frey pain measures from the paws of contralateral control and MMNX injured knees with and without PTH+BMP2 (TAI) treatment. Von Frey measures spanning 11 -weeks from the time of MMNX injury. Rats were treated with or without PTH+BMP2 lx concentration. Statistical analyses indicate that PTH+BMP2 OA treated rats show PWT within one standard deviation of contralateral controls. PTH and BMP2 are conjugated to a PLGA nanocarrier with branched poly (1-lysine) (BPL) and Alendronate. PLGA ratio 50:50 for BMP2. PLGA ratio 75:25 for PTH. lx = BMP2 (lOOOng) + PTH (120ng). One-way ANOVA with Tukey’s multiple comparisons. * indicates within one standard deviation of no OA control; (B) pain mitigation in female severe OA demonstrated by BUD-010.

[0071] FIG. 25 shows data from a rat MMNX model, showing that TAI demonstrated long-term pain reduction in the Rat MMNX model (CL = contralateral control).

[0072] FIGS. 26A-26B show primary cartilage measures on uninjured control and MMNX injured rat knees with and without TA2 agent treatments. The graphs quantify the OARSI scores (A) and SafO area / total cartilage area (B) for control and TA2 drug treated rats following a severe MMNX injury (severe OA model). One-way ANOVA with multiple comparisons. p<0.05. Each data point represents a mean OARSI score for an individual rat.

[0073] FIG. 27 shows representative SafO / Fast Green stained images of uninjured control and MMNX injured rat knees with and without RLP TA2 agent treatments. The cartoon (red dashed lines) shows the 3 levels in the sagittal plane (Outer, Middle, Central) collected for cartilage measures at Levels 1, 2, and 3. Level 2 images are shown at higher magnification.DUKE-45610.601 FIG. 28 shows Von Frey pain measures from the paws contralateral control and MMNX injured knees with and without RLP treatment. Von Frey measures spanning 12-weeks from the time of MMNX severe surgery. Rats were treated at 4-wpi with or without RPL for 8-weeks. Statistical analyses indicate that RPL OA treated rats show MMNX injured limb with PWT that is significantly different than the untreated MMNX injured OA limb and within one standard deviation of CL by 6-wpi.

[0074] FIGS. 29A-29D show data from human knee osteochondral explants (14-day cultures) showing that the final TAI lead formulation (BP) reproducibly induces significant bone formation across ethnicities, sex, and ages of patient derived knee osteochondral explants.

[0075] FIGS. 30A-30B show analysis of sequential treatments for the lead TAI formulation (BP) for human shoulder and hip osteochondral explants (14-day cultures), demonstrating that the formulation reproducibly induces significant bone formation across multiple joints, including shoulder and hip.

[0076] FIGS. 31A-31B show: (A) a schematic description of TAI nanocarrier (PLGA-BPL-Aln) assembly with varying surface functionalization and drug incorporation; and (B) data for the localization of PLGA-BPL-Aln to cartilage and subchondral bone.

[0077] FIGS. 32A-32D show data for the restoration of bone and cartilage outcomes following TAI IA treatment. BMP2+PTH was administered as a single dose at 4- weeks post injury (wpi) and the joint tissues were harvested at 8-wpi and 10-wpi. (A, C) TA1-IA formulation regenerates bone to restore Connectivity Density scores to baseline 10-wpi (equivalent to 1 human year). (B, D) Representative SafO / Fast Green-stained joint sections and quantification from OA rats with and without TAI treatment via intra- articular injection. PTH and BMP2 are conjugated to a PLGA nanocarrier with branched poly (1-lysine) (BPL) and Alendronate. PLGA ratio 50:50 for BMP2. PLGA ratio 75:25 for PTH.

[0078] FIG. 33 shows representative IHC images of subchondral bone from rats subjected to MMNX surgery (OA) or uninjured (control), with (OA+TA1) or without (OA) TAI IA treatment.

[0079] FIG. 34 shows representative IHC images showing delivery of the TAI API PTH to subchondral bone.DUKE-45610.601 FIG. 35 shows that critical sized defects are induced (left) following surgery for severe OA, and these defects are repaired following treatment with TAI IA (right).

[0080] FIGS. 36A-36D show a detailed patient profile analysis of sequential treatments for the lead TA2 formulation (RPE). The data from human knee osteochondral explants (14-day cultures) treated with TA2 drugs showed significant increases in cartilage regeneration compared to untreated controls.

[0081] FIGS. 37A-37B show analysis of sequential treatments for the lead TA2 formulation (RPE) for human shoulder and hip osteochondral explants (14-day cultures). When all patient data was pooled together, an average of 44% increase was observed.

[0082] FIG. 38 shows a schematic illustration of nanocarrier functionalization, API loading, API release and in vivo delivery for TA2 IA.

[0083] FIGS. 39A-39B show that TA2 treatment exhibits effective cartilage regeneration following IA drug delivery. The tibial (bottom) surface appears uneven in the OA+TA2 section due to the commonly observed artifact of folding of the section.

[0084] FIG. 40 shows representative IHC images of joint cartilage from rats with and without TA2 IA treatment. High magnification images of pCREB, JAG1, pSMAD2 / 3, and pSMAD 1 / 5 IHC / IF stained sections of joint cartilage of rats with and without TA2 intraarticular injection. TA2 treatment was delivered at 4-wpi and tissues harvested at 8-wpi. MMNX injured rats without treatment had tissue harvested at time of drug delivery or 4wpi. Contralateral joints are used as controls. One-way ANOVA with Tukey’s multiple comparisons. ** indicates significance with p< 0.01. n=3 rats per group.

[0085] FIG. 41 shows that critical sized defects are induced (left) following surgery for severe OA, and these defects are repaired following treatment with TA2 IA (right). Severe OA surgery induces a ~1.5+mm full thickness, non-healing critical sized defect by 4wpi (time of treatment) that progresses to ~3+mm full thickness by 8wpi with continual cartilage loss over time. TA2 IA treatment reverses this process to regenerate articular cartilage and restore normal joint architecture.

[0086] FIG. 42 shows that combining TAI and TA2 has no detrimental effects during cartilage regeneration. Rats underwent MMNX surgery and were treated with TAI or TA1+TA2.DUKE-45610.601 FIG. 43 shows that increasing the weight ratio of LDN to the nanocarrier polymer precursors resulted in higher loading capacity using the single emulsion method.

[0087] FIG. 44 shows data comparing the loading efficiency of EDN214117 using water vs. PBS as aqueous phase on loading capacity.

[0088] FIG. 45 shows the release profile of EDN from PLGA (75:25) nanocarriers in PBS. N > 3.

[0089] FIG. 46 shows the roflumilast content in the feed on loading capacity using PBS as the aqueous phase. N > 3.

[0090] FIG. 47 shows the release profile of roflumilast from PLGA (50:50) nanocarriers in PBS. N > 3.

[0091] FIG. 48 is a representative TEM image of the PTH loaded sugar glass micelle recorded at 80 kV and a graph showing size distribution determined by Image J software from the TEM image in accordance with one embodiment of the present disclosure.

[0092] FIG. 49 is a graph showing intracellular cAMP levels of free PTH and those incorporated into sugar glass micelles in accordance with one embodiment of the present disclosure.

[0093] FIG. 50 shows the effect of PTH-SG in the feed on loading capacity. PTH concentrations were measured using an ELISA assay per manufacturer’s protocols. N > 3.

[0094] FIGS. 51A-51B show the API Release Profile from Nanocarriers.

[0095] FIG. 52 is a graph showing the release profile of PTH from the nanocarrier in accordance with one embodiment of the present disclosure.

[0096] FIGS. 53A-53B show the effect of BMP2 content in the feed on loading capacity. FIG. 54 shows LDN release from PLGA nanocarriers with different LA: GA ratio. N>3.

[0097] FIG. 55 shows data for an explant assay to assess cartilage penetration and bone binding of nanocarriers disclosed herein. In the images, C is cartilage, B is subchondral bone.

[0098] FIGS. 56A-56B shows cytotoxicity data for nanocarriers disclosed herein.

[0099] FIG. 57 shows that nanocarriers disclosed herein are not cytotoxic to osteochondral explants.DUKE-45610.601 FIG. 58 shows whole body IVIS images that show retention of the labeled nanocarriers without distribution anywhere else in the body.

[0100] FIG. 59 shows knee joint IVIS images over time.

[0101] FIG. 60 shows that fluorescence intensity in the knee joint declines over time. FIG. 61 shows histology slides that show fluorescence signal is localized to the joint.

[0102] FIG. 62 shows knee joint IVIS images of Cy7 labelled nanocarrier or free Cy7 in rat.

[0103] FIG. 63 shows Quantitative Cy7 fluorescence signal over time in the rat joint. FIG. 64 shows that PTH is localized in the subchondral bone following BUD-010 I A delivery.

[0104] FIGS. 65A-65B show that MMNX Rats treated with BUD-010 show improved OARSI scores.

[0105] FIG. 66 shows histology sections of control, OA, and BUD-010 treated animals. FIG. 67 shows that BUD-010 treatment restores subchondral bone connectivity density by MicroCT (OA = MMNX, no OA = contralateral control, PTH+BMP-2 = BUD-010).

[0106] FIG. 68 shows that BUD-010 improves pain response over time.

[0107] DETAILED DESCRIPTION

[0108] Described herein are particles, and pharmaceutical compositions comprising the particles, wherein the particles comprise, consist of, or consist essentially of a polymeric nanocarrier, a branched poly-lysine (e.g., branched poly(L-lysine)) compound covalently attached to a subset of the polymer chains in the polymeric nanocarrier, and one or more pharmaceutically active compounds. Also disclosed herein are pharmaceutical compositions comprising at least two pharmaceutically active compounds. The particles and pharmaceutical compositions can be used for the treatment of arthritis, such as osteoarthritis (e.g., post-traumatic osteoarthritis) and related joint diseases.

[0109] A. Definitions

[0110] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific languageDUKE-45610.601 will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[0111] Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical objects of the article. By way of example, “an element” means at least one element and can include more than one element.

[0112] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.

[0113] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0114] As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0115] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C. or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0116] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what isDUKE-45610.601 specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0117] As used herein, “treatment,” “therapy” and / or “therapy regimen” refer to the clinical intervention made in response to a disease (e.g., a joint disease) manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease (e.g., a joint disease) and / or the remission of the disease (e.g., a joint disease).

[0118] As used herein, the terms “attenuation” or “attenuate” refer to the reduction, diminution, decrease, inhibition, elimination or abatement of the degree, intensity or extent of the disease (e.g., a joint disease), or the symptom of the disease (e.g., a joint disease).

[0119] As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disease in a subject, who does not have, but is at risk of or susceptible to developing a disease (e.g., a joint disease).

[0120] The terms “effective amount” or “therapeutically effective amount” refer to an amount sufficient to effect beneficial or desirable biological and / or clinical results.

[0121] As used herein, the term “administering” an agent / composition, such as a therapeutic composition to a subject or cell, is intended to refer to dispensing, delivering or applying the substance to the intended target. In terms of the therapeutic agent, the term “administering” is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to a subject by any suitable route for delivery of the therapeutic agent / composition to the desired location in the subject, including delivery by either the parenteral or oral route, intramuscular injection, intra-articular, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by the intranasal or respiratory tract route. In some embodiments, administration is by intra-articular injection. In other embodiments, administration is intravenous.

[0122] The term “biological sample” as used herein includes, but is not limited to. a sample containing tissues, cells, and / or biological fluids isolated from a subject. Examples of biological samples include, but are not limited to, tissues, cells, biopsies, blood, lymph,DUKE-45610.601 serum, plasma, urine, saliva, mucus and tears. A biological sample may be obtained directly from a subject e.g., by blood or tissue sampling) or from a third party {e.g., received from an intermediary, such as a healthcare provider or lab technician).

[0123] The term “disease” as used herein includes, but is not limited to, any abnormal physical or psychological condition and / or disorder of a structure or a function that affects a part of an organism. It may be caused by an external factor, such as an infectious disease, or by internal dysfunctions, such as autoimmunity, tissue / cartilage degradation, cancer, cancer metastasis, and the like. In some embodiments, the disease comprises a joint disease.

[0124] As used herein, the term “joint disease” refers to the more than 100 different kinds of diseases and conditions that involve the inflammation, swelling, tenderness, and / or stiffness of one or more of the joints. A joint disease may, for example, be the result of the breakdown of cartilage {e.g., osteoarthritis) or the result of autoimmunity {e.g., rheumatoid arthritis). Suitable examples of joint diseases include, but are not limited to. arthritis, including osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, osteochondritis dissecans, avascular necrosis, post-traumatic osteoarthritis, and psoriatic arthritis, fibromyalgia, gout, lupus, and the like. In other embodiments, a joint disease may comprise joint cartilage or bone damage that has not yet been diagnosed as arthritis or osteoarthritis or post-traumatic OA.

[0125] As used herein, the term “pain” refers to the basic bodily sensation induced by a noxious stimulus, received by naked nerve endings, characterized by physical discomfort {e.g., pricking, throbbing, aching, etc.) and typically leading to an evasive action by the individual. As used herein, the term pain may include “nociceptive pain:” that results from tissue damage or the threat of tissue damage. The term pain may also include chronic and acute neuropathic pain. The terms “neuropathic pain” or “neurogenic pain” can be used interchangeable and refer to pain that arises from direct stimulation of nervous tissue itself, central or peripheral, and can persist in the absence of stimulus. Chronic neuropathic pain often seems to have no obvious cause, however, some common causes may include, but are not limited to, arthritis (including rheumatoid and osteoarthritis), alcoholism, amputation, back, leg and hip problems, chemotherapy, diabetes, facial nerve problems, HIV infection or AIDS, multiple sclerosis, shingles, and spine surgery.DUKE-45610.601 As used herein, the term “subject” and “patient” are used interchangeably and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. The methods and compositions disclosed herein can be used on a sample either in vitro (for example, on isolated cells or tissues) or in vivo in a subject (i.e. living organism, such as a patient). In some embodiments, the subject is a human. In other embodiments, the subject is a human suffering from, or at risk of developing, a joint disease, such as osteoarthritis.

[0126] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Sorrell, Organic Chemistry, 2ndedition, University Science Books, Sausalito, 2006; Smith, March's Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7thEdition, John Wiley & Sons, Inc., New York, 2013; Larock, Comprehensive Organic Transformations, 3rdEdition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modem Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0127] As used herein, the term “alkyl” refers to a radical of a straight or branched saturated hydrocarbon chain. The alkyl chain can include, e.g., from 1 to 24 carbon atoms (C1-C24 alkyl), 1 to 16 carbon atoms (C1-C16 alkyl), 1 to 14 carbon atoms (C1-C14 alkyl), 1 to 12 carbon atoms (C1-C12 alkyl), 1 to 10 carbon atoms (C1-C10 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), 1 to 4 carbon atoms (C1-C4 alkyl), 1 to 3 carbon atoms (C1-C3 alkyl), or 1 to 2 carbon atoms (C1-C2 alkyl). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, secbutyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.DUKE-45610.601 As used herein, the term “nitrogen protecting group” refers to a group intended to protect an amino group against undesirable reactions during synthetic procedures. Common nitrogen protecting groups include acyl groups such as acetyl, benzoyl, 2-bromoacetyl, 4-bromobenzoyl, tert-butylacetyl, carboxaldehyde, 2-chloroacetyl, 4-chlorobenzoyl. a- chlorobutyryl, 4-nitrobenzoyl, o-nitrophenoxyacetyl. phthalyl. pivaloyl, propionyl, trichloroacetyl, and trifluoroacetyl; sulfonyl groups such as benzenesulfonyl andp- toluenesulfonyl; carbamate-forming groups such as benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), p-chlorobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, fluorenylmethyloxycarbonyl (Fmoc); and the like. Nitrogen protecting groups are well-known in the art and include those described in detail in Greene's Protective Groups in Organic Synthesis, P. G. M. Wuts, 5th edition, John Wiley & Sons, Inc., 2014, which is incorporated herein by reference.

[0128] When a group or moiety can be substituted, the term “substituted” indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments 1, 2, or 3; and in other embodiments 1 or 2) hydrogens on the group indicated in the expression using “substituted” can be replaced with a selection of recited indicated groups or with a suitable substituent group known to those of skill in the art (e.g., one or more of the groups recited below), provided that the designated atom's normal valence is not exceeded. Substituent groups include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroaryl, heterocyclyl. hydroxy, hydrazine, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, thione, or combinations thereof.

[0129] As used herein, in chemical structures the indication:

[0130]

[0131] represents a point of attachment of one moiety (e.g., a substituent group to the rest of the compound).

[0132] When groups are specified by their conventional chemical formulae, written from left to right, such indication also encompass substituent groups resulting from writing the structure from right to left. For example, if a bivalent group is shown as -CH2O-, such indication also encompasses -OCH2-; similarly, -OC(O)NH- also encompasses -DUKE-45610.601 NHC(O)O-. When linker moieties are shown, the linkers can be attached to other moieties of the compound in either direction.

[0133] The term “pharmaceutically acceptable salt” refers to salts or zwitterions of the compounds which are water or oil-soluble or dispersible, suitable for treatment of diseases without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio and effective for their intended use. The salts may be prepared during the final isolation and purification of the compounds or separately by reacting an amino group of the compound with a suitable acid. For example, a compound may be dissolved in a suitable solvent, such as but not limited to methanol and water, and treated with at least one equivalent of an acid, such as hydrochloric acid. The resulting salt may precipitate out and be isolated by filtration and dried under reduced pressure. Alternatively, the solvent and excess acid may be removed under reduced pressure to provide a salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloric, hydrobromic, sulfuric, phosphoric, and the like. Amino groups of the compounds may also be quaternized with alkyl chlorides, bromides and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like.

[0134] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0135] B. Particles

[0136] Disclosed herein are particles comprising a polymeric nanocarrier, a branched polylysine compound covalently attached to a subset of polymer chains in the polymeric nanocarrier, and one or more pharmaceutically active compounds.

[0137] i. Polymers

[0138] The core of the polymeric nanocarrier is formed from polymer chains. In some embodiments, the polymeric nanocarrier comprises a polyester. For example.DUKE-45610.601 biocompatible and biodegradable polyesters can result in controlled degradation and release of pharmaceutically active compounds from polymeric nanocarriers. In some embodiments, the polyester is poly(lactic-co-glycolic acid) (PLGA), which is a linear copolymer that can be prepared at different ratios between its monomers, lactic acid (LA) and glycolic acid (GA). PLGA degrades by hydrolysis of its ester linkages in the presence of water to produce its constituent monomers, lactic acid and glycolic acid. These monomers are by-products of various metabolic pathways in the body under normal physiological conditions; lactic acid is metabolized in the tricarboxylic acid cycle and eliminated via carbon dioxide and water, while glycolic acid is metabolized in the same way and is excreted through the kidneys.

[0139] The PLGA in the nanocarrier can include a variety of ranges of lactic acid and glycolic acid. For example, in some embodiments, the PLGA comprises about 40 mol% to about 80 mol% lactic acid and about 20 mol% to about 60 mol% glycolic acid. In some embodiments, the PLGA comprises about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, or about 80 mol% lactic acid, and about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, or about 40 mol% glycolic acid, hi some embodiments, the PLGA comprises about 65 mol% to about 85 mol% lactic acid and about 15 mol% to about 35 mol% glycolic acid. In some embodiments, the PLGA comprises about 70 mol% to about 80 mol% lactic acid and about 20 mol% to about 30 mol% glycolic acid. In some embodiments, the PLGA comprises about 55 mol% to about 75 mol% lactic acid and about 25 mol% to about 45 mol% glycolic acid. In some embodiments, the PLGA comprises about 60 mol% to about 70 mol% lactic acid and about 30 mol% to about 40 mol% glycolic acid. In some embodiments, the PLGA comprises about 40 mol% to about 60 mol% lactic acid and about 40 mol% to about 60 mol% glycolic acid. In some embodiments, the PLGA comprises about 45 mol% to about 55 mol% lactic acid and about 45 mol% to about 55 mol% glycolic acid. In some embodiments, the PLGA comprises about 40 mol% lactic acid and about 60 mol% glycolic acid. In some embodiments, the PLGA comprises about 45 mol% lactic acid and about 55 mol% glycolic acid. In some embodiments, the PLGA comprises about 50 mol% lactic acid and about 50 mol% glycolic acid. In some embodiments, the PLGA comprises about 55 mol% lactic acid and about 45 mol% glycolic acid. In some embodiments, the PLGADUKE-45610.601 comprises about 60 mol% lactic acid and about 40 mol% glycolic acid. In some embodiments, the PLGA comprises about 65 mol% lactic acid and about 35 mol% glycolic acid. In some embodiments, the PLGA comprises about 70 mol% lactic acid and about 30 mol% glycolic acid. In some embodiments, the PLGA comprises about 75 mol% lactic acid and about 25 mol% glycolic acid. In some embodiments, the PLGA comprises about 80 mol% lactic acid and about 20 mol% glycolic acid.

[0140] The PLGA in the nanocarrier can include PLGA having a range of different molecular weights. In some embodiments, the PLGA in the polymeric nanocarrier has a molecular weight of from about 15 kDa to about 150 kDa, or about 20 kDa to about 125 kDa. In some embodiments, the PLGA in the polymeric nanocarrier has a molecular weight of about 20 to about 40 kDa (e.g., about 24 to about 38 kDa), about 40 to about 55 kDa (e.g., about 45 to about 50 kDa), or about 60 to about 110 kDa (e.g., about 66 to about 107 kDa). For example, PLGA is commercially available at a variety of different molecular weights, and is often sold as a polydisperse product having an indicated molecular weight range. For example, commercially available PLGA products may have molecular weight ranges of 24-38 kDa, 30-60 kDa, 45-50 kDa, 50-75 kDa, 40-75 kDa, or 66-107 kDa. ii. B PL-functionalized polymers

[0141] In the polymeric nanocarriers, a subset of polymer chains (e.g., polyester polymer chains, such as PLGA polymer chains) are functionalized with a branched poly-lysine (BPL) compound, such as a branched poly(L-lysine) compound. Poly-lysine, such as poly(L-lysine), is a positively charged synthetic amino acid chain that increases the number of surface positive charges. When used for local administration to cartilage tissue, the positively charged BPL moieties may interact with the anionic cartilage extracellular matrix (ECM) via electrostatic interactions, allowing for retention of the particles in the joint space and transport through the full thickness of the cartilage tissue.

