WO2025049740A1 - Compositions comprising biomimetic proteoglycan constructs - Google Patents

Abstract

Provided herein are novel methods for site-specific delivery of active agents comprising the use of biomimetic proteoglycans. Biomimetic proteoglycan and active agent constructs (BPG constructs) effectively diffuse across cellular membranes enabling intracellular delivery of agents such as pharmaceutical agents. BPG constructs described herein may be engineered for prolonged release of active agents.

Description

COMPOSITIONS COMPRISING BIOMIMETIC PROTEOGLYCAN CONSTRUCTS

PRIORITY CLAIM AND CROSS-REFERENCE

[1] This application claims priority to US Patent Application No. 63/579,393 filed on August 29, 2023. The foregoing application, and all documents cited therein, together with any manufacturer’s instructions, descriptions, mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

TECHNICAL FIELD

[2] Embodiments of this disclosure relate generally to biomimetic proteoglycan constructs and uses thereof for site-specific delivery of active agents.

BACKGROUND OF THE INVENTION

[3] Proteoglycans, defined by a combination of a core protein and attached glycosaminoglycan chains, play a crucial role in proper tissue morphology and function throughout the body. Although they serve a variety of roles, the functions of extracellular proteoglycans can be generally sorted into four categories: modulation of tissue mechanical properties, regulation and protection of the extracellular matrix, sequestering of proteins, and regulation of cell signaling. The loss of proteoglycans can result in significant tissue dysfunction, ranging from poor mechanical properties to uncontrolled inflammation. Because of the key roles they play in proper tissue function and due to their complex synthesis, the past two decades have seen significant research into the development of proteoglycan mimetic molecules to recapitulate the function of proteoglycans for therapeutic and tissue engineering applications. Such strategies have ranged from semisynthetic graft copolymers to recombinant proteoglycan domains synthesized by genetically engineered cells.

[4] Previous discoveries by the inventors herein have enabled the creation of a biomimetic proteoglycan comprising a hybrid synthetic/bio-based macromolecular bottle brush structure specifically synthesized to incorporate chondroitin sulfate. Additional innovation around such proteoglycans includes the aspect of an enzymatically resistant molecular design that can advance the survival of the molecule in vivo, while maintaining molecular function. The resulting structures and their properties, enable, in theory, the creation of a family of tunable biomacromolecules for use in various applications of soft-tissue restoration.

[5] Within synovial joints lies articular cartilage (also called hyaline cartilage), a dense connective tissue covering long bones within diarthrodial joints and providing a low-friction surface during articulation and joint loading. This layer acts to support and distribute the forces from daily activities, such as walking and exercise, across the joint. Cartilage is an avascular, aneural tissue with only one cell type, the chondrocyte. Under normal use and physiological conditions, chondrocytes maintain their crucial environment of the extracellular matrix (ECM) through synthesis and maintenance of a myriad of molecular constituents. The main components of the ECM, collagens and proteoglycans, confer the functional properties of cartilage and therefore are crucial for joint health and use.

[6] Osteoarthritis (OA) is a progressive, degenerative disease that affects all structures within synovial joints with many possible initiation events (e.g. aging, genetic predisposition, trauma, and obesity). The disease is hallmarked by chondrocyte phenotypic shift, loss of proteoglycans leading to decreased biomechanics, and significant matrix remodeling leading to denuding of the cartilage from the bone. Throughout OA pathogenesis, a complex interplay between mechanical, inflammatory, and biochemical factors influence chondrocyte homeostasis causing increased production of inflammatory cytokines, chemokines, matrix degrading enzymes as well as cell surface receptors. Collectively, these factors affect both the extracellular as well as the pericellular matrix, not only damaging bulk cartilage mechanics but also interfering with proper mechanosensing of chondrocytes. Therefore, these changes lead to a vicious cycle of degradation within cartilage tissue and the synovial joint overall.