[0142] Branched poly-lysine compounds can be synthesized according to a general method known in the art. See, e.g., Rodrfguez-Hemandez et al. Biomacromolecules 2003. 4(2), 249-258. See also International Patent Publication No. WO 2025 / 043212 A2, which is incorporated by reference herein in its entirety.

[0143] More specifically, and as further outlined in the Examples, branched poly(L-lysine) compounds can be synthesized by a ring-opening polymerization reaction using Ne-DUKE-45610.601

[0144] benzyloxycarbonyl-L-lysine-N-carboxyanhydride (Cbz-Lys-NCA) as a monomer and an initiator with primary amine groups. First, a lysine oligomer containing a certain number of lysine units, such as 10 lysine units (i.e., nl = 10), termed the core peptide, can be synthesized using Cbz-Lys-NCA and a compound having a primary amino group (e.g., n-hexylamine, or a functionalized alkylamine, or a functionalized PEG-amine compound). Three generations of BPL molecules - generation 0 (GO) with two branches (i.e., each R3is hydrogen), generation 1 (Gl) with four branches (i.e., each R3is a group of formula (A) and each R4is hydrogen), and generation 2 (G2) with eight branches (i.e., each R3is a group of formula (A) and each R4is a group of formula (B)), can then be synthesized by reacting the Cbz-Lys-NCA with either the core peptide, GO, or Gl as initiators to generate GO, Gl, or G2 BPL molecules, respectively (FIG. 1). Increasing the number of branches provides multivalent BPL molecules with increasing functional groups and charge density without significantly increasing their hydrodynamic diameter. A theoretical calculation, based on the monomer-to-initiator feed ratio, suggests that the GO, Gl, and G2 BPL molecules possess approximately 30, 70, and 150 functional amine groups, respectively (Table 1).

[0145] Table 1. Theoretical characterization of branched poly(L-lysine) molecules assuming each arm contains 10 lysine repeating units.

[0146] Poly(L-lysine) Number of arms Theoretical number of lysine units per molecule

[0147] Core 1 10 GO 2 30 Gl 4 70

[0148]

[0149] G2 8 150

[0150] In the BPL moieties of formula (I), complete deprotection of the nitrogen protecting groups (such as carbobenzyloxy, Cbz) results in a compound in which each R2is -CH₂CH₂CH₂CH₂NHR³ and Rais hydrogen. In the event that deprotection is incomplete, however, the BPL moiety of formula (I) may include a certain number of remaining nitrogen protecting groups at each Raposition (e.g., benzyloxycarbonyl groups).

[0151] Accordingly, in the particles disclosed herein, a subset of polymer chains in the polymeric nanocarrier are functionalized with a branched poly-lysine moiety having a structure of formula (I):DUKE-45610.601

[0152]

[0153] or a salt thereof, wherein:

[0154] p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;

[0155] each R² is independently -CH₂CH₂CH₂CH₂NHR³, wherein each Rais independently hydrogen or a nitrogen protecting group;

[0156] each R3is independently hydrogen or a group of formula (A):

[0157]

[0158] each R4is independently hydrogen or a group of formula (B):

[0159]

[0160] nl, n2, n3, n4, n5. n6. and n7 are each independently 5, 6, 7. 8, 9. 10. 11. or 12. In some embodiments, p is 1, 2, 3, or 4.DUKE-45610.601 In some embodiments, each R2is -CH2CH2CH2CH2NHRa, wherein each Rais hydrogen.

[0161] In some embodiments, each R3is hydrogen (i.e., the branched poly-lysine moiety is a GO BPL moiety). In other embodiments, each R3is a group of formula (A) and each R4is hydrogen (i.e., the branched poly-lysine moiety is a G1 BPL moiety). In other embodiments, each R3is a group of formula (A) and each R4is a group of formula (B) (i.e., the branched poly-lysine moiety is a G2 BPL moiety). In particular embodiments, the branched poly-lysine is a G1 BPL moiety.

[0162] The branched poly-lysine moiety of formula (I) can be covalently attached to the polymeric nanocarrier via a direct bond or via a linker. For example, the branched polylysine moiety of formula (I) can be covalently attached to the polymeric nanocarrier by reacting a polymer of the polymeric nanocarrier (e.g., PLGA) having a reactive moiety, with a precursor compound to the moiety of formula (I) that comprises a complementary reactive moiety. For example, in some embodiments, the poly-lysine moiety of formula (I) is attached to a polymer of the polymeric nanocarrier by reacting a poly-lysine precursor compound of formula (II) shown below, which has a reactive group X, with a particle in which a subset of polymer chains in the nanocarrier have a reactive group Y, wherein X and Y react with each other to form a B PL-functionalized polymer. For example, a compound of formula (II) is:

[0163]

[0164] or a salt thereof, wherein:

[0165] p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;

[0166] each R2is independently -CH2CH2CH2CH2NHR', wherein each Rais independently hydrogen or a nitrogen protecting group;

[0167] each R3is independently hydrogen or a group of formula (A):DUKE-45610.601

[0168]

[0169] each R4is independently hydrogen or a group of formula (B):

[0170]

[0171] nl, n2, n3, n4, n5, n6, and n7 are each independently 5, 6, 7, 8, 9, 10, 11, or 12; Lis a linker; and

[0172] X is a reactive group.

[0173] In some embodiments, each R2is -CH2CH2CH2CH2NHRa, wherein each Rais hydrogen.

[0174] In some embodiments, each R3is hydrogen (i.e., the branched poly-lysine moiety is a GO BPL moiety). In other embodiments, each R3is a group of formula (A) and each R4is hydrogen (i.e., the branched poly-lysine moiety is a G1 BPL moiety). In other embodiments, each R3is a group of formula (A) and each R4is a group of formula (B) (i.e., the branched poly-lysine moiety is a G2 BPL moiety). In particular embodiments, the branched poly-lysine is a G1 BPL moiety.

[0175] For the linker L, any suitable linker can be used. In embodiments, the linker comprises any combination of -CH2-, -CH(CH3)-, -C(CH3)2-, -CH=CH-, -C=C-, -O-, -NH-, -S-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, arylene, hetero arylene, cycloalkylene, and heterocyclylene groups, wherein the arylene, heteroarylene, cycloalkylene, and heterocyclylene groups are independently unsubstituted or substituted with 1, 2, or 3DUKE-45610.601 substituents. In some embodiments, the linker comprises any combination of -CH2-, -O-, -C(O)-, and -NH- groups. In some embodiments, the linker comprises one or more alkylene oxide groups, such as ethylene glycol groups. For example, in some embodiments, the linker comprises a group -(CH₂CH₂O)q-, wherein q is 1, 2, 3, 4, 5, or 6.

[0176] For the reactive group X, any suitable reactive group can be used. The group X is selected to react with a complementary reactive group that is included on a polymer chain in the nanocarrier. In some embodiments, X is selected from an alkyne (e.g., a -C=CH group or a dibenzocyclooctyne), an azide, a thiol, an amine, an alkene, a maleimide, or a succinimidyl ester. In some embodiments, X is an azide. In some embodiments, X is a dibenzocyclooctyne.

[0177] In some embodiments, the linker comprises a group that is the reaction product of two reactive moieties. For example, in some embodiments, such a group has a formula selected from:

[0178]

[0179] Hi. Poly (alkylene oxide)-functionalized polymers

[0180] In the polymeric nanocarriers, in some embodiments, a second subset of polymer chains within the polymeric nanocarrier (e.g., polyester chains, such as PLGA chains) are functionalized with a poly(alkylene oxide). In some embodiments, the second subset of polymer chains are functionalized with a polyethylene glycol) moiety. Poly(ethylene glycol) is a hydrophilic compound that is commonly used in drug formulations and other products. When incorporated into the particles disclosed herein, poly(alkylene oxides) such as polyethylene glycol) may be included, e.g., to prevent protein adsorption onto the particles.

[0181] In some embodiments, a second subset of polymer chains within the polymeric nanocarrier are functionalized with a poly(ethylene glycol) moiety of formula:DUKE-45610.601

[0182]

[0183] wherein m is an integer from about 25 to about 100. In some embodiments, m is 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57. 58. 59. 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71. 72. 73. 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. As poly(alkylene oxide) (e.g., polyethylene oxide)) starting materials can be sold as polydisperse products with a molecular weight range, in some embodiments, the particles will include a subset of polymer chains functionalized with poly(alkylene oxides) (e.g., polyethylene glycol)) of different molecular weights, i.e., each m is within a range. For example, in some embodiments, m is about 25 to about 100, about 30 to about 95, about 35 to about 90, about 40 to about 85, or about 45 to about 80.

[0184] The poly(alkylene oxide) (e.g., poly(ethylene glycol) chains) may be attached to the second subset of polymer chains within the polymeric nanocarrier via a direct bond or via a linker. Any suitable linker can be used, including linkers described above regarding the attachment of the BPL moiety to the first subset of polymer chains. For example, in some embodiments, the linker comprises any combination of -CH2-, -CH(CH3)-, -C(CH3)2, -CH=CH-, -CO, -O-, -NH-, -S-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, arylene, heteroarylene, cycloalkylene, and heterocyclylene groups, wherein the arylene, heteroarylene, cycloalkylene, and heterocyclylene groups are independently unsubstituted or substituted with 1, 2, or 3 substituents. In some embodiments, the poly(alkylene oxide) chain (e.g., polyethylene glycol) chain) is attached to the second subset of polymer chains via a direct bond, e.g., by reacting a PLGA polymer chain (which includes a free terminal -COOH) group with a PEG-amine compound, such as mPEG-NH2, in the presence of a suitable coupling agent. In some embodiments, the coupling agent is a carbodiimide coupling agent, such as l-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (EDC) or dicyclohexylcarbodiimide (DCC), which can be used in the presence or absence of a succinimide, such as N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (sulfo-NHS) to effect the coupling reaction.DUKE-45610.601 iv. Polymers functionalized with bone-binding compounds

[0185] In the polymeric nanocarriers, in some embodiments, a third subset of polymer chains within the polymeric nanocarrier (e.g., polyester chains, such as PLGA chains) are functionalized with a compound that binds to bone. This may allow nanocarriers to achieve subchondral bone binding and localization, which may be particularly useful for drug formulations intended for use in bone regeneration. In some embodiments, the compound that binds to bone is selected from the group consisting of bisphosphonates (e.g., alendronate, risedronate, ibandronate, pamidronate, or zoledronic acid), glutamic acid, aspartic acid, and osteoprotegerin (OPG).

[0186] In some embodiments, the compound that binds to bone is alendronate, which is a drug compound approved as Fosamax® and Binosto® for the treatment of osteoporosis in post-menopausal women. Alendronate has been shown in nonclinical studies to enhance binding to hydroxyapatite in vitro and in animal models of osteoporosis. Accordingly, in some embodiments, a third subset of polymer chains are further functionalized with a group of formula:

[0187]

[0188] PO3H2

[0189] wherein L is a linker. As with other components, any suitable linker may be used as group L. In some embodiments, the linker comprises a group -(CH2CH2O)q-, wherein q is 1, 2, 3, 4, 5, or 6. In some embodiments, the linker comprises a group that is the reaction product of two reactive moieties. In some embodiments, such a group has a formula selected from:

[0190]

[0191] For example, in some embodiments, the third subset of polymer chains in the polymeric nanocarrier can be prepared by first conjugating a PEG-containing compound, such as azido-PEG-carboxylic acid, to alendronate (Ain) via an amide coupling reaction using a suitable coupling reagent (e.g., a carbodiimide coupling agent such as EDC or DCC, which can be used in the presence or absence of a succinimide, NHS or sulfo-NHS). The resultingDUKE-45610.601 product, azido-PEG-Aln, can be further reacted with a polymer (e.g., a polyester, such as PLGA) comprising an alkyne functional group, such as dibenzocyclooctyne, in a “click” reaction to generate the PLGA-PEG-Aln, in which the PLGA and the Ain are connected via a linker that includes the PEG moiety and the product of the azide-dibenzocyclooctyne click reaction.

[0192] v. Pharmaceutically active compounds

[0193] The particles also include one or more pharmaceutically active compounds. For example, in some embodiments, the particles include one or more pharmaceutically active compounds that are encapsulated within the polymeric nanocarrier. In particular, pharmaceutically active compounds disclosed herein (also referred to herein as pharmaceutical moiety(ies). drugs, therapeutic molecules, etc.) confer a therapeutic benefit for the prevention, attenuation, and / or treatment of joint diseases, such as osteoarthritis, as well as injury to a joint or cartilage, or pain associated with said injury and / or joint disease. These compounds thus find benefit from use in cartilage- and bone-targeting nanocarriers such as those disclosed herein.

[0194] In a first set of embodiments, the particles comprise one or more pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof, and any combination thereof.

[0195] Parathyroid hormone (PTH) is a hormone released by parathyroid glands to control blood calcium levels. PTH is used in a number of commercially available drugs. For example, recombinant human parathyroid hormone includes the full length native 84-amino acid polypeptide sequence of parathyroid hormone and has been approved for medical use in the U. S. (Natpara®) and in the European Union (Preotact). Additionally, teriparatide is a synthetic parathyroid hormone analog that includes the first 34 amino acids of human parathyroid hormone (i.e., the 34 N-terminal amino acids of human parathyroid hormone) and is sometimes referred to as PTH(l-34); it is currently approved for medical use in the U. S. (Forteo®, Bonsity®) and the E. U. (Forsteo, Terrosa, Movimya). Palopegteriparatide is also approved both the U. S. and the E. U. (Yorvipath®), and is a PEGylated form of PTH(l-34). Abaloparatide is a synthetic peptide analog having 34 amino acids, with the first 21 corresponding to the sequence of the first 21 amino acids ofDUKE-45610.601 human parathyroid hormone-related protein (PTHrP), and certain substitutions compared to amino acids 22-34 of PTHrP to enhance the stability of the peptide. Abaloparatide is approved for medical use in the U. S. (Tymlos®) and in the E. U. (Eladynos). In some embodiments, the parathyroid hormone used in the particles disclosed herein is selected from recombinant human parathyroid hormone and teriparatide. In some embodiments, the parathyroid hormone used in the particles disclosed herein is recombinant human parathyroid hormone. In some embodiments, the parathyroid hormone used in the particles disclosed herein is teriparatide.

[0196] Bone morphogenic protein 2 (BMP-2) is a growth factor in the TGF-0 superfamily of proteins. It plays important roles in developmental processes including cardiogenesis, neurogenesis, and osteogenesis. Recombinant human BMP-2 (rhBMP-2), also known as dibotermin alfa, is approved for medical use in the U. S. (Infuse®) and the E. U. (Inductos) for use with a collagen carrier and an interbody fusion cage for specific lumbar fusion indications. rhBMP-2 is also sold by a number of commercial suppliers. In some embodiments, the BMP-2 used in the particles disclosed herein is recombinant human BMP-2.

[0197] ALK4-Fc is a chimeric protein that includes 95 amino acids of the human ALK-4 protein (also known as Activin RIB), a short amino acid linker, and the Fc fragment of human IgGl. It is commercially available from a number of suppliers, including R& D Systems (catalog no. 808-AR). Inhibiting activity of the ALK4 pathway, such as by using ALK4-Fc, has been reported to protect against age-related bone loss (Maridas et al. doi: 10.1101 / 2025.10.24.684408). Alternatives to ALK4-Fc include CRIPTO antagonist ALK4L75A-FC, ALK5-Fc, MU1700, and other suitable ALK1 / ALK2 antagonists (see, e.g., Nemec. V. et al.. 2024, J. Med. Chem., 67(15): 12632-12659).

[0198] Other agents that may be used in the first set of embodiments include zoledronic acid, adenosine, lithium, and anti-TGF-0 compounds, such as TGF beta- 1,2, 3 antibody or recombinant TGF-0 R1 FC.

[0199] In some embodiments, the pharmaceutically active compound is selected from the group comprising or consisting of PTH and BMP-2, or a pharmaceutically acceptable salt thereof, or a combination thereof. In some embodiments, the particle comprises a single pharmaceutically active compound, and the compound is PTH or a pharmaceuticallyDUKE-45610.601 acceptable salt thereof. In some embodiments, the particle comprises a single pharmaceutically active compound, and the compound is BMP-2 or a pharmaceutically acceptable salt thereof. In some embodiments, the particle comprises two pharmaceutically active compounds, and the compounds are PTH and BMP-2, or pharmaceutically acceptable salts thereof.

[0200] In a second set of embodiments, the particles comprise one or more pharmaceutically active compounds selected from the group consisting of roflumilast, Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-β (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof, and any combination thereof.

[0201] Roflumilast is a phosphodiesterase-4 (PDE4) inhibitor, which is currently approved for use in the treatment of chronic obstructive pulmonary disease (COPD) (e.g., as an oral medication, available in the U. S. as Daliresp®), as well as for plaque psoriasis, seborrheic dermatitis, and atopic dermatitis (e.g., as a topical medication, available in the U. S. as Zoryve®). Its inhibition of PDE leads to accumulation of intracellular cyclic AMP (cAMP). It is a small molecule having the following structure:

[0202]

[0203] As alternatives to roflumilast, other functionally similar cAMP agonists could be used.

[0204] LDN-214117 is a selective inhibitor of bone morphogenic protein (BMP) type I receptor kinases, including ALK2, with high selectivity for BMP versus TGF-β signaling, and low cytotoxicity. It is a small molecule having the following structure:

[0205]

[0206] Use of LDN-214117 for promoting cartilage preservation, repair, and / or regeneration (e.g., in a subject with osteoarthritis) is disclosed in International Patent Publication No. WO 2024 / 182505 Al, which is incorporated by reference herein in its entirety. This compoundDUKE-45610.601 may be referred to simply as “LDN” herein. As alternatives to LDN-214117, other functionally similar ALK-2 antagonist could be used.

[0207] Growth and Differentiation Factor 11 (GDF11) also known as bone morphogenic protein 11 (BMP-11), is a protein that is a member of the Transforming growth factor beta family. GDF11 competes with BMP9 / 10 for binding to BMP type II receptors ACVRIIA or ACVRIIB, thereby preventing the association of BMP type I receptor ALK1 with ACVRIIA / B and decreasing BMP signaling. Additionally, GDF11 can increase TGF-β signaling. Use of GDF11 for promoting cartilage preservation, repair, and / or regeneration (e.g., in a subject with osteoarthritis) is disclosed in International Patent Publication No. WO 2024 / 182505 Al, which is incorporated by reference herein in its entirety.

[0208] Other agents that may be used in the second set of embodiments include dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-P (TGF-β), parathyroid hormone (PTH), and resveratrol.

[0209] In some embodiments, the pharmaceutically active compound is selected from the group consisting of roflumilast, PTH, and LDN-214117, or a pharmaceutically acceptable salt thereof, or a combination thereof. In some embodiments, the particle comprises a single pharmaceutically active compound, and the compound is roflumilast or a pharmaceutically acceptable salt thereof. In some embodiments, the particle comprises a single pharmaceutically active compound, and the compound is PTH or a pharmaceutically acceptable salt thereof. In some embodiments, the particle comprises a single pharmaceutically active compound, and the compound is LDN-214117 or a pharmaceutically acceptable salt thereof. In some embodiments, the particle comprises three pharmaceutically active compounds, and the compounds are roflumilast, PTH, and LDN-214117, or pharmaceutically acceptable salts thereof.

[0210] The pharmaceutically active compounds in the particles disclosed herein include isotopically-labeled compounds, which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds of the disclosure are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to2H,3H,13C,14C,15N,18O,17O,31P,32P,35S,18F, and36C1, respectively. Substitution withDUKE-45610.601 heavier isotopes such as deuterium (2H) can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements, and accordingly may be useful in some circumstances. Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using appropriate isotopically labeled reagents in place of non-isotopically-labeled reagents.

[0211] The pharmaceutically active compounds disclosed herein may also exist in solvated as well as unsolvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the disclosure encompass both solvated and unsolvated forms. In one embodiment, the compound is amorphous. In one embodiment, the compound is a single polymorph. In another embodiment, the compound is a mixture of polymorphs. In another embodiment, the compound is in a crystalline form.

[0212] The pharmaceutically active compounds disclosed herein may also be used in the particles in prodrug forms. Prodrugs of the pharmaceutically active compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the active pharmaceutically active compound, e.g., by hydrolysis. A thorough discussion is provided in Fralish et al. Nat. Rev. Drug Discov. 2024, 23(5):365-380.

[0213] In some embodiments, the pharmaceutically active compound is incorporated into the particle in an encapsulated form. For example, prior to loading the pharmaceutically active compound into the nanocarrier, the pharmaceutically active compound can be encapsulated in, for example, a sugar glass. For example, inverse micelles made of sugar glass can protect certain pharmaceutically active compounds during the nanocarrier fabrication process and during storage. See, e.g., Giri etal. Adv. Mater. 2011, 23(42):4861-4867. Pharmaceutically active compounds that may particularly benefit from incorporation into a sugar glass micelle include peptide- and protein-based pharmaceutically active compounds, such as PTH and BMP-2. Sugar glass micelles can be manufactured using sugars such as trehalose, sucrose, or other disaccharides, and surfactants such as dioctyl sulfosuccinate, hexadecyltrimethylammonium bromide, lecithin, imidazolium-based surfactants, polyoxyethylene ethers or related non-ionic surfactants, sodium di-(n-DUKE-45610.601 octyl)phosphinate, 3,3-dimethyl-1-butysulfosuccinate sodium salt, and the like. In some embodiments, pharmaceutically active compounds disclosed herein are encapsulated in sugar glass micelles comprising trehalose and dioctyl sulfosuccinate.