[7] Key cartilage degrading enzymes include metalloproteinases belonging to the matrix metalloproteinases (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (AD AMTS) families. MMP-1 and MMP-13 are collagenases while MMP-3 is a potent aggrecanse and MMP activator. Other main aggrecanases are ADAMTS-4, and ADAMTS-5 and these enzymes collectively work to destabilize the intricate network of proteoglycan and collagen matrices within both the ECM and PCM. As cartilage matrix constituents are degraded (e.g. SLRPs, aggrecan, and collagens) they can further stimulate production of inflammatory cytokines and chemokines through binding with certain receptors within the tissue, either initiating or amplifying OA disease progression. Cytokines, specifically IL-ip, have been shown to be essential mediators of acute inflammation after traumatic joint injury. Within 24 hrs after injury, concentrations of IL-ip can increase up to 70 times within synovial fluid which directly contributes to post-traumatic OA (PTOA) development due to aggravation of catabolic activities of chondrocytes. Mechanical integrity of PCM is compromised early after injury (< 3 days) with impairment of mechanosensing of chondrocytes, as measured by intracellular calcium signaling ([Ca2+]i), observed shortly after (< 7 days) loss of mechanical stiffness within the region. Loss of mechanotransduction due to compromised PCM integrity contributes to a shift in chondrocytes metabolism towards catabolic outputs thus perpetuating this cycle of degeneration.

[8] Despite decades of research and development into potential disease-modifying OA drugs (DM0 ADs), none have yet to be approved for use in humans partly due to inadequate drug delivery to target joint tissues. A key obstacle to successful drug intervention is poor pharmacokinetics as drugs are rapidly cleared (hours-days) from the joint cavity following intraarticular injection and many have difficulty penetrating the dense, avascular ECM to reach their intended targets (e.g. chondrocytes). Small molecule drugs like the glucocorticoid dexamethasone, and MMP -inhibitors, such as GM6001, are among those DM0 AD candidates that could greatly benefit from targeted delivery in cartilage to ameliorate systemic side effects and increase efficacy by extending cartilage residence time thereby reducing injection frequency and administration concentration. Designing drug delivery systems to increase pharmacokinetics could greatly improve clinical efficacy of potential DMOADs leading to breakthroughs in ameliorating cartilage destruction and altering OA disease trajectory.

[9] As drug delivery for OA therapy remains challenged by rapid joint clearance following intra-articular injection, researchers have developed systems to prolong joint residence time and provide sustained drug delivery. Systems such as hydrogels, micelles, polymeric particles have been gaining clinical success due to their larger size or viscous nature. Classical intra-articular injections of interventions such as corticosteroids or viscosupplements like hyaluronic acid (HA) can temporarily relieve pain and inflammation but fail to initiate any long-term diseasemodifying effects. Due to previous failures for effective drug delivery, cartilage penetrating drug delivery systems targeting intra-cartilage species (e.g. aggrecan, chondrocytes) are being actively investigated to hopefully enable clinical translation of true disease-modifying OA drugs including gene therapy. For these cartilage penetration systems, however, the carrier vehicle is not intended to have any matrix restorative effects and simply seek to increase the efficacy of the therapeutic delivered to the cartilage. Therefore, the delivered therapeutics must be able to halt, and hopefully, reverse the extensive matrix remodeling that occurs during OA. If OA degeneration cannot be reversed, or at the very least halted, total joint replacement (TJR) remains an aggressive operative option undertaken by 13.6% of OA patients. TJR has many complications for patients including, but not limited to, bleeding, infection, implant loosening, reoperation, and even death. Therefore, minimally invasive, non-operative treatment strategies remain a crucial gap in effective treatment for the millions of adults suffering from OA annually.

[10] What is needed are novel compositions and methods for site-specific delivery of active agents in order to obtain desired outcomes such as ameliorating cartilage destruction and altering OA disease trajectory. Ideally, such novel compositions and methods could also be utilized for site-specific delivery or a variety of active agents including therapeutic agents, pharmaceutical agents and cosmeceutical agents. Such compositions and methods should be customizable depending on the condition being treated and should also be customizable for prolonged/extended/sustained release. What is also needed are novel compositions and methods for active agent delivery wherein the effectiveness of the agents is maximized and negative side effects such as toxicity are minimized.

SUMMARY OF THE INVENTION

[11] In an embodiment, the present disclosure relates to novel constructs comprising biomimetic proteoglycans (BPG) and active agents.

[12] In an embodiment, the biomimetic proteoglycan comprises a glycosaminoglycan (GAG) that is attached to a core structure. The GAG may be selected from the group consisting of hyaluronic acid, chondroitin, chondroitin sulfate, heparin, heparin sulfate, dermatin, dermatin sulfate, laminin, keratan sulfate, chitin, chitosan, acetyl-glucosamine, oligosaccharides, and any combination thereof. In an embodiment, the core structure is selected from the group consisting of a synthetic polymer, a protein, a peptide, a nucleic acid, a carbohydrate and any combination thereof. In an embodiment, the core structure is a synthetic polymer selected from the group consisting poly(4-vinylphenyl boronic acid), poly (3,3 '-di ethoxypropyl methacylate), polyacrolein, poly (N -isopropyl acrylaminde-co-glycidyl methacrylate), poly(allyl glycidyl ether), poly(ethylene glycol), poly(aciylic acid), and any combination thereof.

[13] In an embodiment, provided herein are methods for intracellular delivery of an active agent, comprising the use of a BPG, wherein the BPG may be combined with an active agent creating a construct and wherein the construct effectively diffuses across a cellular membrane.

[14] In an embodiment, the BPG constructs of the invention may be used to address and alleviate problems associated with tissue mechanical properties, regulation and protection of the extracellular matrix, sequestering of proteins, and regulation of cell signaling as well as treating tissue dysfunction or uncontrolled inflammation.

[15] In an embodiment, the disease, disorder, or condition treated using the constructs of the invention comprises osteoarthritis and the composition is administered to the mammal by an approach to the diarthrodial joints selected from group consisting of injection, athroscopic implantation, and open implantation.

[16] In an embodiment, the constructs of the invention are utilized to effectively deliver active agents, such as cosmeceuticals, to dermatological cells.

BRIEF DESCRIPTION OF FIGURES

[17] The present disclosure is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not necessarily to scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Like reference numerals denote like features throughout specification and drawings.

[18] Figure 1 is confocal imaging of BPGs (white) inside live chondrocytes (gray). BPGCy5.5 was dosed at 2.5 mg/mL for 24 hours prior to imaging. BPGCy5.5 is shown inside and is retained within the cell. White arrows highlight two of the many cells BPG has entered. [19] Figure 2 provides flow cytometry quantifying entry of fluorescently-tagged BPGs into cells by tracking the fluorescence intensity of the fluorescent marker, Cy5.5, in cells that have passed by the detector.

DETAILED DESCRIPTION

[20] The following detailed description is exemplary and explanatory and is intended to provide further explanation of the present disclosure described herein. Other advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the present disclosure. Texts and references mentioned herein are incorporated in their entirety, including US Patent Application No. 63/579,393, filed on August 29, 2023 and United States Patent Application Publication No. 20190070216A1, filed November 1, 2018 and 20240117158A1, filed September 12, 2023.

[21] For purposes of the description hereinafter, it is to be understood that the embodiments described below may assume alternative variations and embodiments. It is also to be understood that the specific articles, compositions, and/or processes described herein are exemplary and should not be considered as limiting.

[22] Abbreviations a) Pericellular matrix: PCM b) Extracellular matrix: ECM c) Territorial-extracellular matrix: T-ECM d) Interterritorial extracellular matrix: IT-ECM e) Osteoarthritis: OA f) Biomimetic proteoglycan: BPG g) Dexamethasone: Dex or D h) Tritylglycine: TG i) Glycine: Gly or G j) Gly cine-Dexamethasone: Gly-Dex or GD k) BPGIO-Gly cine-Dexamethasone: BPGIO-Gly-Dex or BGD l) Matrix metalloproteinases: MMP m) Glycosaminoglycans: GAG n) Sulfated glycosaminoglycan: sGAG