[0214] vi. Specific exemplary embodiments

[0215] In one exemplary set of embodiments, disclosed herein is a particle comprising: (a) a polymeric nanocarrier, comprising:

[0216] (i) poly(lactic-co-glycolic) acid;

[0217] (ii) poly(lactic-co-glycolic) acid functionalized with a branched poly-lysine compound;

[0218] (iii) poly(lactic-co-glycolic) acid functionalized with a polyethylene glycol) compound; and

[0219] (iv) poly(lactic-co-glycolic) acid functionalized with a polyethylene glycol compound), wherein the polyethylene glycol) compound is further functionalized with alendronate; and

[0220] (b) one or more pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof, and any combination thereof.

[0221] In some embodiments, the particle comprises PTH, or a pharmaceutically acceptable salt thereof. In some embodiments, the particle comprises BMP-2, or a pharmaceutically acceptable salt thereof. In some embodiments, the particle comprises PTH or a pharmaceutically acceptable salt thereof, and BMP-2 or a pharmaceutically acceptable salt thereof.

[0222] In a second exemplary set of embodiments, disclosed herein is a particle comprising:

[0223] (a) a polymeric nanocarrier, comprising:

[0224] (i) poly(lactic-co-glycolic) acid;

[0225] (ii) poly(lactic-co-glycolic) acid functionalized with a branched poly(lysine) compound; and

[0226] (iii) poly(lactic-co-glycolic) acid functionalized with a polyethylene glycol) compound; andDUKE-45610.601 (b) one or more pharmaceutically active compounds selected from the group consisting of roflumilast, Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-β (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof, and any combination thereof.

[0227] In some embodiments, the particle comprises roflumilast, or a pharmaceutically acceptable salt thereof. In some embodiments, the particle comprises PTH, or a pharmaceutically acceptable salt thereof. In some embodiments, the particle comprises LDN-214117, or a pharmaceutically acceptable salt thereof. In some embodiments, the particle comprises roflumilast or a pharmaceutically acceptable salt thereof, PTH or a pharmaceutically acceptable salt thereof, and LDN-214117 or a pharmaceutically acceptable salt thereof.

[0228] C. Pharmaceutical Compositions Comprising Particles Disclosed Herein

[0229] In another aspect, disclosed herein are pharmaceutical compositions comprising a plurality of particles disclosed herein (e.g„ particles as described hereinabove), and a pharmaceutically acceptable carrier. Details regarding pharmaceutically acceptable earners will be discussed further below.

[0230] In some embodiments, the pharmaceutical composition comprises a plurality of particles in which each particle comprises a single pharmaceutically active compound, such as a pharmaceutically active compound disclosed herein. In some embodiments, the single pharmaceutically active compound is selected from the group consisting of PTH, BMP-2, ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof, and any combination thereof. In some embodiments, the single pharmaceutically active compound is BMP-2, or a pharmaceutically acceptable salt thereof. In some embodiments, the single pharmaceutically active compound is PTH, or a pharmaceutically acceptable salt thereof. In some embodiments, the single pharmaceutically active compound is selected from the group consisting of roflumilast, GDF11, LDN-214117, DKK1, IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, TGF-p, PTH, and resveratrol. In some embodiments, the single pharmaceutically active compound is roflumilast, or a pharmaceutically acceptable salt thereof. In some embodiments, the singleDUKE-45610.601 pharmaceutically active compound is PTH, or a pharmaceutically acceptable salt thereof. In some embodiments, the single pharmaceutically active compound is LDN-214117, or a pharmaceutically acceptable salt thereof.

[0231] In some embodiments, the pharmaceutical composition comprises a plurality of particles in which each particle comprises two pharmaceutically active compounds, such as pharmaceutically active compounds disclosed herein. In some embodiments, the two pharmaceutically active compounds are selected from the group consisting of PTH, BMP-2, ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof. In some embodiments, the two pharmaceutically active compounds are BMP-2 or a pharmaceutically acceptable salt thereof, and PTH or a pharmaceutically acceptable salt thereof. In some embodiments, the two pharmaceutically active compounds are selected from the group consisting of roflumilast, GDF11, LDN-214117, DKK1, IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, TGF-0, PTH, and resveratrol, and pharmaceutically acceptable salts thereof. In some embodiments, the two pharmaceutically active compounds are selected from roflumilast or a pharmaceutically acceptable salt thereof, PTH or a pharmaceutically acceptable salt thereof, and LDN-214117 or a pharmaceutically acceptable salt thereof.

[0232] In some embodiments, the pharmaceutical composition comprises a plurality of particles in which each particle comprises three pharmaceutically active compounds, such as pharmaceutically active compounds disclosed herein. In some embodiments, the three pharmaceutically active compounds are selected from the group consisting of PTH, BMP-2, ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof, and any combination thereof. In some embodiments, the three pharmaceutically active compounds are selected from the group consisting of roflumilast, GDF11, LDN-214117, DKK1, IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, TGF-p, PTH, and resveratrol. In some embodiments, the three pharmaceutically active compounds are roflumilast or a pharmaceutically acceptable salt thereof, PTH or a pharmaceutically acceptable salt thereof, and LDN-214117 or a pharmaceutically acceptable salt thereof.

[0233] In some embodiments, in a pharmaceutical composition comprising a plurality of particles disclosed herein, the plurality of particles comprises a first subset of particlesDUKE-45610.601 comprising a first pharmaceutically active compound, and a second subset of particles comprising a second pharmaceutically active compound. In some embodiments, each subset of particles comprises a single pharmaceutically active compound, with the first subset of particles comprising a single first pharmaceutically active compound, and the second subset of particles comprising a single second pharmaceutically active compound. In some embodiments, the first pharmaceutically active compound and the second pharmaceutically active compound are independently selected from the group consisting of PTH, BMP-2, ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof. In some embodiments, the first pharmaceutically active compound is BMP-2 or a pharmaceutically acceptable salt thereof, and the second pharmaceutically active compound is PTH or a pharmaceutically acceptable salt thereof. In some embodiments, the first pharmaceutically active compound and the second pharmaceutically active compound are independently selected from the group consisting of roflumilast, GDF11, LDN-214117, DKK1, IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, TGF-β, PTH, and resveratrol, and pharmaceutically acceptable salts thereof. In some embodiments, the first pharmaceutically active compound and the second pharmaceutically active compound are independently selected from the group consisting of roflumilast or a pharmaceutically acceptable salt thereof, PTH or a pharmaceutically acceptable salt thereof, and LDN-214117 or a pharmaceutically acceptable salt thereof.

[0234] In some embodiments, in a pharmaceutical composition comprising a plurality of particles disclosed herein, the plurality of particles comprises a first subset of particles comprising a first pharmaceutically active compound, a second subset of particles comprising a second pharmaceutically active compound, and a third subset of particles comprising a third pharmaceutically active compound. In some embodiments, each subset of particles comprises a single pharmaceutically active compound, with the first subset of particles comprising a single first pharmaceutically active compound, the second subset of particles comprising a single second pharmaceutically active compound, and the third subset of particles comprising a third pharmaceutically active compound. In some embodiments, the first pharmaceutically active compound, the second pharmaceutically active compound, and the third pharmaceutically active compound are independently selected from the group consisting of PTH, BMP-2, ALK4-Fc, zoledronic acid, adenosine,DUKE-45610.601

[0235] and lithium, and pharmaceutically acceptable salts thereof. In some embodiments, the first pharmaceutically active compound, the second pharmaceutically active compound, and the third pharmaceutically active compound are independently selected from the group consisting of roflumilast, GDF11, LDN-214117, DKK1, IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, TGF-β, PTH, and resveratrol, and pharmaceutically acceptable salts thereof. In some embodiments, the first pharmaceutically active compound is roflumilast or a pharmaceutically acceptable salt thereof, the second pharmaceutically active compound is PTH or a pharmaceutically acceptable salt thereof, and the third pharmaceutically active compound is LDN-214117 or a pharmaceutically acceptable salt thereof.

[0236] In embodiments in which pharmaceutical compositions comprise a plurality of particles disclosed herein (e.g., particles as described hereinabove), and particularly when the pharmaceutical compositions comprise two or three populations of particles wherein each particle comprises a different pharmaceutically active compound, the polymers within the particles may be different between the two populations of particles. This can allow for, e.g., a single pharmaceutical composition comprising multiple populations of particles with different release profiles, so that two or three different pharmaceutically active compounds can be released at different time points. See. e.g., Schemes 1 and 2 below.

[0237] Scheme 1. Release profile for two drugs from a single pharmaceutical composition comprising two populations of particles.

[0238] Drug 1 Drug 2

[0239]

[0240] Ti B Scheme 2. Release profile for three drugs from a single pharmaceutical composition comprising three populations of particles.

[0241]

[0242] DUKE-45610.601

[0243] For example, in some embodiments, in a pharmaceutical composition comprising a plurality of particles disclosed herein, the plurality of particles comprises:

[0244] a first subset of particles comprising a first pharmaceutically active compound, wherein the polymeric nanocarrier in the first subset of particles comprises PLGA having about 70 mol% to about 80 mol% lactic acid and about 20 mol% to about 30 mol% glycolic acid (e.g.. about 75 mol% lactic acid and about 25 mol% glycolic acid); and a second subset of particles comprising a second pharmaceutically active compound, wherein the polymeric nanocarrier in the second subset of particles comprises PLGA having about 45 mol% to about 55 mol% lactic acid and about 45 mol% to about 55 mol% glycolic acid (e.g., about 50 mol% lactic acid and about 50 mol% glycolic acid).

[0245] In some embodiments, in a pharmaceutical composition comprising a plurality of particles disclosed herein, the plurality of particles comprises:

[0246] a first subset of particles comprising a first pharmaceutically active compound, wherein the polymeric nanocarrier in the first subset of particles comprises PLGA having about 70 mol% to about 80 mol% lactic acid and about 20 mol% to about 30 mol% glycolic acid (e.g.. about 75 mol% lactic acid and about 25 mol% glycolic acid);

[0247] a second subset of particles comprising a second pharmaceutically active compound, wherein the polymeric nanocarrier in the second subset of particles comprises PLGA having about 45 mol% to about 55 mol% lactic acid and about 45 mol% to about 55 mol% glycolic acid (e.g., about 50 mol% lactic acid and about 50 mol% glycolic acid); and

[0248] a third subset of particles comprising a third pharmaceutically active compound, wherein the polymeric nanocarrier in the third subset of particles comprises PLGA having about 60 mol% to about 70 mol% lactic acid and about 30 mol% to about 40 mol% glycolic acid (e.g., about 65 mol% lactic acid and about 35 mol% glycolic acid).

[0249] D. Non-Particle Based Pharmaceutical Compositions

[0250] Another aspect of the present disclosure provides pharmaceutical compositions for preventing, attenuating and / or treating a joint disease in a subject, the compositions comprising therapeutically effective amounts of two or more pharmaceutically activeDUKE-45610.601 compounds selected from the group consisting of PTH, BMP-2, ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof, and any combination thereof. In some embodiments, the pharmaceutical composition comprises two pharmaceutically active compounds selected from the group consisting of PTH and BMP-2, and pharmaceutically acceptable salts thereof.

[0251] Another aspect of the present disclosure provides pharmaceutical compositions for preventing, attenuating and / or treating a joint disease in a subject, the compositions comprising therapeutically effective amounts of two or more pharmaceutically active compounds selected from the group consisting of roflumilast, GDF11, LDN-214117, DKK1, IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, TGF-β, PTH, and resveratrol, and pharmaceutically acceptable salts thereof. In some embodiments, the pharmaceutical composition comprises two pharmaceutically active compounds selected from the group consisting of roflumilast, PTH, and LDN-214117, and pharmaceutically acceptable salts thereof. In some embodiments, the pharmaceutical composition comprises three pharmaceutically active compounds selected from the group consisting of roflumilast, PTH, and LDN-214117, and pharmaceutically acceptable salts thereof.

[0252] E. Pharmaceutical Compositions and Modes of Administration

[0253] In some embodiments, the pharmaceutical compositions disclosed herein (e.g., any pharmaceutical composition described herein, such as those described hereinabove) further comprise an appropriate pharmaceutically acceptable carrier, excipient and / or diluent. The term “pharmaceutically acceptable carrier, excipient and / or diluent” as used herein, means any non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose, trehalose, and sucrose; starches such as, but not limited to, com starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to. cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethylDUKE-45610.601 laurate; agar; buffering agents such as, but not limited to, phosphate buffers (e.g., monobasic sodium phosphate and / or dibasic sodium phosphate), magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The exact nature of the carrier will depend upon the desired use for the composition and may range from being suitable or acceptable for veterinary uses to being suitable or acceptable for human use. Techniques and formulations may generally be found in “Remington's Pharmaceutical Sciences,” (Meade Publishing Co., Easton, Pa.). The exact nature of the carrier, excipient or diluent will depend upon the desired use for the composition and may range from being suitable or acceptable for veterinary uses to being suitable or acceptable for human use. The composition may optionally include one or more additional compounds.

[0254] When used to treat, attenuate, or prevent a disease, such as a joint disease {e.g., osteoarthritis), a pharmaceutical composition disclosed herein may be administered singly, as mixtures of one or more compounds or in mixture or combination with other agents {e.g., therapeutic agents) useful for treating such diseases and / or the symptoms associated with such diseases. Such agents may include, but are not limited to, NS AIDS, antiinflammatory compounds {e.g., paracetamol, ibuprofen, IL-ip antagonists), corticosteroids (e.g., prednisone), hyaluronic acid injections, disease-modifying antirheumatic drugs (DMARDs), biologic response modifiers {e.g., infliximab, abatacept, etc.), β2-adrenergic receptor antagonists {e.g., propranolol), and janus kinase (JAK) inhibitors, to name a few. The compounds may be administered in the form of compounds per se, or as pharmaceutical compositions comprising a compound.

[0255] Pharmaceutical compositions comprising the particles disclosed herein may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping or lyophilization processes. The compositions may be formulated in conventional manner using one or moreDUKE-45610.601 physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the compounds into preparations which can be used pharmaceutically.

[0256] The therapeutic compositions and / or pharmaceutically active compound(s) as provided herein may be formulated in the pharmaceutical composition per se, or in the form of a hydrate, solvate, N-oxide or pharmaceutically acceptable salt, as previously described. Typically, such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases may also be formed.

[0257] Pharmaceutical compositions may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, intra-articular, systemic, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation.

[0258] In some embodiments, the therapeutic compositions and / or pharmaceutically active compound(s) as provided herein are formulated for intra-articular injection. For example, in some embodiments, useful injectable preparations include sterile suspensions, solutions or emulsions of the compounds or delivery systems in aqueous or oily vehicles. The preparations may also contain formulating agents, such as a suspending, stabilizing and / or dispersing agent. The formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives. Alternatively, the injectable formulation may be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer (e.g., phosphate-buffered saline), dextrose solution, etc., before use.

[0259] For topical administration, the therapeutic compositions and / or pharmaceutically active compound(s) as provided herein may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.

[0260] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.DUKE-45610.601 For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art with, for example, sugars, films or enteric coatings.

[0261] Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g.. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, Cremophor™ or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring and sweetening agents as appropriate.

[0262] Preparations for oral administration may be suitably formulated to give controlled release of the compound, as is well known. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For rectal and vaginal routes of administration, the compound(s) may be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides.

[0263] For nasal administration or administration by inhalation or insufflation, the therapeutic compositions and / or pharmaceutically active compound(s) as provided herein can be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro tetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator (for example capsules and cartridges made of gelatin) may be formulatedDUKE-45610.601 containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0264] For ocular administration, the therapeutic compositions and / or pharmaceutically active compound(s) as provided herein may be formulated as a solution, emulsion, suspension, etc. suitable for administration to the eye. A variety of vehicles suitable for administering compounds to the eye are known in the art.

[0265] For prolonged delivery, the therapeutic compositions and / or pharmaceutically active compound(s) as provided herein can be formulated as a depot preparation for administration by implantation, intra-articular injection, systemic injection, or intramuscular injection. The therapeutic compositions and / or pharmaceutically active compound(s) as provided herein may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g.. as a sparingly soluble salt. Alternatively, transdermal delivery systems manufactured as an adhesive disc or patch which slowly releases the compound(s) for percutaneous absorption may be used. To this end, permeation enhancers may be used to facilitate transdermal penetration of the compound(s).

[0266] Alternatively, other pharmaceutical delivery systems may be employed. Liposomes and emulsions are well-known examples of delivery vehicles that may be used to deliver compound(s). Certain organic solvents such as dimethyl sulfoxide (DMSO) may also be employed, although usually at the cost of greater toxicity.

[0267] The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the compound(s). The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

[0268] The therapeutic compositions and / or pharmaceutically active compound(s) as provided herein described herein, or pharmaceutical compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat, attenuate or prevent the particular disease (e.g., joint disease) being treated. By therapeutic benefit is meant eradication, attenuation, or prevention of the underlying disease being treated or the attenuation, treatment or prevention of one or more of the symptoms associated with the underlying disease such that the patient reports anDUKE-45610.601 improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disease. Therapeutic benefit also generally includes halting or slowing the progression of the disease, regardless of whether improvement is realized.

[0269] The amount of therapeutic compositions and / or pharmaceutically active compound(s) as provided herein, or pharmaceutical compositions thereof, administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular therapeutic compositions and / or pharmaceutically active compound(s), or pharmaceutical compositions thereof as provided herein, the conversation rate and efficiency into active drug compound under the selected route of administration, etc.

[0270] Determination of an effective dosage of the therapeutic compositions and / or pharmaceutically active compound(s), or pharmaceutical compositions thereof, as provided herein for a particular use and mode of administration is well within the capabilities of those skilled in the art. Effective dosages may be estimated initially from in vitro activity and metabolism assays. For example, an initial dosage of compound for use in animals may be formulated to achieve a circulating blood or serum concentration of the metabolite active compound that is at or above an IC50 / EC50 of the particular therapeutic composition(s) and / or pharmaceutically active compound(s), or pharmaceutical compositions thereof, as provided herein as measured in as in vitro assay. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compound via the desired route of administration is well within the capabilities of skilled artisans. Initial dosages of compound can also be estimated from in vivo data, such as animal models. Animal models useful for testing the efficacy of the active metabolites to treat or prevent the various diseases described above are well-known in the art. Animal models suitable for testing the bioavailability and / or metabolism of compounds into active metabolites are also well-known. Ordinarily skilled artisans can routinely adapt such information to determine appropriate dosages suitable for human administration.DUKE-45610.601 Dosage amounts will typically be in the range of from about 0.0001 mg / kg / day, 0.001 mg / kg / day or 0.01 mg / kg / day to about 100 mg / kg / day, but may be higher or lower, depending upon, among other factors, the activity of the pharmaceutically active compound, the bioavailability of the pharmaceutically active compound, its metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors, discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the compound(s) and / or active metabolite compound(s) which are sufficient to maintain therapeutic or prophylactic effect. For example, the therapeutic compositions and / or pharmaceutically active compound(s), or pharmaceutical compositions thereof, as provided herein may be administered at the same time or sequentially (i.e., in a particular order). In other examples, they may be administered once per year, semi-annually, quarterly, once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of the pharmaceutically active compound(s) and / or active metabolite compound(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective dosages without undue experimentation.

[0271] In some embodiments, the pharmaceutically active compounds and / or therapeutic compositions as provided herein, or pharmaceutical compositions thereof, are administered with a delivery vehicle. Examples of such delivery vehicles may include, but are not limited to, hydrogels (e.g., Hyaluronic Acid (HA), biomimetic lubricant compounds, liposomes, polymeric nanoparticles, solid lipid nanoparticles (SLNs), micelles, dendrimers, carbon nanotubes, virus-like particles (VLPs), protein-based nanoparticles, nanoemulsions, and the like. In some embodiments, the delivery vehicle comprises a hydrogel. In one embodiment, the delivery vehicle comprises a biomimetic lubricant compound, such as hyaluronic acid (HA). In certain embodiments, the lubricant comprises a functionalized hyaluronic acid compound as described in International Patent Publication No. WO 2022 / 099059 Al, the contents of which are incorporated herein by reference. In one embodiment, the therapeutic composition further comprises a functionalized hyaluronicDUKE-45610.601 acid compound, the HA comprising a hyaluronic acid backbone with one or more side chains attached thereto, wherein at least one side chain comprises a ureidopyrimidinone moiety.

[0272] F. Methods of Treatment

[0273] The present disclosure is based, in part, on the discovery that particular combinations of pharmaceutically active compounds, when administered together or in a certain order, or when released from a single pharmaceutical composition in a certain order, can confer a therapeutic benefit for the prevention, attenuation and / or treatment of joint diseases, such as osteoarthritis, by exhibiting a significant anabolic effect on osteoarthritic cartilage tissue and subchondral bone, with no detrimental effects on joint tissues.

[0274] It is estimated that osteoarthritis (OA) affects more than 30 million Americans and is the leading cause of disability in American adults. OA is one of the most common causes of disability in the Western world with the age of onset typically between 25 and 50, although it can occur at any age. It is a painful and disabling disease associated with chronic inflammation of the joints. The knee is one of the most commonly affected joints; approximately 14 million Americans have symptomatic OA of the knee, including 8 million people under the age of 65. It has been estimated that 45 percent of people are at risk of developing knee OA in their lifetime. Knee OA symptoms often develop slowly and may become markedly worse over time or with specific activities. It is the most common cause of limitations in the activities of daily living, such as walking and climbing stairs, and the functional impairment associated with OA is a significant risk factor for total knee arthroplasty (TKA).