[23] In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “an antibody” or “an antibody fragment” is a reference to one or more of such structures and equivalents thereof known to those skilled in the art, and so forth. When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. As used herein, “about X” (where X is a numerical value) preferably refers to ±10% of the recited value, inclusive. For example, the phrase “about 8” preferably refers to a value of 7.2 to 8.8, inclusive; as another example, the phrase “about 8%” preferably (but not always) refers to a value of 7.2% to 8.8%, inclusive. Where present, all ranges are inclusive and combinable. For example, when a range of “1 to 5” is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, “2-5”, and the like. In addition, when a list of alternatives is positively provided, such listing can be interpreted to mean that any of the alternatives may be excluded, e.g., by a negative limitation in the claims. For example, when a range of “1 to 5” is recited, the recited range may be construed as including situations whereby any of 1, 2, 3, 4, or 5 are negatively excluded; thus, a recitation of “1 to 5” may be construed as “1 and 3-5, but not 2”, or simply “wherein 2 is not included.” It is intended that any component, element, attribute, or step that is positively recited herein may be explicitly excluded in the claims, whether such components, elements, attributes, or steps are listed as alternatives or whether they are recited in isolation.

[24] The abbreviations or acronyms used in the present disclosure have corresponding meanings as those skilled in the art understand. [25] As used herein, the terms “patient,” “subject,” “individual” and the like are used interchangeably, and refer to any animal, or organs, tissues or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain embodiments, the patient, subject or individual is a vertebrate. In other embodiments, the patient, subject or individual is a mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, equine and murine mammals. In yet other embodiments, the patient, subject or individual is a human.

[26] As used herein, the term “binding” typically refers to a non-covalent association between or among two or more entities, unless expressed indicated otherwise, for example, referring to covalent bonding. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts — including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell).

[27] As used herein, “linker” refers to a component that attaches an active agent to a biomimetic proteoglycan. Additional linkers may include, but are not limited to, succinic anhydride, maleic anhydride, maleamide, alkyl polymer chains (of varying links thereby enabling modification of distances between the BPG and active agent in order to address aspects such as steric and cleavage kinetic control), non-degradable linkers (so that chelating drugs (such as MMP inhibitors or antibodies) may be retained longer in the tissue as the BPGs have long residence time in matrix), hydroxyl PEG amines of varying size to selectively control for nanoparticle formation, linkers having properties such that they can be cross-linked (for example under UV with themselves for either a degradable or non-degradation gel that could be intra- articulary injected then released over an extended period enabling prolonged deliver of both the BPG and active agent into the site, such as cartilage), peptide sequences as linkers (altering amino acid sequence for controlling cleavage rate by introduction of hydrophobicity or hydrophilicity, net charges or zwitterionic nature or other physiological conditions), mRNA (or ASOs) that would be cleavable from both an active agent as well as BPGs (thereby making the construct (or delivery system) functional, including the linker) multi-valent linkers (enabling the addition of more than one active agent, per BPG molecule). Though not wishing to be bound by the following theory, active agents may be conjugated to BPG molecules utilizing methods known to those skilled in the art, including but not limited to carbodiimide chemistry, solidphase synthesis, reversible addition fragmentation-chain transfer (RAFT) polymerization, click reactions (CU(I)-catalyzed azide-alkyne cycloaddition, Cu(II)-catalyzed [3+2] azide-alkyne cycloaddition, and Diels Alder cycloaddition), and 2-iminothiolane chemistry.

[28] Synthetic constructs of native proteoglycans that are able to resist enzymatic destruction while also imparting critical mechanical, biochemical, and structural outcomes during OA progression have been widely sought after.

[29] The inventors herein utilized a terminal-end functionalized natural CS bristles and a grafting-to approach on polymer backbones, to synthesize a suite of enzymatically resistant biomimetic proteoglycans (BPGs) that closely mimic the structural and functional characteristics of native proteoglycans. Mimicking small, leucine-rich proteoglycans (SLRPs), such as biglycan, BPG0.5 was synthesized from either polyethylene glycol)-diglycidyl ether (PEG-DEG) or ethylene glycol-diglycidyl ether (ED-DGE) momoner backbones and end-on attachment of CS bristles. The structures were found to incorporate -2 CS side-chains per backbone and exhibited cytocompatibility equivalent to or better than CS-only controls. The next size up in the BPG family is BPGIO, a -180 kDa molecule comprised of 7-8 CS bristles attached to a 10 kDa poly(acrylic acid) (PAA) core. AFM imaging suggests a 3D brush-like macroarchitecture and bristle density calculations indicate that these molecules have bristle-to-bristle spacing comparable to native aggrecan (-3-4 nm for BPG10 vs. -2-3 nm for aggrecan). Water uptake experiments indicated BPG10 had a -50% increased water uptake over native aggrecan or CS alone and cytocompatibility was demonstrated for a physiological range of concentrations. The largest member of the BPG suite is BPG250, a -1.6 MDa molecule synthesized from PAA and natural CS side-chains. Chemical, physical, and structural analysis confirmed the 3D bottle-brush architecture, superior water uptake to that of natural aggrecan, and general cytocompatibility for a range of dosages. When fluorescently labeled BPG250 was injected into the NP space of a lumbar bovine intervertebral disc, the molecule distributed throughout the NP and was retained following short-term static loading.

[30] In pursuit of understanding the diffusion capability of these molecules through the cartilage matrix, fluorescently labeled CS, BPG10, and BPG250 were diffused through normal bovine and human OA cartilage explants. All molecules displayed diffusion rates dependent on their size and starting concentration. Most interestingly, the BPGs were found localized around chondrocytes in both bovine and human tissue in the study. In ex vivo bovine cartilage, BPG10 was localized to the pericellular matrix (PCM), as indicated by collagen VI staining of the PCM. As BPGs have been shown to permeate throughout all zones of the cartilage ECM, interactions of BPGs with collagen, a leading component of ECM was investigated. BPG0.5, BPG10, and BPG250 had an effect on type I collagen fibrillogenesis in vitro and further elucidation of this phenomenon demonstrated that BPGs aided in lateral growth and enhanced fibril banding periodicity of collagen I. Surprisingly, BPGs were found attached to the collagen fibers despite the lack of protein core; a process likely mediated by the CS side-chains.

[31] In certain embodiments, BPG comprises a core structure and at least one glycosaminoglycan (GAG), wherein the core structure and the GAG are covalently linked. In other embodiments, the GAG is at least one selected from the group consisting of hyaluronic acid, chondroitin, chondroitin sulfate, heparin, heparin sulfate, dermatan sulfate, laminin, keratin sulfate, chitin, chitosan, acetyl-glucosamine, and oligosaccharides. In other embodiments, the core structure is at least one selected from the group consisting of a synthetic polymer, a protein, a peptide, a nucleic acid, and a carbohydrate. In yet other embodiments, the synthetic polymer is at least one selected from the group consisting poly(4-vinylphenyl boronic acid), poly(3,3'- di ethoxy propyl methacylate), polyacrolein, poly(N-isopropyl acrylamide-co-glycidyl methacrylate), poly(allyl glycidyl ether), poly(ethylene glycol), poly(acrylic acid), poly(acryloyl chloride) and epoxides. In yet other embodiments, the core structure comprises polyacrylic acid (PAA). In certain embodiments, the biomimetic proteoglycan comprises chondroitin sulfate and PAA, wherein the chondroitin sulfate is covalently linked to the PAA. In certain embodiments, the biomimetic proteoglycan is resistant to the breakdown of an endogenous enzyme. In other embodiments, the endogenous enzyme is at least one selected from the group consisting of hyaluronidases, aggrecanases and matrix metalloproteinases (MMPs). In certain embodiments, the biomimetic proteoglycan is susceptible to the breakdown of an exogenous enzyme. In other embodiments, the exogenous enzyme is chondroitinase ABC (ChABC).