[0275] Accordingly, in one aspect, the present disclosure provides a method of preventing, treating and / or attenuating a joint disease in a subject in need thereof, the method comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of one or more pharmaceutical compositions described herein, such that the joint disease is prevented, treated and / or attenuated in the subject.

[0276] In some embodiments, the subject is suffering from a joint disease. In some embodiments, the joint disease comprises arthritis. In some embodiments, the joint disease comprises osteoarthritis. In some embodiments, the joint disease comprises post-traumaticDUKE-45610.601 osteoarthritis. In some embodiments, the joint disease comprises joint cartilage or bone damage, e.g.. joint cartilage or bone damage that has not yet been diagnosed as arthritis or osteoarthritis or post-traumatic OA.

[0277] In particular embodiments, the one or more therapeutic compositions are administered to the subject via intra-articular injection.

[0278] In some embodiments, when more than one pharmaceutical composition disclosed herein is administered to the subject, the pharmaceutical compositions are administered to the subject simultaneously. In other embodiments, the pharmaceutical compositions are administered to the subject sequentially.

[0279] While the methods of treatment disclosed herein can make use of any pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition comprising a plurality of particles disclosed herein, or a non-particle based pharmaceutical composition disclosed herein), in another aspect, the disclosure provides methods of preventing, treating and / or attenuating a joint disease in a subject in need thereof, comprising administering to the subject a series of pharmaceutically active compounds, in a particular order. In such embodiments, the pharmaceutical compositions administered to the subject may each comprise a single pharmaceutically active compound.

[0280] For example, in one aspect, disclosed herein is a method of preventing, treating and / or attenuating a joint disease in a subject in need thereof, comprising administering to the subject a first pharmaceutical composition comprising a first pharmaceutically active compound, and a second pharmaceutical composition comprising a second pharmaceutically active compound, wherein the first and second pharmaceutical compositions are administered sequentially, and wherein the first and second pharmaceutically active compounds are independently selected from PTH, BMP-2, ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof. In some embodiments, the first pharmaceutically active compound is BMP-2, and the second pharmaceutically active compound is PTH. Accordingly, the method comprises first administering to the subject the pharmaceutical composition comprising BMP-2, and subsequently administering to the subject the pharmaceutical composition comprising PTH.DUKE-45610.601 In another aspect, disclosed herein is a method of treating preventing, treating and / or attenuating a joint disease in a subject in need thereof, comprising administering to the subject a first pharmaceutical composition comprising a first pharmaceutically active compound, a second pharmaceutical composition comprising a second pharmaceutically active compound, and a third pharmaceutical composition comprising a third pharmaceutically active compound, wherein the first, second, and third pharmaceutical compositions are administered sequentially, and wherein the first, second, and third pharmaceutically active compounds are independently selected from roflumilast, GDF11, LDN-214117, DKK1, IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, TGF-β, PTH, and resveratrol. In some embodiments, the first pharmaceutically active compound is roflumilast, the second pharmaceutically active compound is PTH, and the third pharmaceutically active compound is LDN-214117. Accordingly, the method comprises: (a) first administering to the subject the pharmaceutical composition comprising roflumilast; (b) second administering to the subject the pharmaceutical composition comprising PTH; and (c) third administering to the subject the pharmaceutical composition comprising LDN-214117.

[0281] G. Kits

[0282] The present disclosure further provides kits comprising the pharmaceutical compositions described herein and for carrying out the methods described herein. For example, in one embodiment, a kit may comprise, consist of, or consist essentially of one or more pharmaceutical compositions described herein.

[0283] Another aspect of the present disclosure provides a kit for the prevention, attenuation and / or treatment of a joint disease, the kit comprising, consisting of, or consisting essentially of: (i) one or more pharmaceutical compositions described herein; (ii) a syringe; and (iii) instructions for use.

[0284] Another aspect of the present disclosure provides a kit for the treatment or attenuation of an injury to a joint or cartilage of a subject, the kit comprising, consisting of, or consisting essentially of: (i) one or more therapeutic compositions as provided herein; (ii) a syringe; and (iii) instructions for use.DUKE-45610.601 In other embodiments, a kit may further include other components. Such components may be provided individually or in combinations, and may be provided in any suitable container such as a vial, a bottle, or a tube. Examples of such components include, but are not limited to, one or more additional reagents, such as one or more buffers, one or more delivery vehicles as provided herein, or the like.

[0285] In addition to above-mentioned components, a subject kit can further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0286] H. Clauses

[0287] For reasons of completeness, various aspects of the disclosure are set forth in the following numbered clauses.

[0288] Clause 1. A particle comprising a polymeric nanocarrier, a branched poly-lysine compound covalently attached to a subset of polymer chains in the polymeric nanocarrier, and one or more pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof, and any combination thereof.DUKE-45610.601 Clause 2. The particle of clause 1, wherein the one or more pharmaceutically active compounds are selected from the group consisting of PTH and BMP-2, and a combination thereof.

[0289] Clause 3. The particle of clause 2, wherein the particle comprises PTH.

[0290] Clause 4. The particle of clause 3, wherein the PTH is teriparatide.

[0291] Clause 5. The particle of clause 2, wherein the particle comprises BMP-2.

[0292] Clause 6. The particle of clause 5, wherein the BMP-2 is recombinant human BMP-2.

[0293] Clause 7. The particle of clause 2, wherein the particle comprises PTH and BMP-2.

[0294] Clause 8. The particle of clause 7, wherein the PTH is teriparatide and the BMP-2 is recombinant human BMP-2.

[0295] Clause 9. A particle comprising a polymeric nanocarrier, a branched poly-lysine compound covalently attached to a subset of polymer chains in the polymeric nanocarrier, and one or more pharmaceutically active compounds selected from the group consisting of roflumilast, Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-β (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof, and any combination thereof.

[0296] Clause 10. The particle of clause 9, wherein the one or more pharmaceutically active compounds are selected from the group consisting of roflumilast, PTH, and LDN-214117, and any combination thereof.

[0297] Clause 11. The particle of clause 10, wherein the particle comprises roflumilast. Clause 12. The particle of clause 10. wherein the particle comprises PTH.

[0298] Clause 13. The particle of clause 12, wherein the PTH is teriparatide.

[0299] Clause 14. The particle of clause 10, wherein the particle comprises LDN-214117. Clause 15. The particle of clause 10, wherein the particle comprises roflumilast, PTH, and LDN-214117.

[0300] Clause 16. The particle of clause 15, wherein the PTH is teriparatide.

[0301] Clause 17. The particle of any one of clauses 1-16, wherein the branched polylysine compound comprises a branched poly(L-lysine) compound.DUKE-45610.601 Clause 18. The particle of any one of clauses 1-17, wherein the branched polylysine compound is covalently attached to the polymeric nanocarrier via a direct bond.

[0302] Clause 19. The particle of any one of clauses 1-17, wherein the branched polylysine compound is covalently attached to the polymeric nanocarrier via a linker.

[0303] Clause 20. The particle of any one of clauses 1-19, wherein the polymeric nanocarrier comprises a polyester.

[0304] Clause 21. The particle of clause 20, wherein the polyester is poly(lactic-co-glycolic) acid (PLGA).

[0305] Clause 22. The particle of clause 21, wherein the PLGA comprises about 70 mol% to about 80 mol% lactic acid and about 20 mol% to about 30 mol% glycolic acid.

[0306] Clause 23. The particle of clause 22, wherein the PLGA comprises about 75 mol% lactic acid and about 25 mol% glycolic acid.

[0307] Clause 24. The particle of clause 21, wherein the PLGA comprises about 60 mol% to about 70 mol% lactic acid and about 30 mol% to about 40 mol% glycolic acid.

[0308] Clause 25. The particle of clause 24, wherein the PLGA comprises about 65 mol% lactic acid and about 35 mol% glycolic acid.

[0309] Clause 26. The particle of clause 21, wherein the PLGA comprises about 45 mol% to about 55 mol% lactic acid and about 45 mol% to about 55 mol% glycolic acid.

[0310] Clause 27. The particle of clause 26, wherein the PLGA comprises about 50 mol% lactic acid and about 50 mol% glycolic acid.

[0311] Clause 28. The particle of any one of clauses 1-27, wherein a second subset of polymer chains within the polymeric nanocarrier are functionalized with a polyalkylene oxide.

[0312] Clause 29. The particle of clause 28, wherein the polyalkylene oxide is polyethylene glycol).

[0313] Clause 30. The particle of any one of clauses 1-29, wherein a third subset of polymer chains within the polymeric nanocarrier are functionalized with a polyalkylene oxide and a compound that binds to bone.

[0314] Clause 31. The particle of clause 30, wherein the polyalkylene oxide is polyethylene glycol) and the compound that binds to bone is alendronate.DUKE-45610.601 Clause 32. The particle of any one of clauses 1-31, wherein the one or more pharmaceutically active compounds is encapsulated within the polymeric nanocarrier.

[0315] Clause 33. The particle of any one of clauses 1-32, wherein at least one pharmaceutically active compound is encapsulated in a sugar glass micelle, and the sugar glass micelle is encapsulated within the polymeric nanocarrier.

[0316] Clause 34. The particle of clause 33, wherein the sugar glass micelle comprises a sugar selected from trehalose and sucrose, and a surfactant selected from dioctyl sulfosuccinate, hexadecyltrimethylammonium bromide, lecithin, imidazolium-based surfactants, polyoxyethylene ethers, sodium di-(n-octyl)phosphinate, and 3, 3 -dimethyl- 1-butysulfosuccinate sodium salt.

[0317] Clause 35. The particle of any one of clauses 1-34, wherein the particle comprises the one or more pharmaceutically active compounds in an amount of about 0.01 pg / mg to about 5.0 pg / mg.

[0318] Clause 36. A pharmaceutical composition comprising a plurality of particles of any one of clauses 1-35, and a pharmaceutically acceptable carrier.

[0319] Clause 37. The pharmaceutical composition of clause 36, wherein each particle in the plurality of particles comprises a single pharmaceutically active compound.

[0320] Clause 38. The pharmaceutical composition of clause 37, wherein the single pharmaceutically active compound is BMP-2.

[0321] Clause 39. The pharmaceutical composition of clause 37, wherein the single pharmaceutically active compound is PTH.

[0322] Clause 40. The pharmaceutical composition of clause 37, wherein the single pharmaceutically active compound is roflumilast, or a pharmaceutically acceptable salt thereof.

[0323] Clause 41. The pharmaceutical composition of clause 37, wherein the single pharmaceutically active compound is LDN-214117, or a pharmaceutically acceptable salt thereof.

[0324] Clause 42. The pharmaceutical composition of clause 36, wherein each particle in the plurality of particles comprises two pharmaceutically active compounds.

[0325] Clause 43. The pharmaceutical composition of clause 42, wherein the two pharmaceutically active compounds are BMP-2 and PTH.DUKE-45610.601 Clause 44. The pharmaceutical composition of clause 36, wherein each particle in the plurality of particles comprises three pharmaceutically active compounds.

[0326] Clause 45. The pharmaceutical composition of clause 44, wherein the three pharmaceutically active compounds are roflumilast, PTH, and LDN-214117, or pharmaceutically acceptable salts thereof.

[0327] Clause 46. The pharmaceutical composition of clause 36, wherein the plurality of particles comprises a first subset of particles comprising a first pharmaceutically active compound, and a second subset of particles comprising a second pharmaceutically active compound.

[0328] Clause 47. The pharmaceutical composition of clause 46, wherein the first pharmaceutically active compound is BMP-2, and the second pharmaceutically active compound is PTH.

[0329] Clause 48. The pharmaceutical composition of clause 36, wherein the plurality of particles comprises a first subset of particles comprising a first pharmaceutically active compound, a second subset of particles comprising a second pharmaceutically active compound, and a third subset of particles comprising a third pharmaceutically active compound.

[0330] Clause 49. The pharmaceutical composition of clause 48, wherein the first pharmaceutically active compound is roflumilast or a pharmaceutically acceptable salt thereof, the second pharmaceutically active compound is PTH, and the third pharmaceutically active compound is LDN-214117 or a pharmaceutically acceptable salt thereof.

[0331] Clause 50. The pharmaceutical composition of any one of clauses 36-49, further comprising a delivery vehicle.

[0332] Clause 51. The pharmaceutical composition of clause 50, wherein the delivery vehicle comprises hyaluronic acid.

[0333] Clause 52. The pharmaceutical composition of clause 50, wherein the delivery vehicle comprises a functionalized hyaluronic acid compound, wherein the functionalized hyaluronic acid compound comprises a hyaluronic acid backbone with one or more side chains attached thereto, wherein at least one side chain comprises a ureidopyrimidinone moiety.DUKE-45610.601 Clause 53. A method of treating a joint disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of clauses 36-52.

[0334] Clause 54. A method of treating pain associated with a joint disease or joint tissue damage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of clauses 36-52.

[0335] Clause 55. A method of delivering a pharmaceutically active compound to cartilage and / or subchondral bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of clauses 36-52.

[0336] Clause 56. The method of clause 55, wherein the subject is suffering from a joint disease.

[0337] Clause 57. A method of treating an injury to a joint or cartilage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of clauses 36-52.

[0338] Clause 58. The method of any one of clauses 53-57, wherein the joint disease comprises arthritis.

[0339] Clause 59. The method of any one of clauses 53-57, wherein the joint disease comprises osteoarthritis.

[0340] Clause 60. The method of any one of clauses 53-57, wherein the joint disease comprises post-traumatic osteoarthritis.

[0341] Clause 61. The method of any one of clauses 53-57, wherein the joint disease comprises joint cartilage or bone damage.

[0342] Clause 62. The method of any one of clauses 53-61, wherein the pharmaceutical composition is administered via intra-articular injection.

[0343] Clause 63. A kit comprising the pharmaceutical composition of any one of clauses 36-52.

[0344] Clause 64. The kit of clause 63, further comprising a syringe.

[0345] Clause 65. The kit of clause 63 or clause 64, further comprising instructions for using the pharmaceutical composition to treat a joint disease, to treat pain associated withDUKE-45610.601 a joint disease or joint tissue damage, to deliver a pharmaceutically active compound to cartilage and / or subchondral bone, or to treat an injury to a joint or cartilage in a subject.

[0346] Clause 66. A pharmaceutical composition of any one of clauses 36-52, for use as a medicament.

[0347] Clause 67. A pharmaceutical composition of any one of clauses 36-52, for use in treating a joint disease, treating pain associated with a joint disease or joint tissue damage, delivering a pharmaceutically active compound to cartilage and / or subchondral bone, and / or treating an injury to a joint or cartilage.

[0348] Clause 68. The pharmaceutical composition for use of clause 67, wherein the joint disease comprises arthritis.

[0349] Clause 69. The pharmaceutical composition for use of clause 67, wherein the joint disease comprises osteoarthritis.

[0350] Clause 70. The pharmaceutical composition for use of clause 67, wherein the joint disease comprises post-traumatic osteoarthritis.

[0351] Clause 71. The pharmaceutical composition for use of clause 67, wherein the joint disease comprises joint cartilage or bone damage.

[0352] Clause 72. A pharmaceutical composition comprising at least two pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier.

[0353] Clause 73. The pharmaceutical composition of clause 72, wherein the pharmaceutical composition comprises PTH and BMP-2.

[0354] Clause 74. A pharmaceutical composition comprising at least two pharmaceutically active compounds selected from the group consisting of roflumilast, Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-(TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier.

[0355] Clause 75. The pharmaceutical composition of clause 74, wherein the pharmaceutical composition comprises roflumilast, PTH, and LDN-214117.DUKE-45610.601 Clause 76. The pharmaceutical composition of any one of clauses 72-75, wherein the pharmaceutical composition is formulated for intra-articular injection.

[0356] Clause 77. A method of treating a joint disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof.

[0357] Clause 78. A method of treating pain associated with a joint disease or joint tissue damage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof.

[0358] Clause 79. A method of delivering a pharmaceutically active compound to cartilage and / or subchondral bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof.

[0359] Clause 80. The method of clause 79, wherein the subject is suffering from a joint disease.

[0360] Clause 81. A method of treating an injury to a joint or cartilage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof.

[0361] Clause 82. The method of any one of clauses 77-81, comprising administering to the subject a therapeutically effective amount of PTH and BMP-2.

[0362] Clause 83. A method of treating a joint disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of roflumilast,DUKE-45610.601 Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-P (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof.

[0363] Clause 84. A method of treating pain associated with a joint disease or joint tissue damage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of roflumilast, Growth and Differentiation Factor 11 (GDF11), EDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-p (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof.

[0364] Clause 85. A method of delivering a pharmaceutically active compound to cartilage and / or subchondral bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of roflumilast, Growth and Differentiation Factor 11 (GDF11), EDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-p (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof.

[0365] Clause 86. The method of clause 85, wherein the subject is suffering from a joint disease.

[0366] Clause 87. A method of treating an injury to a joint or cartilage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least two pharmaceutically active compounds selected from the group consisting of roflumilast. Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-P (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof.

[0367] Clause 88. The method of any one of clauses 83-87, comprising administering to the subject a therapeutically effective amount of roflumilast, PTH, and LDN-214117.DUKE-45610.601 Clause 89. The method of any one of clauses 77-88, wherein the joint disease comprises arthritis.

[0368] Clause 90. The method of any one of clauses 77-88, wherein the joint disease comprises osteoarthritis.

[0369] Clause 91. The method of any one of clauses 77-88, wherein the joint disease comprises post-traumatic osteoarthritis.

[0370] Clause 92. The method of any one of clauses 77-88, wherein the joint disease comprises joint cartilage or bone damage.

[0371] Clause 93. The method of any one of clauses 77-92, wherein the at least two pharmaceutically active compounds are administered via intra- articular injection.

[0372] Clause 94. The method of any one of clauses 77-93, wherein the at least two pharmaceutically active compounds are administered simultaneously.

[0373] Clause 95. The method of any one of clauses 77-93, wherein the at least two pharmaceutically active compounds are administered sequentially.

[0374] Clause 96. A kit comprising at least two pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof.

[0375] Clause 97. The kit of clause 96, wherein the kit comprises PTH and BMP-2.

[0376] Clause 98. A kit comprising at least two pharmaceutically active compounds selected from the group consisting of roflumilast, Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid, transforming growth factor-β (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof.

[0377] Clause 99. The kit of clause 98, wherein the kit comprises roflumilast, PTH, and LDN-214117.

[0378] Clause 100. The kit of any one of clauses 96-99, further comprising a syringe. Clause 101. The kit of any one of clauses 96-100, further comprising instructions for using the pharmaceutically active compounds to treat a joint disease, to treat pain associated with a joint disease or joint tissue damage, to deliver a pharmaceutically activeDUKE-45610.601 compound to cartilage and / or subchondral bone, or to treat an injury to a joint or cartilage in a subject.

[0379] The following Examples are provided by way of illustration and not by way of limitation.

[0380] I. Examples

[0381] Example 1

[0382] Polymer Syntheses and Nanocarrier Fabrication

[0383]

[0384] Thiol-functionalized BPL was synthesized via ring-opening polymerization (ROP) of / Ve-benzyloxycarbonyl-L-lysine / V-carboxy anhydride (Cbz-Lys-NCA) using a disulfide- containing initiator. 2,2'-dithiobis(ethanamine) (cystamine). First, AC-benzyloxycarbonyl- L-lysine / V-carboxyanhydride (Cbz-Lys-NCA) was synthesized by reacting NE- benzyloxycarbonyl-L-lysine (Cbz-Lys) with triphosgene at 50 °C in anhydrous tetrahydrofuran (THF) for 3 h. The reaction mixture was then precipitated in excess hexane, washed three times with hexane, and vacuum-dried overnight to obtain Cbz-Lys-NCA. Cbz-Lys-NCA (20 equivalents) was dissolved in dry N, N-dimethylformamide (DMF, 100 mg / mL) in a round-bottomed flask fitted with a drying tube. 2,2'-Dithiobis ethanamine (1 equivalent) was added to the reaction mixture and the reaction was carried out at room temperature for about 5 days under continuous stirring. The reaction mixture was added to a ~20-fold excess of cold water to precipitate the oligo(Cbz-L-lysine) core peptide. The precipitate was filtered, washed with water, and freeze-dried. A solution of the oligo(Cbz- L-lysine) core peptide in DMF was then reacted with N“, NE-di(9- fluorenylmethoxycarbonyl-L- lysine) (N“, N£-diFmoc-Lys, 8 equivalents) using 2-(lH- benzo-triazole-l-yl)-oxy-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU, 4 equivalents), and 1-hydroxy benzotriazole (HOBt, 24 equivalents) and N, N- diisopropylethylamine (DIPEA, 20 equivalents) at room temperature for 3 days. The product was precipitated in excess of cold water, filtered, washed three times with water, and freeze-dried. The freeze-dried powder was washed 4 times with diethyl ether, and vacuum-dried to obtain N“, Ne-diFmoc-Lys conjugated core peptide. A solution of theDUKE-45610.601 N“, NE-diFmoc-Lys end-functionalized core peptide in DMF (-200 mg / mL) was reacted with 20% (v / v) piperidine under continuous stirring for an hour at room temperature and subsequently precipitated in water. The precipitate was filtered, washed repeatedly with water, and freeze-dried. The freeze-dried product was washed 4 times with diethyl ether, and vacuum-dried to obtain lysine core peptide end-functionalized with thiol group. The lysine end-functionalized core peptide was reacted with the Cbz-Lys-NCA monomer to obtain next-generation branched peptide. This include repeating the above steps of ringopening polymerization, end functionalization, and subsequent removal of Fmoc protecting groups. The Cbz protecting group was completely removed from BPL molecules to achieve free amine groups in the BPL molecules. The reaction scheme is shown in FIG.