[32] Provided herein are constructs comprising biomimetic proteoglycans (BPG) and active agents. As used herein, active agents comprise agents that have a desired effect on target tissues or organs in living organisms including animals. Such active agents may include for example, therapeutic, pharmaceutical, cosmeccutical, or other effective agents. The constructs of the invention, are unique in that they enable site-specific intracellular delivery of active agents. Though not wishing to be bound by the following theory, it is thought that the BPGs of the invention can diffuse easily across cell membranes.

[33] As noted above, biomimetic proteoglycans comprise a structure consisting of a core and bristles, wherein the core comprises a polymer backbone, including but not limited to polyethylene glycol or polyacrylic acid, wherein the size of the polymer backbone can be modified, wherein the bristles comprise chrondroitin sulfate, dermatan sulfate, heparin sulfate, keratin sulfate or Small Leucine-Rich Proteoglycans (SLRP) mimics, and wherein the bristle density can be modified. BPG may comprise BPGIO, BPG250, BPG0.5 or Small Leucine-Rich Proteoglycans (SLRP) mimics.

[34] As used herein, active or therapeutic agents, include, but are not limited to: pharmaceutical agents, immunomodulatory agents, cytotoxic agents, enzymes, proteins, antimicrobial agents, interferons, vitamins, nucleic acids, steroids, aminoglycosides, antioxidants, stem cells, anesthetic agents, analgesics, anti-inflammatories or hormonal agents, disease modifying osteoarthritic drugs (DMOAD), tumor necrosis factor, endostatin, angiostatin, thalidomide, taxol, melphalan, paclitaxel, vinblastin, vincristine, doxorubicin, acyclovir, cisplatin, tacrine, 5 -fluorouracil, mitaxantrone, VM-16, etoposide, VM-26, teniposide, or taxanes.

[35] As used herein, disease modifying osteoarthritic drugs (DMOAD) include but are not limited to, bone active drugs glucocorticoids (including, but not limited to, dexamethasone, cortisol, cortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamicinolone, deflazacort, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, beclometasone) strontium ranelate, Zoledronic acid, Risedronate, anticytokine therapy, antibodies, statins (atorvastatin, pravastatin, fluvastatin, Tanezumab, nerve growth factor antibody, AMG 108, interleukin-1 (IL-1 ) receptor type 1 antibody, adalimumab, tumor necrosis factor alpha antibody, bispecific antibodies, Lutikizumab, bispecific antibody for IL- la and IL- 1(3), Canakinumab, receptor antagonists, Anakinra, a IL-1 receptor antagonist)), enzyme inhibitors (including but not limted to, nanobodies, M6495 (Anti-ADAMTS5), small molecules inhibitors, doxycycline, Cindunistat (SD-6010)), growth factors (including, but not limited to, transforming growth factor beta family, bone morphogenetic protein 7, fibroblast growth factor (FGF) family, FGF-18 (sprifermin), nucleic acids (gene therapy), antisense oligonucleotides, peptides, Calcitonin, SM04690 (a Wnt inhibitor), UBX101 (Senolitic), Transient receptor potential vanilloid 4, Neural EGFL-like 1, TPCA-1 (a KB kinase inhibitor) and tofacitinib (a Janus kinase inhibitor), Lorecivivint, and Quercitrin.

[36] The constructs of the invention are linked via a linker that is suitable for the environment wherein the construct is to have its intended effect. The linker is selected for various qualities, such as for enabling extended release of the therapeutic agent. For example, the linker may comprise a glycine linker and wherein the glycine linker is pH sensitive in the range of inflammatory cartilage. In certain embodiments, for example, the BPG consists of BPGIO and the DMO AD consists of dexamethasone wherein the BPGIO and DM0 AD: wherein the BPGIO-DMOAD construct is comprised of ester-linked glycine-dexamethasone (GD) which is conjugated to BPGIO via an amide bond (referred to as BPGIO-Gly-Dex or BGD). In certain embodiments, the linker may comprise matrix metalloproteinase (MMP) sensitive linkers.

[37] In certain embodiments, the constructs of the invention provide long term release of the DMOAD (or other active agent) including for example, 1-7 days, 1-20 weeks, 1-12 weeks, 1-10 weeks, 1-8 weeks, 1-6 weeks, 1-4 weeks and/or 1-2 weeks, including intervals therebetween.

[38] The constructs of the invention useful for the treatment of arthritis including osteoarthritis, display minimal drug side effects wherein the side effects include, but are not limited to, bone resorption, systemic organ toxicity, hypertension. Furthermore, advantages of utilizing the present constructs for treating osteoarthritis include, but are not limited to, decreased drug administration frequency, extended release in situ, increased drug efficacy, decreased cartilage degeneration, and decreased joint inflammation. Additional, the constructs have anti- catabolic or pro-anabolic chondrocyte metabolism, inhibit MMP activity, inhibit aggrecanase activity and/or other catabolic enzymatic activity.

[39] The disease modifying therapeutic effects of the constructs may comprise mechanical and mechanobiological support for the extracelluar matrix (ECM) of cartilage, the pericellular matrix (PCM) of cartilage, rescue of chondrocyte catabolism, and/or arrested cartilage degeneration. The constructs may also modify the PCM to modulate mechanobiological pathways.

[40] As noted, in certain embodiments, constructs of the invention may be administered to a subject having arthritis, including osteoarthritis. The construct may be administered directly into intra-articular joint space and may diffuse directly into the cartilage, having a site-specific disease modifying therapeutic effect. The intra-articular joint space may comprise joint space in the hip, knee, ankle, spine, neck, shoulder, elbow, and/or wrist. As used herein, the term “joint” comprises “any place where two or more bones meet” and includes, but is not limited to fibrous joints, cartilaginous joints, synovial joints, hinge joints, condyloid joints, saddle joints, planar joints and pivot joints. Included herein are metacarpo-phalangeal joints, proximal interphalangeal joints of the hands, interp halangeal joints of the thumbs, interphalangeal joints of the feet, temporomandibular joints (TMJ) and intervertebral discs.

[41] In certain embodiments, the constructs of the invention may be used to deliver cosmeceutical agents for purposes of improving dermatological conditions. Such conditions may include improving skin appearance, repairing dermatological issues caused by factors such scarring, burns or sun damage, and for providing protection. In certain embodiments, the BPG constructs of the invention may be conjugated or otherwise linked with select agents that provide positive effects in aspects of skin care such as moisturizing, sunscreen, spot removal, hyperpigmentation, antiaging or other pigmentation enhancements. As used herein, the term “cosmeceutical” includes agents, substances, pharmaceuticals useful for the enhancement of the health and beauty of skin. Representative cosmeceuticals include, but are not limited to vitamins (Vitamins A or retinoids tretinoin, adapalene, tazarotene, retinaldehyde, retinol, and retinyl esters; Vitamins B (B3 nicotinamide or niacinamide), C (L-ascorbic acid), D, and E(alpha- tocopherol)), hydroxyacids (a-hydroxyacids, [3-hydroxyacids, polyhydroxyacids, a-hydroxyacid glycolic acid, and bionic acids, peptides (including peptide fragments of collagen and elastin, pal-KTTKS (Matrixyl), Ac-EEMQRR (Argireline), and Cu-GHK), growth factors, botanicals, selenium, lycopene, pycnogenol zinc and copper. The constructs of the invention may be used to effectively deliver therapeutic and/or cosmeceutical agents to the cells of the epidermis (including keratinocytes, melanocytes, and Langerhans and Merkel cells), dermis (fibroblasts) or subcutitis. [42] In certain embodiments, the constructs of the invention may be used to deliver therapeutic agents to osteoblasts, stem cells, fibroblasts, cancer cells, dermatological cells, retina cells and other cells as would be known to those skilled in the art that would serve as appropriate targets for receiving active agents.

[43] In an embodiment, provided herein are methods for intracellular drug delivery, comprising: site-specific and targeted delivery of biomimetic proteoglycans (BPG). In certain embodiments, the BPG may be combined with a therapeutic agent creating a construct, and the construct effectively and easily diffuses across a cellular membrane. In certain embodiments, the construct may be comprised of an ester-linked glycine-drug (or active agent) which is conjugated to BPG via an amide bond.