[0385] 1. The product was characterized via1H NMR (500 MHz, DMSO-d6): 5 4.80 (s), 4.35-4.23 (m), 3.04-3.01 (m), 1.92 (m), 1.79-1.70 (m), 1.48-1.45 (m) ppm.

[0386] Synthesis of Azide -Functionalized Branched Poly-lysine (BPL}

[0387] A terminal azide functionalized BPL was synthesized via ring opening polymerization (ROP) reaction of Ne-benzyloxycarbonyl-L-lysine N-carboxy anhydride (Cbz-Lys-NCA) with an azide containing initiator, azide-PEG4-amine. First, NE-benzyloxycarbonyl-L-lysine N-carboxyanhydride (Cbz-Lys-NCA) was synthesized by reacting NE-benzyloxycarbonyl-L-lysine (Cbz-Lys) with triphosgene at 50 °C in anhydrous tetrahydrofuran (THF) for 3 hours. The reaction mixture was precipitated in excess of hexane, washed thrice with hexane, and vacuum-dried overnight to obtain Cbz-Lys-NCA. Next, Cbz-Lys-NCA (10 equivalents) was dissolved in dry N, N-dimethylformamide (DMF, 100 mg / mL) in a round-bottomed flask fitted with a drying tube. Azide-PEG4-amine (1 equivalent) was added to the Cbz-Lys-NCA solution and the reaction (ring opening polymerization) was continued at room temperature for 5 days under continuous stirring. The reaction mixture was added to a ~20-fold excess of cold water to precipitate the product, oligo(Cbz-L-lysine) core peptide with terminal azide group. The precipitate was filtered, washed with water, and freeze-dried. A solution of the oligo(Cbz-L-lysine) peptide in DMF was then reacted with N“, NE-di(9-fluorenylmethoxycarbonyl-L-lysine) (N“, NE-diFmoc-Lys, 4 equivalents) using 2-(lH-benzo-triazole-l-yl)-oxy-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU, 4 equivalents), 1-hydroxy benzotriazole (HOBt, 12DUKE-45610.601 equivalents) and N, N-diisopropylethylamine (DIPEA, 10 equivalents) at room temperature for 3 days. The product was precipitated in excess of cold water, filtered, washed three times with water, and freeze-dried. A solution of the N“, NE-diFmoc-Lys end-functionalized core peptide in DMF (-200 mg / mL) was reacted with 20% (v / v) piperidine under continuous stirring for an hour at room temperature to remove the Fmoc group. The product was precipitated in water, filtered, washed repeatedly with water, and freeze-dried. The freeze-dried product was washed with diethyl ether multiple times, and vacuum-dried to obtain lysine end-functionalized with azide group. Finally, the lysine end-functionalized core peptide was used as initiator and subsequently reacted with the Cbz-Lys-NCA monomer to obtain next-generation branched peptides. Another generation of the branched peptides was then synthesized by repeating the above reaction steps of ring-opening polymerization, end functionalization, and subsequent removal of Fmoc protecting groups. The Cbz protecting group was completely removed to generate free amine groups in BPL molecules. The reaction scheme is given in FIG. 2. The product was characterized via1H NMR (500 MHz, D2O): 54.77, 4.25-4.15, 3.63-3.42, 2.94, 1.77-1.36 ppm.

[0388] Synthesis of PLGA-PEG

[0389] PLGA (20 kDa - 150 kDa) was dissolved in DCM and EDC (10 equivalent) was added to the solution and the mixture was stirred for 15 minutes. NHS (10 equivalent) was added to the reaction mixture. After 15 minutes, mPEG-NH2 (Mol. Wt. of PEG = 2 kDa; 5 equivalent) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 h following the addition of DIPEA (10 equivalent). The product was precipitated in a mixture of diethyl ether and methanol (1:1). The precipitate was washed repeatedly in the same solvent mixture and dried under vacuum. The percentage of PEG modification was quantified by proton NMR, and over 95% of the PEG modification into the PLGA was observed. The reaction scheme is given in FIG. 3. The product was characterized via1H NMR (500 MHz, CDCl3): 5 5.23-5.16, 4.90-4.71, 3.64, 1.59-1.55 ppm.

[0390] Synthesis of PLGA-DBCODUKE-45610.601 PLGA (20 kDa - 150 kDa) was dissolved in dichloromethane (DCM) and N-(3-dimethylaminopropyl)-A'-ethyl carbodiimide hydrochloride (EDC, 20 equivalent) was added to the solution and the mixture was stirred for 15 minutes. N-hydroxy succinimide (NHS, 20 equivalent) was added to the reaction mixture. After 15 minutes, DBCO amine (10 equivalent), dissolved in DCM, was added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 h following the addition of DIPEA (20 equivalent). The product was precipitated in a mixture of diethyl ether and methanol (1:1). The precipitate was washed repeatedly in the same solvent mixture and dried under vacuum. The percentage of DBCO modification was quantified by proton NMR as the spectrum showed the appearance of aromatic protons at 7.26-7.40 ppm corresponding to benzene protons of DBCO ring. Over 98% of the DBCO modification into the PLGA was observed. The reaction scheme is given in FIG. 4. The product was characterized via1H NMR (500 MHz, CDCl3): δ7.41-7.26, 5.22-5.16, 4.82-4.70, 3.78-3.77, 1.59-1.55 ppm.

[0391]

[0392] -BPL

[0393] Disulfide group containing BPL molecules were dissolved in methanol. Tris(2-carboxyethyl) phosphine (TCEP, 2 equivalents), dissolved in methanol, was then added to the BPL solution. The mixture was stirred for 6 h following the addition of a small amount of water. The BPL solution was added to a solution of PLGA-PEG-maleimide, dissolved in a mixture of acetone and methanol. DIPEA (10 equivalent) was subsequently added to the reaction mixture and stirred for 48 h. After the reaction, the solvent was evaporated at reduced pressure and the reaction mixture was washed repeatedly with methanol to remove unreacted BPL and other reagents. The degree of BPL conjugation to PLGA-PEG-BPL was determined by proton NMR spectroscopy, which suggests ~ 85 % BPL conjugation.

[0394] Alternatively, PLGA-BPL was synthesized by direct conjugation of PLGA-DBCO with azide-BPL via a SPAAC reaction. Briefly, PLGA-DBCO (1 equivalent), synthesized as described previously, was dissolved in dichloromethane (DCM). Azide-BPL (1.1 equivalents), dissolved in DMSO, was then added to the PLGA-DBCO solution. The reaction mixture was stirred for approximately 2 hours at room temperature to produce PLGA-BPL via the SPAAC reaction between DBCO and azide groups. The product was precipitated in a 1:1 cold diethyl ether / methanol mixture, collected by centrifugation,DUKE-45610.601 washed repeatedly with the same solvent mixture, and vacuum-dried. The reaction scheme is given in FIG. 5. The product was characterized via1H NMR (500 MHz, CDCl3): δ5.05-4.70, 4.35-4.05, 3.60-3.35, 3.05-2.70, 2.10-1.85, 1.80-1.35 ppm.

[0395] Synthesis of PLGA-alendronate (Ain)

[0396] Incorporation of the alendronate to the PLGA nanocarrier was achieved in two steps. First, azido-PEG-Aln was synthesized by conjugating azido-PEG-carboxylic acid (molecular weight of PEG is 3.4 kDa) with alendronate via amide coupling reaction. Briefly, azido-PEG-carboxylic acid (Biopharma PEG, catalog number HE017006-3.4K) was dissolved in water. EDC (20 equivalent) and NHS (20 equivalent) was subsequently added to the azido-PEG-carboxylic acid solution. Next, alendronate (5 equivalent), dissolved in water, was added to reaction mixture and the reaction continued for 48 hours at room temperature. After 48 hours, the reaction mixture was dialyzed against DI water for 3-4 days and freeze-dried to obtain azido-PEG-Aln. Next, PLGA-DBCO (1 equivalent), as synthesized previously, was dissolved in dichloromethane (DCM) and azido-PEG-Aln (1.5 equivalent), dissolved in DCM, was added to the solution of azido-PEG-Aln. The reaction mixture was stirred for about 2 hours at room temperature to produce PLGA-PEG-Aln via SPAAC reaction between DBCO and azide groups. The product was precipitated in a 1: 1 cold diethyl ether and methanol mixture, centrifuged, washed repeatedly with 1:1 diethyl ether and methanol mixture and vacuum dried. The synthesis of PLGA-PEG-Aln was confirmed via proton NMR spectroscopy as the spectra confirmed the presence of the -OCH2CH2O- peaks at 3.64 ppm in addition to the characteristic peaks for lactic acid proton at 5.18-5.25 ppm. The reaction scheme is given in FIG. 6. The product was characterized via

[0397]

[0398] NMR (500 MHz, CDCh): 85.26-5.15, 4.89-4.71, 3.64, 1.59-1.55 ppm.

[0399] 2. Nanocarrier Fabrication

[0400] To synthesize cartilage penetrating, bone binding nanocarriers (NC), the PLGA polymer is modified with a cartilage-binding branched poly-L- lysine (BPL) peptide, which facilitate penetration across cartilage, and optionally alendronate (Ain), which enables binding to the bone (see FIG. 7 and FIG. 8). (Alendronate was not included for nanocarriers intended only for delivery to cartilage.) Briefly, the BPL molecules are functionalized withDUKE-45610.601 either thiol or azide and reacted with PLGA molecules that have complementary functional groups (maleimide or DBCO). In the case of Ain functionalization, Ain was directly conjugated to PLGA molecules. To prevent protein adsorption, a small percentage of polyethylene glycol (PEG) conjugation (<5 wt% of the NCs) is used. The PLGA nanocarriers are prepared via a water-in-oil emulsification method by adding an organic solution of PLGA, PLGA-PEG, PLGA-BPL, and PLGA-PEG-Aln to an aqueous solution of polyvinyl alcohol dissolved in PBS, where polyvinyl alcohol acts as an emulsifier. Nanocarriers from different PLGA precursors with varying LA to GA ratios have been tested, 65:35; 75:25; 85:15 and 50:50 (see, e.g., FIG. 7 and FIG. 8). For protein-based drugs, such as PTH and BMP-2, the unique properties of sugar glass micelles were utilized to load protein molecules. The sugar glass micelle stabilizes the proteins and protects them from denaturation during formulation, transport, and storage. The sugar-glass micelle stabilized proteins were subsequently loaded into the PLGA-BPL-Aln nanocarrier. Each drug molecule is loaded into the NCs independently and the NCs are mixed to generate the combination formulations with the desired spatio-temporal drug release profiles and concentration ratios.

[0401] Example 2

[0402] Formulation Selection and Data

[0403] In this example, experiments are described that led to selection of the final drug combinations for treatment of joint diseases, with two particular areas of focus: drug formulations that target subchondral bone and cartilage regeneration are referred to as “TAI,” and drug formulations for cartilage regeneration are referred to as “TA2.” The initial “lead” formulations identified were as follows: for TAI, a combination of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), and ALK4-Fc; and for TA2, a combination of roflumilast, LDN-214117, and Growth and Differentiation Factor 11 (GDF11). Following the extensive formulation selection experiments described below, the final “lead” formulations were as follows: for TAI, a combination and sequence of BMP-2 and PTH; and for TA2, a combination and sequence of roflumilast, PTH, and LDN-214117.DUKE-45610.601 a. Ex Vivo Data

[0404] Testing of Pharmaceutical Agents

[0405] All primary / lead agents were evaluated individually and in combinations using osteochondral (OC) explants, harvested from the joint surfaces that were surgically removed from osteoarthritic patients during a medically necessary total joint arthroplasty surgery. Lead and alternative agents that showed less than a 10% improvement over baseline in key parameters (PSR for TAI; SafO for TA2) were down selected. The best regenerative outcomes were compared against as well as in combination with other agents to down select those with a worse regenerative outcome. When outcomes were equivalent, combinations with fewer components and / or lowest costs were prioritized. All TAI and TA2 agents were studied individually and in combination with dosing guided by the initial screening studies. All lead agents, their combinations, and alternative agents have been tested and the data is described below.

[0406] The following additional alternative agents were evaluated: Salvianolic acid (Sigma Aldrich Cat#: 1609818), Kaempferol (Sigma Aldrich Cat# K0133), binimetinib (MedChemExpress Cat#: HY-15202), Givinostat (MedChemExpress Cat#: HY-14842), Human DKK-1 Recombinant Protein (PeproTech SKU: 120-30), Recombinant TGF-β (BioTechne). TGF beta-1.2,3 antibody (Fisher Scientific, Cat# MA5-23795), Recombinant TGF-β R1 FC (Fisher Scientific, Cat#: 3025-BR-050), Resveratrol (SIGMA ALDRICH, Cat# R5010), Adenosine (SIGMA ALDRICH, Cat# A4036), Lithium Carbonate (SIGMA ALDRICH, Cat# 203629), Zoledronic Acid (SIGMA ALDRICH, Cat# SML0223), Exenatide Acetate (Exendin-4) (SELLECK CHEMICALS LLC Cat# P1046), Anakinra / Raleukin (MedChemExpress: Cat# HY-108841), ALK5-FC (R& D Systems, Cat#3025-BR).

[0407] Overall description of testing of alternative agents'. Cell culture-based prescreening assays were conducted to identify anabolic molecules that could be advanced in the pipeline to osteochondral (OC) explant assays and in vivo studies. Only agents that demonstrate a superior response or exhibit an equivalent response while being more cost-effective than the lead agents proceed to ex vivo and subsequently in vivo testing. There were no TAI alternative agents that resulted in PSR+ scores of greater than 10% over control OC explants, thus all were downselected. (Table 2, FIG. 9). For TA2, a cell culture-DUKE-45610.601

[0408] based pre-screening studies identified binimetinib, a MAP2K inhibitor, TGF-beta, and PTH as potential TA2 agents with chondro-anabolic functions (not shown). Further studies using OC explants from OA patients confirmed the chondroanabolic function of PTH as having an increase of >10% SafO staining compared to control OC explants (FIG. 10). The data of the evaluation of alternative agents in prescreening, if applicable, and in ex vivo explants is summarized in Table 2 and 3.

[0409] Table 2. Summary of TAI Alternative Agent Evaluation

[0410] TAI Compounds Ex vivo OC explant Up-select to in vivo studies Adenosine Fail No

[0411] Lithium Fail No

[0412] ALK5-Fc Fail No Zoledronic Acid unable to assess Not available Anakinra Fail No

[0413]

[0414] Table 3. Summary of TA2 Alternative Agent Evaluation

[0415] TA2 Prescreening Passed / Failed Up-select / OC explant Compounds done? down-select

[0416] DKKl / Wnt Yes Fail Down- select N / A inhibitor IWP2

[0417] PTH Yes Pass Up- select Pass Quercetin Yes Fail Down- select N / A Kaempferol Yes Fail Down- select N / A Binimetinib Yes Pass Up- select Fail Salvianolic Yes Fail Down- select N / A

[0418] acid

[0419] TGFB Yes Pass Up- select Fail (despite cost)

[0420] Resveratrol Yes Fail Down- select N / A Givinostat Yes Fail Down- select N / A Exenatide Yes Fail Down- select N / A

[0421]

[0422] Anakinra Yes Fail Down- select N / A

[0423] OC explant harvest, treatment and histological preparation: OC explants were harvested in a pairwise manner (adjacent to each other) from excised joint surfaces such that cartilage thickness and visible degeneration was similar prior to treatment (FIG. 11). One explant was treated with a drug cocktail while the paired explant was cultured for theDUKE-45610.601 same period of time (14 days) without drugs as a control. A 14-day duration for treatment was established as optimal (FIGS. 12A-12C). Explants were either exposed to individual compounds, a combined drug regimen continuously throughout the 14-day experiment or treated with compounds sequentially. Human OC explants were fixed for 2-days in 10% NBF, rinsed in IX PBS multiple times, decalcified in 20% EDTA solution for 5-days. and processed for paraffin sectioning. Histological sections were stained with Picrosirius red or safranin O with fast green counterstain and scored by reviewers blinded to treatment.

[0424] Principle of Primary Bone Measures in Human OC Explants: Picrosirius red (PSR) stain was used to calculate the ratio of PSR+ subchondral plate / bone surface per total subchondral plate / bone surface for all treated and control explants. Histological sections were stained with 0.04% picrosirius red (PSR), marking areas of osteoid (newly formed woven bone) as red, while more mature lamellar bone stained yellow or pink in bright field images. The color discrimination is based on the enhanced uptake of PSR by the thicker and more loosely packed Type I collagen fibers in osteoid compared to the thinner and more densely packed fibers in lamellar bone, which inhibits uptake of the dye. Bone anabolic matrix changes would be represented by an enhanced ratio of PSR+ bone surface perimeter / total bone surface perimeter.

[0425] Principle of Primary Cartilage Measures in Human OC Explants: Safranin-O / Fast Green (SafO) staining identifies matrix that is rich in sulfated glycosaminoglycans (sGAGs), or in other words, a proteoglycan rich cartilage matrix. Anabolic activity of articular chondrocytes during development and regeneration produces a matrix rich in proteoglycans marked by SafO, while proteoglycans and SafO staining are lost during PTOA or OA-associated articular cartilage degeneration. SafO+ stained articular cartilage area / total articular cartilage area in human OC explants provides a measure of healthy and / or regenerating cartilage.

[0426] Pharmaceutical Agent Optimization (ex vivo)

[0427] The selection of candidates prior to and during animal studies was determined based on: 1) optimal and reproducible regeneration potential; and 2) fewer agents (a two-agent approach would be simpler than a three-agent approach); and 3) Cost - if two approaches were similar, lowest cost would be selected.DUKE-45610.601 Primary bone and cartilage measures, PSR and SafO staining respectively, were performed, and PSR+ bone perimeter / total bone perimeter and SafO+ area / total cartilage area were recorded. All measures were completed for all treatments and combinations for at least 3-5 patient derived samples with at least 3-4 technical replicates for each sample. A 10% improvement over baseline was utilized as an indicator of regeneration, with PSR indicating regeneration of bone (TAI) and SafO indicating regeneration of cartilage (TA2).

[0428] TAI: Patient derived osteochondral explants were utilized for testing of single and co-administered drug treatments that included PTH, BMP2, ALK4-Fc for TAI primary agents. None of the TAI alternative agents were up-selected to be evaluated in vivo because they failed to induce anabolic effects on bone in the ex vivo explants (FIG. 9).

[0429] PTH+BMP2 treatments and PTH+BMP2+ALK4Fc each demonstrated a greater than 10% increase in the mean primary outcome measure of PSR+ bone perimeter / total bone perimeter ratio from 5 different patient derived osteochondral explants (FIG. 13 A). However, when assessing the secondary SafO cartilage measures for these TAI agents, PTH+BMP2 treatments elicited a more positive SafO+ area / total cartilage area ratio as compared to PTH+BMP2+ALK4Fc and all other treatments (FIG. 13B). Representative images of PSR and SafO stained human osteochondral explants treated with and without PTH+BMP2 demonstrate these changes (FIGS. 14A-14B). Based on these data, PTH+BMP2 treatment was prioritized for in vivo rat studies assessing OA and joint associated bone, cartilage, and pain parameters following MMNX surgery (OA surgery).

[0430] Further studies aimed at determining the optimal sequence of the TAI agents resulted in the final formulation of a faster release profile of BMP2, followed by a slower release profile of PTH (BP) (FIGS. 15A-15B, FIG. 16, Final formulation, left). The sequential order of the compounds was determined ex vivo. In particular, it was found that BMP treatment before PTH resulted in a superior regenerative result, and removing ALK4-Fc did not reduce the regenerative effect over BMP and PTH alone.

[0431] TA2: Given that the approach to regenerating articular cartilage is to provide a combination of drugs that result in the greatest regenerative potential, alternative agents were evaluated for the ability to regenerate cartilage in ex vivo osteochondral explants. PTH was the only alternative agent that caused >10% increase in the percentage of SafO+ cartilage area (FIG. 10) and was therefore up-selected to be evaluated against and inDUKE-45610.601 combination with lead TA2 agents. Tests were conducted to determine whether PTH, when utilized in combination with roflumilast and LDN214117, would outperform the proposed lead formulation of GDFll+roflumilast+LDN214117 (RGL) in the ex vivo explants. Roflumilast, GDF11, LDN-214117, PTH and combinations thereof were tested in the explants. Several treatments led to increases in the percentage of SafO-i- cartilage area (RGL, R, P, RL, and RPL) (FIG. 17). The combination of RPL resulted in the highest increase of SafO+ area (17%) over RGL (7%) and R alone (14%) and thus RPL became the lead formulation for TA2. Representative images of SafO stained human osteochondral explants treated with and without roflumilast+GDF11+LDN or roflumilast+PTH+LDN are examples of TA2 treatments demonstrating the enhancement in SafO area / total cartilage area indicated in FIG. 17 and FIGS. 18A-18B).

[0432] Further studies aimed at determining the optimal sequence of the TA2 agents resulted in the final formulation of a faster release profile of Roflumilast. followed by a sustained PTH release and finally a slower release profile of LDN (RPL, lead) (FIGS. 19A-19B, FIG. 16, Final formulation, left). The sequential order of the compounds was determined ex vivo. In particular, it was found that the sequence RPL resulted a superior regenerative result.

[0433] b. In Vivo Data

[0434] Evaluation of Agents with the Rat MMNX Models of Mild, Moderate, and Severe PTOA

[0435] Rat Model of Post-Traumatic Osteoarthritis (PTOA): To study the progression of PTOA and the specific effects that the lead agents have on regenerating joint tissues following joint injury and degeneration, a modification of the medial meniscus transection (MMNX) surgery was utilized on rats. MMNX surgeries lead to a steady PTOA disease progression. Briefly, the MMNX surgeries were performed on 12-week old male rats. Rats were anesthetized and hair is shaved from the right knee and sterilized with betadine. An incision was made on the medial aspect of the right knee and the joint space is opened slightly. To induce a mild OA phenotype, a scalpel was used to partially transect the medial meniscus on the anterior 1 / 3 of the meniscus near the medial meniscotibial ligament (MMTL). To induce a moderate OA phenotype, a scalpel was used to completely transectDUKE-45610.601 the medial meniscus on the anterior 1 / 3 of the meniscus near the MMTL. To induce a severe OA phenotype, a scalpel was used to completely transect the medial meniscus on the anterior 1 / 3 of the meniscus near the MMTL, followed by its clear destabilization from the joint that involves incisions through the synovium above and below the medial meniscus. Post-surgery, rats were rested on a heated pad. administered a slow-release analgesia (Buprenorphine SR at 0.5-1.0mg / kg), and monitored until they recovered sufficiently to ambulate and obtain food and water. Some cohorts of rats then received at 4-wpi (weeks post injury), either: 1) a single intra-articular (IA) injection of the primary TAI or TA2 single agent or combination treatments, or 2) no treatment, and are then sacrificed at 8-wpi for tissue harvest of injured joint and contralateral control. Some cohorts of rats were also sacrificed at 4-wpi (no treatment) to serve as a control at the time of treatment. Pain measurements were performed prior to and after the injury with or without treatment. Following tissue harvests, all samples undergo microCT and histological analyses. All surgical procedures were performed in accordance with approved Duke IACUC protocol and NIH guidelines.

[0436] Gross Tissue Preparation, Fixation, and MicroCT Methodology to Assess Subchondral Plate and Subchondral Bone: The knee joint was dissected and most soft tissue was removed using surgical scissors. The proximal 2 / 3 of the femur and 2 / 3 of the tibia were removed to open the marrow space for effective penetration of the fixative. Specimens were placed in appropriately labeled tissue cassettes and are fixed in 10% neutral buffered formalin (NBF) for 72-hours. Samples were then submitted for microCT scanning and analysis. When using microCT to assess bone changes rodents during PTOA, joint-associated bone regions of interest were first selected within the tibia and femur of rats with and without MMNX injury. Briefly, for subchondral plate analysis 2D microCT images and contoured regions spanning the medial tibial and femoral condyles from the medial cortex to the intercondylar notch and from the subchondral bone to the articular cartilage were segmented. The parameters measured for the subchondral plate included: Bone Fraction (bone volume / total volume; BV / TV), Connectivity Density, SMI, Bone Density (mg HA / cm3), and Maximal Subchondral Plate Thickness. For subchondral bone analysis, 2D microCT images and contoured regions spanning the medial tibial and femoral condyles from the medial cortex to the intercondylar notch and from the growth plate toDUKE-45610.601 the subchondral plate were segmented. The parameters measured for the subchondral bone included: Bone Fraction (bone volume / total volume; BV / TV), Connectivity Density, SMI, Trabecular Number (Tb. N.), Trabecular Separation (Tb. Sp.), Trabecular Thickness (Tb. Th.), and Bone Density (mg HA / cm3). The Connectivity Density parameter were previously demonstrated to be the most sensitive to MMNX induced OA bone changes.

[0437] Decalcification, Processing, Embedding, and Microtomy of Rat Knee Tissue:

[0438] Upon completion of fixation and microCT analyses, samples were rinsed and then decalcified while agitating in a 20% EDTA decalcification solution for 14 to 21-days. This protocol was also modified to speed decalcification times. The new methodology utilized the Immunocal reagent, a proprietary mild formic acid decalcifier, with agitation for 10-days. Samples were then rinsed prior to processing through a graded series of ethanol and xylene followed by a series of paraffin infiltration steps. Following paraffin processing, the samples were embedded in paraffin prior to sectioning. Decalcified joint tissues were sagittally sectioned at 5pm through 3 major levels of the rat knee joint within the medial femoral condyle. Sections from each of the different levels are stained (SafO / Fast Green; as described previously) and scanned for OARSI scoring and SafO area / total cartilage area measurements.

[0439] OARSI Scoring of Rat Joints following MMNX Surgery and Treatments with TAI and TA2 Agents: The rodent OARSI histopathology grading system was utilized to measure the cartilage degeneration that occurs over the course of 8-weeks following MMNX surgeries and compare these values to various controls described above. SafO / Fast Green-stained sections were reviewed and scored by 3-6 independent, blinded reviewers. Direct assessment of SafO area / total cartilage area measurements was added to overcome shortcomings of the OARSI scoring system that have become apparent, notably the broad range of cartilage degeneration that is encompassed by each score within the scale (0-6 OARSI scale). For example, an OARSI score of 3 represents anywhere from 1% to 25% joint cartilage erosion to the level of the calcified cartilage; potentially masking significant gains in cartilage regeneration induced by IA injection of TAI or TA2 agents.

[0440] SafO+ Cartilage Area Measurements of Rat Joints following MMNX Surgery and Treatments with TAI and TA2 Agents: SafO staining identifies a proteoglycan rich cartilage matrix. Anabolic activity of articular chondrocytes during the regenerativeDUKE-45610.601 process produces a matrix rich in proteoglycans marked by SafO, while proteoglycans and SafO staining are lost during PTOA or OA-associated articular cartilage degeneration. Strong SafO+ stained articular cartilage area / total articular cartilage area of the (combined) medial femoral condyle and tibial plateau thus provides an additional measure of healthy and / or regenerating cartilage.

[0441] Pain Measurements: The pain-related behaviors were assessed at regular intervals prior to sacrifice via the Von Frey assay, which measures mechanical allodynia, or sensitivity to innocuous mechanical stimulation. All measurements were performed by a blinded, experimenter in a controlled procedure room, maintaining consistent conditions: temperature (71 °F), dim lighting, low noise, and standardized testing times. The animals were exposed to a 60-minute habituation period for two consecutive days before the behavioral measurements. In the Von Frey assay, the rat paws were probed with filaments of different sizes, each corresponding to a particular force when bent. The up-down method was used to get a 50% paw withdrawal threshold (PWT), or a force at which the animal is expected to respond 50% of the time. PWT was used as a surrogate pain measure, with a lower PWT corresponding to more pain.

[0442] Evaluation of TAI agents for treating PTOA-associated bone and cartilage effects, and pain

[0443] FIGS. 20-24 summarize in vivo PTOA-associated bone, cartilage, and pain parameters for TAI agents individually and in combinations. microCT assessments were performed on rat knees that had received severe MMNX OA surgeries at 4-wpi, and 4 weeks after IA injection of TAI agents and combinations. These data indicate that the BMP2+PTH combination treatment restores connectivity density of the subchondral plate to levels observed in normal joints (no treatment), and significantly below that of the OA joint at 4-wpi (no treatment), while PTH alone, as well as other treatments, did not restore connectivity density or other subchondral bone / plate parameters to within one standard deviation of no OA control levels (FIG. 20). Representative 2D microCT images in the frontal plane at 3 levels (Anterior, Middle, and Posterior) of the tibia demonstrate that the structure of the medial compartment of the subchondral plate / bone of the MMNX operated knee shows thickening and a sclerotic bone phenotype at 4-wpi as compared to the no OADUKE-45610.601 contralateral control (FIG. 21). Importantly, BMP2+PTH treatment restores a near normal (no OA) structure to the subchondral plate and bone at most all levels (FIG. 21).

[0444] Secondary cartilage measures for TAI were assessed next by performing OARSI scoring using the rodent histopathology grading system, as well as measuring SafO area / total cartilage area (FIGS. 22A-22B). OARSI scoring shows that BMP2+PTH significantly reduces the OARSI score as compared to 8-wpi MMNX injured controls (no treatment). SafO area / total cartilage area measurements were performed on the same sections used for OARSI scoring to more directly assess the presence of healthy / regenerating and anabolic joint cartilage. These data demonstrate that BMP2+PTH induces a cartilage anabolic response resulting in not only a significant increase in the ratio of SafO area / total cartilage area stained as compared to the 4-wpi OA rat knees, but also show no significant differences when compared to the ratio of SafO area / total cartilage area of contralateral control joints (FIG. 22B). Representative images of SafO / Fast Green stained sections in the sagittal plane at 3 levels (Outer, Middle, and Central) of the medial compartment of the rat knee are shown for uninjured contralateral control knees, MMNX injured knees at 4-wpi and 8-wpi, and BMP2+PTH treated knees at 8-weeks following MMNX surgeries (FIG. 23). BMP2+PTH treatments, even at a slightly later timepoint, show substantially more regenerative or anabolic (strong SafO+) joint cartilage as compared to either stage of OA following MMNX surgery and to levels similar to (tibia; bottom) or even exceeding (femoral; top) uninjured control knee joints (FIG. 23).

[0445] Prior to and following MMNX knee surgeries, pain was monitored using the Von Frey assay on both the contralateral control paw and the paw of the limb associated with the MMNX knee injury described previously. Following injury, there was a rapid decline in the PWT of rats with MMNX injured knees (OA) as compared to their contralateral controls (CL), as expected (lower numbers indicate higher pain). This initial rapid decline in PWT then maintains a relatively steady state in MMNX injured rats over the course of at least 13-wpi. These data show that BMP2+PTH treatment (single IA injection) results in a restoration of the PWT (lowering pain) to near normal contralateral control levels or within one standard deviation of controls by 10-wpi (FIG. 24A). Pain was also mitigated in female rats with severe injury (FIG. 24B). These data show that BMP2+PTH treatment (single IA injection) results in a time-dependent restoration of the PWT to near normalDUKE-45610.601 contralateral control levels or within one standard deviation of controls by 10 wpi. The pain relief was maintained up to 26-wpi suggesting long-term pain relief following a single IA injection of BMP2+PTH (FIG. 25).

[0446] Identification of Lead TA2 agent for treating PTOA-associated bone, cartilage, and pain:

[0447] Rats were treated with intraarticular injections containing the lead TA2 agents (GDF-11, roflumilast, LDN) as well as the alternative agent that was upselected based on ex vivo experiments (PTH). The primary cartilage outcome measures were assessed for TA2 agent(s) by performing OARSI scoring using the rodent histopathology grading system, as well as measuring SafO area / total cartilage area to more accurately document the cartilage degeneration and regeneration that occurs by 4 weeks post treatment (8-wpi). Rats with mild OA were treated with an I A injection of TA2 agents individually or in combination at 4- wpi or left untreated as controls.

[0448] OARSI and SafO area / total cartilage area measures in the male severe MMNX OA model treated with GDFll+roflumilast+LDN (GRL), roflumilast+LDN (RL), LDN alone, and the alternative agent PTH alone, or the combination of roflumilast+LDN+PTH (RLP) (FIGS. 26A-26B). OARSI scoring of this severe MMNX OA model demonstrates that the OA phenotype results in mean OARSI scores ranging from 3-5 at 4- wpi and 8-wpi (FIG.

[0449] 26A). These data further show that both PTH alone and RLP treatments significantly reduces the OARSI score as compared to both 8-wpi and 4-wpi MMNX injured OA controls (no treatment). The other treatments tested did not alter the progression or reverse the phenotype observed in the severe OA model according to OARSI scoring. SafO area / total cartilage area measurements were performed on the exact same sections used for OARSI scoring to more directly assess the presence of healthy / regenerating and anabolic joint cartilage. These data demonstrate that GRL, RL, LDN alone, PTH alone, and RLP treatments induce a cartilage anabolic response resulting in a significant increase in the ratio of SafO area / total cartilage area stained as compared to the 4-wpi OA rat knees (FIG.

[0450] 26B, blue asterisks). The GRL, RL, and PTH show enhancements in the ratio of SafO area / total cartilage area that were not statistically significantly different contralateral no OA control joints (FIG. 26B). These data further demonstrate that RLP treatment inducesDUKE-45610.601 a cartilage anabolic response resulting in a SafO area / total cartilage area measure that is significantly greater than no OA contralateral control measures (FIG. 26B; red asterisk). Representative images of SafO / Fast Green stained sections in the sagittal plane at 3 levels (Outer, Middle, and Central) of the medial compartment of the rat knee are shown for uninjured contralateral control knees, MMNX injured knees at 4-wpi and 8-wpi, and RLP treated knees at 8-wpi following MMNX surgeries (FIG. 27). RLP treatments show substantially more regenerative or anabolic (strong SafO+) joint cartilage as compared to both stages of OA following the severe MMNX OA surgery and to levels similar to (tibia; bottom) or even exceeding (femoral; top) uninjured control knee joints (FIG. 27).

[0451] Prior to and following severe MMNX OA knee surgeries, pain was monitored using the Von Frey assay on both the contralateral control paw and the paw of the limb associated with the MMNX knee injury described previously. Following injury, there was a rapid decline in the PWT of rats with MMNX injured knees (OA) as compared to their contralateral controls (CL). This initial rapid decline in PWT then maintained a relatively steady state in MMNX injured rats over the course of at least 8-wpi in the study. This mirrors the early rapid progression of joint damage observed in the severe MMNX model that slows in its progression following 4-wpi. These data also show that the alternative RPL treatment (single IA injection) results in a time-dependent increase in the PWT (or lessening of the pain response) beginning at 6-wpi and continuing through 12-wpi (FIG.

[0452] 28).

[0453] Selection of final formulations

[0454] Based on in vivo results, the final formulation of BMP2+PTH: 1) demonstrated a connectivity density of the subchondral plate in MMNX injured OA rats that was more similar to contralateral controls when compared to the original BMP2+PTH+ALK4-Fc treatment, 2) significantly reduced OARSI scores in OA treated rats and normalized the SafO area / total cartilage area measurements comparable to contralateral control levels, and 3) reduced pain measures to levels similar to uninjured contralateral controls. Therefore, the alternate combination of BMP2+PTH has shown to be the most efficacious and final TAI formulation.DUKE-45610.601 Due to the net positive effects of roflumilast+PTH+LDN (RPL) alternative formulation treatments on: 1) the significant reduction in OARSI scores in OA treated rats and the normalization of SafO area / total cartilage area measurements to contralateral control levels and beyond, and 2) the reduction in pain measures as compared to untreated severe MMNX OA rats. RPL became the most efficacious and final TA2 formulation.

[0455] Final TAI Summary

[0456] FIG. 16 (left, bottom) shows a schematic of release profiles of final TAI APIs BMP2 and PTH. These APIs were selected based on their ability to induce an anabolic response in bone in human OA osteochondral explants. Subsequent studies showed superior performance of the BMP2+PTH combination compared to each API administered singly, and that delivery of BMP2 prior to PTH resulted in the optimal anabolic activity, resulting in the final formulation shown in FIG. 16 (left, bottom).

[0457] Representative data from human OA osteochondral explants verifying the efficacy of the TAI APIs are shown in FIG. 13. Explants treated with BMP2 + PTH exhibited significant increases in bone regeneration compared to untreated controls. Bone regeneration was quantified as PSR+ (picrosirius red positive) bone perimeter / total bone perimeter for paired control and treated explants. PSR+ surface area is a direct measure of newly formed bone. Explants from patients from different ethnicities, ages, and sex were examined. Across all demographics, TAI -treated explants showed increased bone regeneration compared to untreated controls. When all patient data are pooled together, an average 30.5% increase was observed (FIGS. 29A-29D). Osteochondral explants from the hip and shoulder joints also demonstrated a regenerative response following treatment with TAI (FIGS. 30A-30B).

[0458] A major roadblock in drug delivery for cartilage regeneration and access to the underlying subchondral bone is the limited bioavailability and distribution of therapeutics. This challenge stems from the dense, negatively charged cartilage matrix and the rapid clearance of drugs from the joint space. Thus, disclosed herein is a nanocarrier system to deliver different drugs to the subchondral bone in a spatiotemporal manner. FDA-approved poly(lactic-co-glycolic acid) (PLGA) nanocarriers were customized by functionalizing them with positively charged branched poly-lysine (BPL) molecules designed to optimizeDUKE-45610.601 interaction with cartilage. To ensure bone binding and access of TAI therapeutics to the subchondral bone, the TAI nanocarriers were further functionalized with alendronate, which binds to the apatite minerals present in bone. While hydrophobic drugs can be encapsulated into PLGA nanocarriers, the unique properties of sugar glass micelles were utilized to stabilize protein APIs and protect them from denaturation during formulation, transport, and storage. To modulate temporal drug release, the PLGA nanocarriers were designed with varying lactate (LA) to glycolate (GA) ratios (eg for TAI, 50:50, and 75:25). The schematic in FIG. 31A summarizes fabrication of the cartilage penetrating NCs with subchondral bone binding (PLGA-BPL-Aln). The localization to cartilage and bone was determined by coupling a fluorescent dye (Cy7) to the TAI nanocarrier (FIG. 31B).

[0459] Efficacy of TAI -I A treatment in regeneration of bone and cartilage, and in pain mitigation. TAI APIs were encapsulated in nanocarriers for in vivo testing. For TA1-IA formulation, BMP2 was encapsulated into a PLGA-BPL-Aln 50:50 nanocarrier for a more rapid release, while PTH was encapsulated into a PLGA-BPL-Aln 75:25 nanocarrier for a slower and sustained release. A surgical model (MMNX surgery) was used to induce OA of varying severity in rat knees. Once OA of the appropriate severity was established in operated rats, the injured knee joints were treated by TAI- IA injection or left untreated as controls. The treated rats were maintained for at least 4 weeks post treatment. MicroCT was used to quantify changes to subchondral bone structure. OARSI scoring and SafO-quantification was used to quantify cartilage regeneration. TAI IA treatment leads to a significant bone and cartilage response (FIGS. 32A-32D). This regenerative result was observed in male and female rats, with mild, moderate, and severe OA.

[0460] Pain was assessed using the Von Frey assay on both the contralateral control paw and the paw of the limb associated with the surgical knee injury of control and TAI -treated rats. Following injury, these data show a rapid increase in pain (decline in the percent (paw) withdrawal threshold (PWT)) of rats with injured knees (MMNX surgery) (OA) as compared to their contralateral controls (CL). This initial rapid decline in PWT is then maintained in a relatively steady state in injured rats over the course of at least 26-wpi. This mirrors the early rapid progression of joint damage observed in the severe knee injury model that slows in its progression following 4-wpi. Importantly, these data also show that TA1-IA treatment (single IA injection) results in a time-dependent increase in the PWT orDUKE-45610.601 lessening of the pain response) beginning at 10-wpi and continuing through 26-wpi (FIG.

[0461] 24 and FIG. 25). A dose-response study including 0.5X, IX, and 5X doses confirmed that the IX dose leads to optimal bone regeneration as monitored by microCT and pain mitigation assessed by von Frey analysis (data not shown).

[0462] Biological activity of TAI loaded nanocarriers. The biological activity of each of the TAI APIs was assessed following their encapsulation in NCs and IA administration into OA knee joints using immunohistochemical (IHC) and immunofluorescent (IF) staining for signaling pathway targets that are impacted by each drug. pSMADl / 5 is a target of BMP signaling and JAG1 is a target of PTH / PTHR signaling. These agents are known to regulate osteoblast differentiation from progenitors, as well as osteoblast / osteocyte activity, which are critical to the regulation of bone mass and homeostasis. FIG. 33 shows that pSMADl / 5 is significantly upregulated in osteocytes of the OA rat knee subchondral bone at 4-wpi compared to controls. The TAI IA treatment restored the elevated pSMADl / 5 levels to those observed in controls (FIG. 33, top). A substantial decrease in JAG1 expression was also observed in cells lining the bone surface of the subchondral bone in OA rats compared to controls (FIG. 33, bottom). The TAI IA treatment restored the reduced levels of IAG1 to levels observed in contralateral controls. This reflects a PTH-mediated restoration of JAG1 expression and suppression of osteoblast differentiation from skeletal progenitors lining the subchondral bone surface. BMP2 has been shown to activate the PTH pathway, suggesting a model in which early exposure to BMP2 enhances expression of PTH receptors, sensitizing osteoprogenitors to the PTH administered in the TAI formulation, resulting in induction of IAG1 and restoration of bone homeostasis.

[0463] As verification that the effects of TAI are direct on subchondral bone, localization of the TAI component PTH was examined using an antibody that detects the human PTH protein but not the native rat protein. As shown in FIG. 34, the TAI nanocarriers deliver the APIs to subchondral bone.

[0464] TAI heals critical size defects. TAI is designed to promote regeneration of joints affected by all severity levels of osteoarthritis (OA), including critical sized defects. Traditionally, studies that introduce critical-size defects into the joint in animal models do so to determine the efficacy of cartilage repair products / implants and not true regeneration.DUKE-45610.601 Further, such osteochondral defects do not invariably progress to whole knee OA in either animal models or humans (e.g., Chihab et al. J Orthop Res. 2024, 42(11):2461-2472). To accurately represent this scale of a defect in an OA-relevant scenario, a surgically induced severe OA model in rats that produces full-thickness, non-healing defects within the articular cartilage in all (100%) of these animals was utilized. As these are non-healing defects in the rat model, they represent critical sized defects. Treatment with TAI agents in this model resulted in near-complete restoration of articular cartilage architecture, approaching baseline morphology. Critical sized defects / cartilage loss are no longer present in treated animals, whereas they persist in untreated severe OA animals. This demonstrates the capacity of TAI to repair a critical-size defect in a model directly relevant to osteoarthritis pathophysiology. FIG. 35 shows that critical sized defects are induced (left) following surgery for severe OA, and these defects are repaired following treatment with TAI IA (right). Severe OA surgery induces a ~1.5+mm wide full thickness, nonhealing critical sized defect by 4wpi (time of treatment) that progresses to ~3+mm full thickness by 8wpi with continual cartilage loss over time. TAI IA treatment reverses this process to regenerate articular cartilage and restore normal joint architecture.

[0465] In summary, TAI restores the subchondral bone architecture, regenerates cartilage, and results in pain mitigation in OA. The therapeutic effects persist beyond the rat equivalent of 1 human year.

[0466] Final TA2 Summary

[0467] FIG. 16 (bottom left) shows a schematic of release profiles of TA2 APIs. These APIs were selected based on their ability to induce an anabolic response in cartilage in human OA osteochondral explants. Studies in explants in which these APIs were added singly, or in pairwise combinations confirmed the efficacy of the triple combination, resulting in the final formulation shown in FIG. 16.

[0468] Human OA osteochondral explants treated with TA2 drugs showed significant increases in cartilage regeneration compared to untreated controls. The primary outcome measure was the ratio of SafO-i- cartilage area / total cartilage area in treated explants compared to their pairwise untreated control (FIGS. 36A-36D). Across all demographics, TA2-treated explants showed increased cartilage regeneration compared to untreatedDUKE-45610.601 controls. When all patient data was pooled together, an average of 44% increase was observed. Osteochondral explants from the hip and shoulder joints also demonstrated a regenerative response following treatment with TA2 (FIGS. 37A-37B).

[0469] Development and validation of cartilage penetrating nanocarriers for intraarticular drug delivery. Nanocarriers functionalized with BPL but not with Ain were used to deliver TA2 drugs to cartilage. Data documenting the ability of the PLGA-BPL nanocarriers to deliver the APIs to cartilage is summarized in FIG. 38. These and related studies demonstrate that varying the PLGA ratio alters the release profiles, showing the rapid release of API from PLGA 50:50, and more sustained release from the 65:35 and 75:25 formulations. IVIS studies and confocal analysis confirmed the retention of TA2 nanocarriers in the joint space and their uptake and distribution throughout the articular cartilage.

[0470] Efficacy of TA2-IA treatment for regeneration of cartilage and pain mitigation.

[0471] TA2 APIs were encapsulated in nanocarriers for in vivo testing. Roflumilast was encapsulated into a PLGA-BPL 50:50 nanocarrier for a more rapid release, PTH is encapsulated into a PLGA-BPL-65:35 nanocarrier for a slower and sustained release, and LDN-214117 was encapsulated into a 75:25 nanocarrier for delayed but sustained release.

[0472] The efficacy of the TA2 formulation was assessed by performing OARSI scoring and measuring SafO area / total cartilage area. Rats were treated the TA2 IA injection at 4-wpi or left untreated as OA controls. Tissues were harvested 8 to 12 wpi. TA2 IA treatment showed substantial regenerative activity (strong SafO+) in the joint cartilage as compared to OA and reached to levels similar to uninjured control knee joints. A representative experiment is shown in FIGS. 39A-39B. OARSI scoring showed that OA joints receiving TA2 IA treatment exhibited joint cartilage regeneration. These effects were similarly observed in male and female rats, with mild, moderate or severe OA.

[0473] Pain was assessed using the Von Frey assay as described above for TAI on both the contralateral control paw and the paw of the limb associated with the OA injury of control and TA2-IA treated rats. Treatment with TA2-IA (single IA injection) results in a time-dependent reduction in pain to baseline levels observed in contralateral control joints. (FIG. 28).DUKE-45610.601 In summary, therapeutic effects persisted to 42 days (the rat equivalent to 1 human year), and thus the expected dosing frequency is <l / year.

[0474] Biological activity of TA2 loaded nanocarriers. IHC / IF was used to assess the biological activity in cartilage of each of TA2 APIs following IA administration of TA2. CREB is a target of PI3K / AKT signaling stimulated by ROF (roflumilast). JAGGED 1 is a target of PTH / PTHR signaling. SMAD2 / 3 and SMAD 1 / 5 are targets of LDN214117 activity. FIG. 40 shows IHC / IF staining of articular cartilage tissue sections from MMNX-injured knees collected at 4 weeks post-injury (4 wpi), their contralateral knee controls, and MMNX-injured rats treated with TA2 via intra- articular injection at 4 wpi, with tissues collected at 8 wpi. The elevation in pCREB and JAG1 provides evidence that ROF and PTH are exerting effects on cAMP signaling consistent with cartilage regeneration. A significant upregulation of pSMADl / 5 in MMNX OA at 4-wpi was observed as compared to controls, and this active pSMADl / 5 signaling is significantly diminished at 8-wpi in TA2 treated MMNX OA rats. These data further demonstrate the effects of LDN on restoring the balance of TGFb superfamily signaling by not only enhancing pSMAD2 / 3 in cartilage but also reducing pSMADl / 5.

[0475] TA2 heals critical size defects. TA2 is designed to promote regeneration of joints affected by all severity levels of osteoarthritis (OA), including critical sized defects. Traditionally, studies that introduce critical-size defects into the joint in animal models do so to determine the efficacy of cartilage repair products / implants and not true regeneration. Further, such osteochondral defects do not invariably progress to whole knee OA in either animal models or humans (e.g., Chihab et al. J Orthop Res. 2024, 42(11):2461-2472). To accurately represent this scale of a defect in an OA-relevant scenario, a surgically induced severe OA model in rats was utilized, which produces full-thickness, non-healing defects within the articular cartilage in all of these animals. As these are non-healing defects in the rat model, they represent critical sized defects. Treatment with TA2 agents in this model resulted in near-complete restoration of articular cartilage architecture, approaching baseline morphology. Critical sized defects / cartilage loss are no longer present in treated animals, whereas they persist in untreated severe OA animals. This demonstrates the capacity of TA2 to repair a critical-size defect in a model directly relevant to osteoarthritis pathophysiology (FIG. 41).DUKE-45610.601 Combination of TAI and TA2

[0476] TAI and TA2 are compatible. A series of studies were performed to document that TAI and TA2 can be used in combination. Subchondral bone by was assessed by microCT, cartilage by OARSI scoring and SafO staining, and pain using the von Frey assay. No evidence of detrimental effects were seen comparing TA1+TA2 treatment to either treatment alone. Representative data for cartilage structure are shown in FIG. 42.

[0477] Example 3

[0478] Drug Loading and Release

[0479] Pharmaceutical compositions disclosed herein comprise a plurality of particles. For example, as discussed herein, in some embodiments, a pharmaceutical composition comprises a single pharmaceutically active compound. In some embodiments, a pharmaceutical composition comprises two or more pharmaceutically active compounds (e.g., PTH, BMP-2, roflumilast. and LDN-214117). Accordingly, discussed below are studies whereby the loading efficiency and time release profiles were optimized for pharmaceutical compositions disclosed herein. Critical optimization parameters included API weight / nanocarrier polymer precursor weight ratios, solvent used (PBS vs. water), and sonication frequency.

[0480] Hydrophobic drugs (e.g., LDN214117 and roflumilast) were directly incorporated into the nanocarriers by co-dissolving the APIs with the polymer precursors in an organic solvent. For protein drugs (e.g., PTH and rhBMP2), the properties of sugar glass micelles were leveraged to facilitate successful loading. Sugar glass micelles have been demonstrated to stabilize protein or peptide drugs and protect them from denaturation during loading, transport, and storage, thereby increasing their shelf-life. See, e.g., Giri et al. Adv. Mater. 2011, 23(42):4861-4867. The APIs were incorporated into the nanocarriers using the oil-in-water single emulsion method. The feed ratios for the TAI and TA2 APIs were varied to optimize loading efficiency while maintaining encapsulation efficiency. Each API was incorporated into its respective nanocarrier (detailed below). Loading capacities were also optimized by using PBS as the solvent instead of water. The drug-loaded nanocarriers were then combined to achieve the desired therapeutic formulation. For example, for the TAI therapeutic formulation, PTH-loaded PLGA-BPL-AlnDUKE-45610.601 nanoparticles are mixed with BMP- loaded PLGA-BPL-Aln nanocarriers at a ratio necessary to achieve the therapeutic dose. The optimal API ratios and sequence were determined in explant cultures and verified using in vivo studies. In addition to characterizing the total amount of each API incorporated in the nanocarriers, in vitro release studies were conducted to evaluate the release profile of the encapsulated drugs as described below.

[0481] LDN Loading and Release: The LDN loading efficiency was optimized initially using PLGA with an LA: GA ratio of 65:35. The LDN-loaded PLGA(65:35)-BPL nanocarrier was synthesized using a combination of polymer precursors, PLGA(65:35), PLGA(65:35)-PEG, and PLGA(65:35)-BPL, in a weight ratio of 40:30:30, respectively. These weight ratios were selected to ensure the fabrication of PLGA nanocarriers with minimal yet effective, chemical modifications for cartilage penetration and in vivo functionality. Briefly, PLGA(65:35) and PLGA(65:35)-PEG were dissolved in chloroform. PLGA(65:35)-BPL and LDN were dissolved in DMSO. The PLGA(65:35) and PLGA-PEG(65:35) solutions were mixed by stirring, followed by the addition of the PLGA(65:35)-BPL and LDN214117 mixtures. The resulting mixture of PLGA precursors and LDN214117 was then added to an aqueous solution (water or PBS) containing 0.4% (w / v) polyvinyl alcohol (PVA) and emulsified using a probe sonicator for 180 seconds at 4 °C with an output frequency of 15-18 kHz. The energy output of the sonication plays a key role in determining the size of the nanoparticles and was optimized through testing multiple frequencies to yield particles with an average hydrodynamic diameter of 160 ± 20 nm and a narrow size distribution as described.

[0482] Following emulsification, the mixture was stirred using a magnetic stirrer for 6 hours at room temperature and centrifuged at 3000 rpm for 10 minutes to remove large particles and aggregates. The supernatant was then collected and subjected to high-speed ultracentrifugation (100,000 x g) for 45 minutes. The resulting pellets containing the LDN-loaded PLGA nanocarriers were washed repeatedly with deionized water, re-suspended in deionized water, flash-frozen in liquid nitrogen, and lyophilized.

[0483] The amount of LDN214117 loaded in the nanocarriers was quantified by UV absorbance at 300 nm. In brief, the dried LDN-loaded nanocarriers were dissolved in DMSO and incubated at 37 °C for 2 hours to ensure complete nanocarrier dissolution andDUKE-45610.601 release of LDN214117, and absorbance was recorded. A standard calibration curve of LDN214117 prepared in DMSO was used to establish the drug concentration loaded into the nanocarriers. The effect of the feed ratio (drug-to-polymer) on loading efficiency was determined using this assay. Keeping the polymer weight constant, the LDN214117 content was varied from 5 to 40 wt%. As the LDN214117 content in the feed increased, the LDN entrapment in the nanocarrier also increased (FIG. 43).

[0484] The effect of modulating LDN solubility and / or protonation on loading efficiency was also examined. Consistent with this, using PBS instead of water as the aqueous phase increased the loading capacity by approximately two-fold (FIG. 44). This may be attributed to the higher degree of protonation of the secondary amine groups in LDN214117 in water compared to PBS, which can lead to greater diffusion into the aqueous phase. The higher salt concentration in PBS compared to water may also act to limit LDN214117 diffusion into the aqueous phase. Given that PBS limits the diffusion of drugs into the aqueous phase, and due to its isotonic properties, PBS was used as the aqueous phase for subsequent drug loading. The loading efficiency in PBS was reproduced independently with a sample size of n>3.

[0485] Next, LDN214117 was encapsulated into PLGA with a 75:25 LA-to-GA ratio using the optimized fabrication and loading conditions. PLGA 75:25 exhibited a loading capacity of 40 ± 5 pg / mg in PBS at a 20% LDN-to-PLGA feeding ratio. The release profile was determined from the respective nanocarrier over time (FIG. 45). For these in vitro drug release studies, a dialysis-based protocol was followed. Briefly, LDN214117-loaded PLGA (75:25) nanocarriers were dispersed in PBS at a concentration of 2 mg / ml, and 500 pl of the suspension was loaded into dialysis bags (MWCO = 12-14 kDa). The sealed bags were placed in a container with 4.5 ml of PBS at 37°C. At predetermined time intervals, 1 ml of the external medium was withdrawn for measurement and replaced with an equal volume of fresh PBS. The drug release profile was generated by quantifying the LDN214117 content in the collected medium over time using UV / Vis absorption spectroscopy at 300 nm. A standard calibration curve (0.39-25 pg / ml) was generated and used to estimate the concentration of LDN214117. As evident from FIG. 45, LDN-PLGA(75:25)-BPL exhibited a slow, sustained and continuous release profile, with approximately 50% of the drug released by day 84 (12 weeks) in vitro.DUKE-45610.601 Roflumilast loading and Release: Since roflumilast (ROF) is a hydrophobic drug, it was directly encapsulated into the nanocarrier similarly to LDN214117 using the oil-in-water single emulsion method. ROF loading was examined by varying the feed ratios of ROF to the PLGA(65:35) precursors (PLGA, PLGA-PEG, and PLGA-BPL). ROF-loaded nanocarriers were prepared as described earlier for LDN214117. Increasing the ROF content in the feed initially enhanced the loading capacity; and no significant difference was observed between 20 and 40 wt(%) ROF (FIG. 46). ROF loading in the nanocarriers was quantified using UV-Vis spectroscopy at a wavelength of 285 nm and converted to a drug concentration using a standard calibration curve as described above for LDN. These data were used to optimize ROF loading into PLGA (50:50)-BPL nanocarriers, leading to a loading capacity of 60 ± 5.8 pg / mg in PBS with a 20% ROF-to-PLGA feeding ratio. Similar to LDN, ROF release from the PLGA (50:50)-BPL nanocarriers was determined in vitro (FIG. 47). Roflumilast from PLGA (50:50) showed an initial rapid release followed by sustained release with over 80% of the drug released from the nanocarrier by day 35; measurements beyond 35 days were not reliable due to the presence of only trace amounts of the drug remaining in the nanocarriers. Since in vitro drug release in PBS is primarily driven by chemical potential and diffusion rather than by nanocarrier degradation, a complete (100%) release of the drug is not expected using this in vitro assay. The majority of the drug was released within 35 days, consistent with the use of PLGA (50:50), which is known to release cargo more rapidly, as well as the relatively low quantity of drug loaded within the nanocarriers.

[0486] PTH loading and Release: Sugar glass micelles (SGMs) were harnessed to stabilize the protein (PTH and BMP-2) prior to loading into the PLGA-BPL-Aln nanocarrier. To encapsulate the proteins into the sugar glass, the protein was suspended in a trehalose sugar solution, where the solvent was chosen based on the targeted protein. The micelles (i.e., reverse micelle) were formed with the help of the surfactant solution of dioctyl sulfosuccinate. The reverse micelles have a hydrophilic core which enables trapping and quenching of the protein molecules during freeze drying as schematically depicted in FIG. 8. An aqueous solution of PTH in trehalose was added dropwise to an isooctane solution containing dioctyl sulfosuccinate (AOT), while continuously vortexing for approximately 2 minutes until a clear suspension formed. The inverse micelles wereDUKE-45610.601 flash-frozen in liquid nitrogen and subsequently lyophilized. The dried micelles were washed multiple times with isooctane. The resulting SGMs loaded with PTH were resuspended in isooctane and stored at -20 °C until further use. The molar ratio of water to the AOT, ([H₂O] / [AOT]), was varied (5:1 to 15:1) to optimize the size and uniformity of sugar-glass micelles. The optimal ratio of 15:1 ([H₂O] / [AOT]) resulted in micelles with an average diameter of 7.5-13 nm with a low polydispersity index and was used in all subsequent sugar-glass micelle preparations. TEM imaging was used to determine potential aggregation of PTH loaded sugars glass, which shows no aggregation (FIG. 48). The TEM characterization confirmed that the glass micelles did not aggregate during freeze-drying, which could otherwise adversely affect loading efficiency and, in some cases, could even affect the protein function.

[0487] The drug loading efficiency was determined by disassembly of the micelles in PBS followed by ELISA assay. In brief, the micellar suspension was vortexed and kept on an orbital shaker for ~2 hrs at a speed of 200 rpm, which ensures complete disassembly of the micelles at or below 0.5 mg / mL and release of PTH into the solution. ELISA measurements were then performed on the solution to quantify the PTH. Bioactivity of the released PTH was determined using intracellular cAMP measurements in MC3T3-E1 osteoblasts. The encapsulated PTH in sugar glass micelles (PTH-SG) elicited similar cAMP production levels as free PTH, indicating the retention of bioactivity following encapsulation and release from the micelles (FIG. 49). PTH-SG nanostructures were encapsulated within PLGA-based nanocarriers — PLGA(75:25)-BPL-Aln for the TAI and PLGA (65:35)-BPL for the TA2 formulation. The drug loaded nanocarriers were prepared via the single emulsification method as described above. PLGA, PLGA-PEG, and PLGA-DBCO were dissolved in chloroform at a weight ratio of 40:30:30 as described above. PTH-SG was centrifuged from isooctane, resuspended in chloroform, and added to the nanocarrier precursor solution. This mixture was subsequently added to a phosphate buffer containing polyvinyl alcohol and emulsified using a probe sonicator as described above. Subsequent fabrication was carried out as described above.

[0488] Functionalization of the PTH-SG-loaded PLGA nanocarriers was performed by suspension in deionized water followed by reaction with terminal azide-functionalized branched poly-L-lysine (BPL) for 2 hours to introduce chemical modification. FollowingDUKE-45610.601 BPL modification, the PTH-SG-loaded nanocarriers were washed repeatedly with deionized water, flash-frozen in the presence of trehalose, lyophilized, and stored at -20 °C. During optimization, the amount of PTH-SG in the feed was varied to determine the loading efficiency. SG-encapsulated PTH within the PLGA-BPL nanocarriers was measured by ELISA following their dissolution into a DMSO / water mixture. As shown in FIG. 50, increasing the amount of PTH-SG in the feed initially enhanced the loading capacity, and loading was only incrementally improved upon increase in PTH-SG from 40% to 60% thus indicating near saturation in drug loading at 40 wt% of PTH-SG. Nanocarriers composed of PLGA (75:25) and PLGA (65:35) were subsequently optimized to achieve loading capacities 280 ± 120 ng / mg in PBS with a 40% feed ratio of IX PTH-SG to PLGA (where IX PTH-SG corresponds to a PTH loading of 2.2 ± 0.2 pg / mg of SG). A sample size of > 3, with technical duplicates, was used for all measurements. PTH release from the nanocarrier was assayed as described above, which showed a sustained release of PTH over the monitored time period (FIG. 51B). The PTH release measurements were stopped at 21 days, as the experiment was performed at 37 °C, which leads to a loss of PTH bioactivity over time in vitro. In the ELISA assays, the values decreased to minimal or non-detectable levels over time, as anticipated for inactive PTH molecules. While the PTH release could be measured for up to 21 days in vitro, the release profile suggested a gradual and sustained release of PTH from the nanocarrier. While the in vitro assays described above can only be performed over a limited timeframe, the sustained release of PTH in vivo was documented previously, by immunostaining with an antibody specific for biologically active rhPTH.

[0489] Bioactivity of the encapsulated PTH was examined by using intracellular cAMP measurements in cell culture involving MC3T3-E1 osteoblasts. The encapsulated PTH in sugar glass micelles (PTH-SG) elicited similar cAMP production as free PTH, indicating the retention of bioactivity following encapsulation in the micelles (FIG. 49). The protein incorporated sugar glass micelles were further loaded into the cartilage penetrating nanocarriers. The PTH content in the cartilage-penetrating nanocarriers was optimized to a range of 180-320 ng / mg based on the dosage required for the rat studies. Further optimizations were performed to improve loading, and the results demonstrate a significant increase in loading efficiency, reaching >960 ng / mg of the nanocarrier. The higher loadingDUKE-45610.601 is attributed to increased incorporation of PTH into the sugar-glass micelle. As shown in FIG. 52, the release profile of PTH from the nanocarrier in PBS at 37°C was a sustained, continuous release. Additional data for the release profile of PTH is provided in FIG. 5 IB. The data demonstrates cumulative -60% PTH release in 21 days.

[0490] BMP-2 Loading and Release: BMP-2 was incorporated into the nanocarriers similar to PTH as described above. After optimizing, a loading of 480 ± 20 ng / mg of the nanocarrier was chosen for the rat studies. Further optimizations have been made to improve loading and the results demonstrate a significantly improved loading reaching up to >4800 ng / mg of the nanocarrier. The higher loading was due to the higher incorporation of the BMP-2 into the sugar-glass micelle.

[0491] An aqueous solution of BMP2 in trehalose was added dropwise to an isooctane solution containing AOT. The resulting inverse micelles were flash-frozen in liquid nitrogen, lyophilized, washed multiple times with isooctane, resuspended in isooctane, and stored at -20 °C until further use. The BMP2 complexed SG micelles were encapsulated into PLGA nanoparticles via the single emulsification method as optimized above. Since near saturation was observed in loading of PTH at 40 wt% sugar glass micelles (FIG. 5 IB), the SG-BMP2 to precursor ratio was maintained at 40 wt% for BMP2 loading. The feed ratio of BMP2 to trehalose was varied and its effect on BMP2 loading capacity into SG was evaluated, which showed a BMP2-dependent increase in loading efficiency (FIG.

[0492] 53A). The SG-BMP2 loading within the nanocarriers was further optimized using SG-BMP2 with varying BMP2 content, while maintaining a constant 40 wt% ratio of BMP2-SGM to nanocarrier precursor. ELISA measurements showed increased BMP2 loading with BMP2-SG formulation containing higher BMP2 content (FIG. 53B). Together, these studies established a range of loading capacities that enable BMP2 loading at the desired therapeutic dose. The PLGA (50:50)-BPL-Aln nanocarrier was then optimized to achieve a loading capacity of 480 ± 80 ng / mg in PBS using a 40% feed ratio of IX BMP-2 SG to PLGA (where IX BMP-2 SG contains 3.3 ± 0.28 pg / mg of BMP-2. The BMP-2 release profile was also determined as described earlier using standard protocol. FIG. 51A shows the representative BMP2 release profile. Similar to PTH, the release measurements were stopped at 21 days, as the experiment was performed at 37 °C, leading to a loss of BMP2DUKE-45610.601 bioactivity over time. In the ELISA, the values decreased to minimal or non-detectable levels over time, as anticipated for inactive BMP2 proteins. Consistent with the release kinetics expected from PLGA (50:50), BMP-2 showed a rapid and sustained release with approximately 75% of the drug released by day 21.

[0493] Since all the encapsulated molecules, irrespective of their chemical structure, exhibited similar release kinetics — first-order or pseudo-first order (to be precise) — the long-term release profiles from various PLGA nanocarriers with different LA-to-GA ratios can be determined using any of the APIs under investigation as a model system. To this end, the long-term release profile was assessed using LDN as the model API (FIG. 54). The LA-to-GA ratio of PLGA can be used to regulate the release profile, with 50:50 formulations exhibiting a rapid, sustained release, while 75:25 formulations show a slower, sustained release over three months of duration. This is consistent with the hydrophobic nature of the LA moieties, as a higher LA content increases hydrophobicity resulting in reduced water absorption, slower hydrolysis, and consequently slower drug release. Since in vitro drug release in PBS is primarily driven by chemical potential and diffusion, a complete (100%) release of the drag is not expected using this in-vitro assay.

[0494] The release profile for BMP-2 is provided in FIG. 51 A. The data demonstrates cumulative -70% BMP2 release in 17 days.

[0495] Example 4

[0496] Preparation and Characterization of Nanocarrier Drug Substances Following identification of the final TAI and TA2 drug combinations described above, the pharmaceutically active compounds were incorporated into nanocarriers. In this and subsequent examples, “DPIl” refers to particles comprising the nanocarriers loaded with recombinant human BMP-2 (rhBMP-2), and “DPI2” refers to particles comprising the nanocarriers loaded with parathyroid hormone (in particular, teriparatide). Reference to “BUD-010” is the final drug product comprising a mixture of DPIl particles and DPI2 particles.

[0497] The nanocarriers drug substances were prepared via an oil-in-water emulsification method by adding an organic solution of PLGA, PLGA-PEG, PLGA-DBCO, PLGA-PEG-Aln, and an organic suspension of sugar glass micelles to an aqueous solution of polyvinylDUKE-45610.601 alcohol (PVA) dissolved in phosphate buffered saline (PBS), where PVA acts as an emulsifier. Nanocarriers were fabricated using PLGAs at LA: GA ratios of 75:25 and 50:50 for BUD-010. The drug load is described in Table 5.

[0498] Table 5. Drug Loading

[0499] Drug Nanocarrier

[0500] API PLGA Ratio Drug Load Concentration Weight

[0501] DPI1 rhBMP-2 50:50 233 pg 155-310 mg 0.75-1.5pg / mg DPI2 PTH 75:25 28 pg 93.5-187 mg 0.15-0.30 pg / mg

[0502]

[0503] PLGA, PLGA-PEG, PLGA-DBCO, and PLGA-PEG-Aln (all described above) were dissolved at a fixed ratio in chloroform. API-loaded sugar glass micelles prepared as discussed above were washed, resuspended in chloroform, and added to the polymer solution. PBS and PVA were combined. The polymer-micelle solution was added dropwise, sonicated, and held with constant stirring for 6 hours. The solvent was evaporated; nanoparticles are collected via centrifugation and washed in PBS. BPL in PBS was added and allowed to react with the nanoparticles for 2 hours. Nanoparticles are collected via centrifugation again and washed in deionized water repeatedly. Particles are resuspended in deionized water and trehalose, freeze dried and stored at -20°C.

[0504] Example 5

[0505] Supporting Data - Nanocarrier Fabrication and Characterization

[0506] The size and surface charge of the nanocarriers were characterized by dynamic light scattering (DLS) and zeta potential measurements, respectively. The size of the PLGA-based nanocarriers was measured by determining the hydrodynamic diameter of the carrier particles upon resuspending in water using a zeta-sizer instrument. Particles are suspended in deionized water with vortex and sonication to ensure separation. Three independent measurements are averaged. The surface charge of nanocarriers is measured by determining the zeta potential of the particles. Particles are suspended in deionized water with vortex and sonication to ensure separation. The suspension is transferred to aDUKE-45610.601 disposable folded capillary cell and placed in a zeta sizer instrument. Three independent measurements are averaged.

[0507] The average size of the nanocarrier is found to be ~ 180 ± 30 nm, and the surface charge to be ~ +40 ± 10 mV (Table 4). The positive zeta potential is due to the presence of the cationic BPL molecules.

[0508] Table 4. Particle Size and Surface Charge Characterization

[0509] Component Particle Size Surface Charge

[0510] DPI1 180 + 30 nm 40 ± 10 mV DPI2 180 ± 30 nm 40 ± 10 mV

[0511]

[0512] BUD-010 180 ± 30 nm 40 ± 10 mV

[0513] Example 6

[0514] Explant Assay

[0515] Human osteochondral plug explants were incubated with nanocarriers (NCs) with and without BPL for 48 hours to demonstrate penetration of NCs throughout the cartilage layer (FIG. 55, PLGA-BPL panel). Bone binding was shown through similar human cartilage explant incubation with fluorescently labeled nanocarriers (FIG. 55, PLGA-BPL-Aln panel).

[0516] Example 7

[0517] Cytotoxicity Assays

[0518] Cartilage (human OA) explants were exposed to nanocarriers (without API) (500 pg / mL) suspended in chondrocyte medium for 24 and 48 hours (FIGS. 56A-56B). Live / dead assay (FIG. 56A) was used to determine the toxicity of the nanocarrier, where green indicates live cells and red indicates dead cells. Cartilage explants in chondrocyte medium without the nanocarriers (control), with nanocarriers consisting of the polymers alone without Ain or BPL (NC) and with nanocarriers prepared with BPL (NC-BPL) were evaluated in comparison to nanocarriers functionalized with BPL and Ain (NC-BPL-Aln). The live / dead assay showed no cytotoxicity following exposure to any of the nanocarriers. Cell viability was also equivalent for all groups tested (FIG. 56B). Additional testing wasDUKE-45610.601 performed with DPI1 and DPI2 using chondrocyte cell culture, confirming no cellular cytotoxicity (FIG. 57).

[0519] Example 8

[0520] In Vivo Studies

[0521] a. Nanocarriers are retained within the joint following 1A administration Retention of nanocarriers following IA delivery was evaluated. PLGA was labeled with cyanine7 (Cy7), a dye similar to Cy5. Nanocarriers were formulated without API according to the methods described above. Nanocarriers were injected into the joint space of SKH hairless mice. Mice were imaged with an In Vivo Imaging System (IVIS), a high-sensitivity optical imaging platform capable of detecting fluorescent signaling in vivo, over time. IVIS imaging shows retention of the labeled nanocarriers without distribution anywhere else in the body (FIG. 58). Furthermore, this imaging allowed demonstration of nanocarrier degradation by reduced IVIS signal throughout day 50 (FIGS. 59-60). In addition to IVIS imaging, histology was performed on the injected knee joints with evaluation of the fluorescent signal. Cy5 signal is present but more diffuse by day 28 in the knee joints (FIG. 61). DAPI (4’,6-diamidino-2-phenylindole) staining indicates cells, Cy7 indicates fluorescent PEGA, with the merged stain images in the right panel. An additional study was conducted in rats, with the limitation of IVIS imaging to the knee joint in the larger animal model. Free Cy7 label was injected into healthy and OA rat knee joints as well as the PLGA-Cy7 nanocarriers (FIG. 62). IVIS imaging showed Cy7 free label cleared more rapidly than the labeled nanocarriers, confirming the attachment of the Cy7 label to the PLGA remains intact with the nanocarrier, confirmed with quantitative signal intensity measurements (FIG. 63). This study also confirms localization of the nanocarriers in the joint space with decreasing signal intensity over time demonstrating nanocarrier clearance likely due to degradation.

[0522] b. PTH Remains Within the Joint Following Intra-articular Delivery PTH is an endogenous molecule released by the thyroid gland into circulation to regulate calcium levels {see, e.g., Lourida etal., PLoS One 2015, 10(5):e0127574). PTH is not detectable in the joint space under normal circumstances (see, e.g., Sun et al. Elife 2021 10:e66532). BUD-010 was injected intra-articularly in the rat OA injury model described below. At 4 and 8 weeks after treatment, joint spaces were sectioned and stained for PTH.DUKE-45610.601 PTH was detected in the subchondral bone at both time points with diminished intensity at 8 weeks (FIG. 64). Similar analysis is not possible for rhBMP-2 due to natural protein presence within bone.

[0523] c. Post-Traumatic Osteoarthritis Rat Model

[0524] The post-traumatic rat model for osteoarthritis was chosen as the model is (1) highly published and regarded as a consistent model of OA disease progression in 4- to 12- weeks (mild to severe), (2) demonstrates both cartilage and subchondral bone damage over time, and (3) allows measurement of both pain and tissue impact of therapeutics. The model is a modification of the medial meniscus transection (MMNX) surgery on rats.

[0525] The MMNX surgery was performed on 12-week-old rats. Rats were anesthetized, hair was shaved from the right knee, and the skin was cleaned with 3 applications of alternating betadine and ethanol. An incision was made on the medial aspect of the right knee, and the joint space was opened slightly. A scalpel was used to transect the medial meniscus near the medial meniscotibial ligament (MMTL), thereby destabilizing the medial meniscus. Post-surgery, rats were rested on a heated pad, administered a slow-release analgesic (Buprenorphine slow release at 0.5-1.0mg / kg), and monitored until they recover sufficiently to ambulate and obtain food and water. At 4 weeks post-injury (wpi), the drug substances were administered, mimicking treatment of OA patients. Rats were then sacrificed at 8 weeks or 12 weeks post-injury for collection of injured and contralateral control joints.

[0526] d. Histopathological Scoring

[0527] Osteoarthritis Research Society International (OARSI) scoring was used as the histopathology grading system to measure the cartilage degeneration that occurs over the course of 8-weeks following MMNX surgery and values are compared to controls. Safranin-O / Fast Green-stained sections are also utilized to calculate the ratio of Safranin-O (SafO+) positive cartilage area / total cartilage area to assess the anabolic activity and quality of the articular cartilage. These sections were reviewed and scored by 3 independent, blinded reviewers.

[0528] e. MicroCT Analyses

[0529] The rat subchondral plate was assessed by MicroCT to evaluate joint-associated bone changes. MicroCT analyses were performed at 4- and 8-wpi using the followingDUKE-45610.601 measures for subchondral plate and / or subchondral bone: maximal subchondral plate thickness, bone fraction (bone volume / total volume; BV / TV), connectivity density (Conn. D.; 1 / mm3), structural model index (SMI), trabecular number (Tb. N.), trabecular separation (Tb. Sp.), trabecular thickness (Tb. Th.), and bone density (mg HA / cm3). Early studies have indicated that several of these parameters, and particularly Conn. D, are relatively sensitive microCT measures that can be used to detect MMNX associated bone and joint changes.

[0530] f. Pain Assessment

[0531] To assess pain in the rat MMNX model, withdrawal threshold due to a pain stimulus was conducted by using increasing weight von Frey (vF) filaments applied to each hind limb. The pain response is reported as the highest weight filament the animal can bear without a pain response.

[0532] g. Results

[0533] At 8-wpi, MMNX injury alone induced a significant increase in OARSI scores (mean 2.58) as compared to the contralateral control group (mean 0.52), while the BUD-010 (30.8 ng) group showed a significant decrease in OARSI scores (mean 1.33) compared to the MMNX alone group at either 4-wpi or 8-wpi (FIG. 65A). loint cartilage SafO staining has also shown a significant increase following BUD-010 treatment as compared to MMNX injured alone group, and it was restored to levels similar to uninjured contralateral controls (FIG. 65B). Collectively, these data are consistent BUD-010 eliciting a cartilage regenerative and anabolic response following joint injury.

[0534] Representative Safranin-O / Fast- green stained images show that a single BUD-010 treatment of MMNX OA animals at 4-wpi demonstrates an ability to regenerate articular cartilage to levels comparable with uninjured control animals (FIG. 66).

[0535] At 8-wpi, BUD-010 treatment resulted in a Connectivity Density (Conn. D.) measurement of the subchondral plate similar to that seen with contralateral controls (no MMNX) (FIG. 67). In FIG. 67, OA = MMNX, no OA = contralateral control, PTH+BMP-2 = BUD-010. At 4-wpi, the MMNX injured rats exhibited a significant increase in subchondral plate Conn. D. as compared to uninjured contralateral controls (p=0.01). The MMNX groups receiving intra-articular BUD-010 exhibited a significant decrease in subchondral plate Conn. D. at 8-wpi (p=0.03), as compared to MMNX injury alone at 4-DUKE-45610.601 wpi. Furthermore, the subchondral plate Conn. D. in MMNX groups receiving intraarticular BUD-010 was not significantly different at 8-wpi (p=0.64) as compared to MMNX injury alone at 4- wpi; consistent with a normalization in this microCT parameter following BUD-010 treatment.

[0536] Rats were administered BUD-010 5-wpi following MMNX injury and maintained for a total of 13 weeks to monitor for the therapeutic impact on pain. Rats with or without MMNX induced OA (•) were compared to OA knees administered BUD-010 (•) (FIG. 68). By 13-weeks post-injury, which is 8-weeks post BUD-010 administration, there was a statistically significant improvement in pain response for treated animals compared to OA control animals. Furthermore. BUD-010 treated animals showed pain responses within one standard deviation of control rats without injury.

Claims

DUKE-45610.601CLAIMS1. A particle comprising a polymeric nanocarrier, a branched poly-lysine compound covalently attached to a subset of polymer chains in the polymeric nanocarrier, and one or more pharmaceutically active compounds selected from the group consisting of parathyroid hormone (PTH), bone morphogenic protein 2 (BMP-2), ALK4-Fc, zoledronic acid, adenosine, and lithium, and pharmaceutically acceptable salts thereof, and any combination thereof.

2. The particle of claim 1, wherein the one or more pharmaceutically active compounds are selected from the group consisting of PTH and BMP-2, and a combination thereof.

3. The particle of claim 2, wherein the particle comprises PTH.

4. The particle of claim 3, wherein the PTH is teriparatide.

5. The particle of claim 2, wherein the particle comprises BMP-2.

6. The particle of claim 5, wherein the BMP-2 is recombinant human BMP-2.

7. The particle of claim 2, wherein the particle comprises PTH and BMP-2.

8. The particle of claim 7, wherein the PTH is teriparatide and the BMP-2 is recombinant human BMP-2.

9. A particle comprising a polymeric nanocarrier, a branched poly-lysine compound covalently attached to a subset of polymer chains in the polymeric nanocarrier, and one or more pharmaceutically active compounds selected from the group consisting of roflumilast, Growth and Differentiation Factor 11 (GDF11), LDN-214117, dickkopf-related protein 1 (DKK1), IWP-2, quercetin, kaempferol, binimetinib, salvianolic acid,DUKE-45610.601 transforming growth factor-p (TGF-β), parathyroid hormone (PTH), and resveratrol, and pharmaceutically acceptable salts thereof, and any combination thereof.

10. The particle of claim 9, wherein the one or more pharmaceutically active compounds are selected from the group consisting of roflumilast, PTH, and LDN-214117, and any combination thereof.

11. The particle of claim 10, wherein the particle comprises roflumilast.

12. The particle of claim 10, wherein the particle comprises PTH.

13. The particle of claim 12, wherein the PTH is teriparatide.

14. The particle of claim 10, wherein the particle comprises LDN-214117.

15. The particle of claim 10, wherein the particle comprises roflumilast, PTH, and LDN-214117.

16. The particle of claim 15, wherein the PTH is teriparatide.

17. The particle of any one of claims 1-16, wherein the branched poly-lysine compound comprises a branched poly(L-lysine) compound.

18. The particle of any one of claims 1-17, wherein the branched poly-lysine compound is covalently attached to the polymeric nanocarrier via a direct bond.

19. The particle of any one of claims 1-17, wherein the branched poly-lysine compound is covalently attached to the polymeric nanocarrier via a linker.

20. The particle of any one of claims 1-19, wherein the polymeric nanocarrier comprises a polyester.DUKE-45610.60121. The particle of claim 20, wherein the polyester is poly(lactic-co-glycolic) acid (PLGA).

22. The particle of claim 21, wherein the PLGA comprises about 70 mol% to about 80 mol% lactic acid and about 20 mol% to about 30 mol% glycolic acid.

23. The particle of claim 21, wherein the PLGA comprises about 60 mol% to about 70 mol% lactic acid and about 30 mol% to about 40 mol% glycolic acid.

24. The particle of claim 21, wherein the PLGA comprises about 45 mol% to about 55 mol% lactic acid and about 45 mol% to about 55 mol% glycolic acid.

25. The particle of any one of claims 1-24, wherein a second subset of polymer chains within the polymeric nanocarrier are functionalized with a polyalkylene oxide.

26. The particle of claim 25, wherein the polyalkylene oxide is polyethylene glycol).

27. The particle of any one of claims 1-26, wherein a third subset of polymer chains within the polymeric nanocarrier are functionalized with a polyalkylene oxide and a compound that binds to bone.

28. The particle of claim 27, wherein the polyalkylene oxide is polyethylene glycol) and the compound that binds to bone is alendronate.

29. The particle of any one of claims 1-28, wherein the one or more pharmaceutically active compounds is encapsulated within the polymeric nanocarrier.

30. The particle of any one of claims 1-29, wherein at least one pharmaceutically active compound is encapsulated in a sugar glass micelle, and the sugar glass micelle is encapsulated within the polymeric nanocarrier.DUKE-45610.60131. The particle of claim 30, wherein the sugar glass micelle comprises a sugar selected from trehalose and sucrose, and a surfactant selected from dioctyl sulfosuccinate, hexadecyltrimethylammonium bromide, lecithin, imidazolium-based surfactants, polyoxyethylene ethers, sodium di-(n-octyl)phosphinate, and 3,3-dimethyl-1-butysulfosuccinate sodium salt.

32. The particle of any one of claims 1-31, wherein the particle comprises the one or more pharmaceutically active compounds in an amount of about 0.01 pg / mg to about 5.0 pg / mg.

33. A pharmaceutical composition comprising a plurality of particles of any one of claims 1-32, and a pharmaceutically acceptable earner.

34. The pharmaceutical composition of claim 33, wherein each particle in the plurality of particles comprises a single pharmaceutically active compound.

35. The pharmaceutical composition of claim 34, wherein the single pharmaceutically active compound is BMP-2.

36. The pharmaceutical composition of claim 34, wherein the single pharmaceutically active compound is PTH.

37. The pharmaceutical composition of claim 34, wherein the single pharmaceutically active compound is roflumilast, or a pharmaceutically acceptable salt thereof.

38. The pharmaceutical composition of claim 34, wherein the single pharmaceutically active compound is LDN-214117, or a pharmaceutically acceptable salt thereof.

39. The pharmaceutical composition of claim 33, wherein each particle in the plurality of particles comprises two pharmaceutically active compounds.DUKE-45610.60140. The pharmaceutical composition of claim 39, wherein the two pharmaceutically active compounds are BMP-2 and PTH.

41. The pharmaceutical composition of claim 33, wherein each particle in the plurality of particles comprises three pharmaceutically active compounds.

42. The pharmaceutical composition of claim 41, wherein the three pharmaceutically active compounds are roflumilast, PTH, and LDN-214117, or pharmaceutically acceptable salts thereof.

43. The pharmaceutical composition of claim 33, wherein the plurality of particles comprises a first subset of particles comprising a first pharmaceutically active compound, and a second subset of particles comprising a second pharmaceutically active compound.

44. The pharmaceutical composition of claim 43, wherein the first pharmaceutically active compound is BMP-2, and the second pharmaceutically active compound is PTH.

45. The pharmaceutical composition of claim 33, wherein the plurality of particles comprises a first subset of particles comprising a first pharmaceutically active compound, a second subset of particles comprising a second pharmaceutically active compound, and a third subset of particles comprising a third pharmaceutically active compound.

46. The pharmaceutical composition of claim 45, wherein the first pharmaceutically active compound is roflumilast or a pharmaceutically acceptable salt thereof, the second pharmaceutically active compound is PTH, and the third pharmaceutically active compound is LDN-214117 or a pharmaceutically acceptable salt thereof.

47. A method of treating a joint disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 33-46.DUKE-45610.60148. A method of treating pain associated with a joint disease or joint tissue damage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 33-46.

49. A method of delivering a pharmaceutically active compound to cartilage and / or subchondral bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 33-46.

50. The method of claim 49, wherein the subject is suffering from a joint disease.

51. A method of treating an injury to a joint or cartilage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 33-46.

52. The method of any one of claims 47-51, wherein the joint disease comprises arthritis.

53. The method of any one of claims 47-51, wherein the joint disease comprises osteoarthritis.

54. The method of any one of claims 47-51, wherein the joint disease comprises post-traumatic osteoarthritis.

55. The method of any one of claims 47-51, wherein the joint disease comprises joint cartilage or bone damage.

56. The method of any one of claims 47-55, wherein the pharmaceutical composition is administered via intra-articular injection.DUKE-45610.601 57. A kit comprising the pharmaceutical composition of any one of claims 33-46.

58. The kit of claim 57, further comprising a syringe.

59. The kit of claim 57 or claim 58, further comprising instructions for using the pharmaceutical composition to treat a joint disease, to treat pain associated with a joint disease or joint tissue damage, to deliver a pharmaceutically active compound to cartilage and / or subchondral bone, or to treat an injury to a joint or cartilage in a subject.

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