[44] In an embodiment, BPG comprises BPGIO, BPG250, BPG0.5 or Small Leucine-Rich Proteoglycans (SLRP) mimics. The drug or active agent comprises a therapeutic agent such as a pharmaceutical agent, or disease modifying osteoarthritic drugs (DMOAD). In certain embodiments, the construct comprises a BPGlO-dexamethasone construct, comprised of ester- linked glycine-dexamethasone (GD) conjugated to BPGIO via an amide bond (BGD). In an embodiment, the construct is delivered to a subject via injection into intra-articular joint space and the construct diffuses into cartilage and into chondrocytes. Such targeted delivery results in the decrease of symptoms of osteoarthritis, wherein the symptoms of osteoarthritis comprise pain, stiffness, tenderness, loss of flexibility, swelling, grating sensation, or bone spurs.

[45] The drug from the constructs described herein may be released over an extended period of time, for example, 1-7 days, 1-20 weeks, 1-12 weeks, 1-10 weeks, 1-8 weeks, 1-6 weeks, 1-4 weeks and/or 1-2 weeks, including intervals therebetween. The drug constructs may be specifically engineered to enable specific rates of release depending on the condition to be treated and other such factors.

[46] The drug construct may be administered, for example, in a dose of BPG10 at lOmg/mL, dexamethasone at 25mM and BGD at lOmg/mL. Delivery of the construct may result in mechanobiological support of PCM, and arrest cartilage degeneration. The constructs described herein may be administered as single or multiple doses and the doses may be administered as needed, for example, they may be administered twice daily, daily, every other day, every third day, three times per week, twice per week, weekly, biweekly, monthly, or bimonthly. The dose amount may be determined based on factors known to those skilled in the art (such as clinical factors, weight, pharmacokinetic profiles and conditions to be treated and may vary from about 1 pg/mL to about 10 iig/mL, about 10 pg/mL to about 50 pg/mL, about 50 pg/mL to about 150 pg/mL, about 150 pg/mL to about 250 pg/mL, about 250 pg/mL to about 500 pg/mL, about 500 pg/mL to about 750 pg/mL, about 750 pg/mL to about 1 mg/mL, about 1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 100 mg/mL, about 50 mg/mL to about 100 mg/mL.

[47] In certain embodiments, the constructs comprise BPG and an ester-linked glycinetherapeutic agent conjugated to BPG via an amide bond, wherein delivery of the construct results in extended release of the therapeutic agent and wherein the construct diffuses across a cellular membrane enabling intracellular drug delivery. Furthermore, administration of the construct results in site-specific and intracellular targeted delivery of dexamethasone to chondrocytes. The effective delivery of dexamethasone to chondrocytes enables superior disease modifying therapeutic effects including mechanical and mechanobiological support for the extracelluar matrix (ECM) of cartilage, the pericellular matrix (PCM) of cartilage, rescue of chondrocyte catabolism, arrested cartilage degeneration and/or arrested inflammatory response.

[48] In an embodiment, the constructs described above may be administered directly into intra-articular joint space and may diffuse into the cartilage, wherein administration of the construct results in a disease modifying therapeutic effect. The intra-articular joint space may comprise the joint space in the hip, knee, ankle, shoulder, elbow, or wrist. The construct may provide long term release of the DMOAD 1-7 days, 1-20 weeks, 1-12 weeks, 1-10 weeks, 1-8 weeks, 1 -6 weeks, 1-4 weeks and/or 1 -2 weeks, and the disease modifying therapeutic effect comprises mechanical and mechanobiological support for the extracelluar matrix (ECM) of cartilage, the pericellular matrix (PCM) of cartilage, rescue of chondrocyte catabolism, and/or arrested cartilage degeneration. In an embodiment, administration of the construct results in minimal drug side effects wherein the side effects include, but are not limited to, bone resorption, systemic organ toxicity, hypertension. Advantages of utilizing the constructs described herein for treating osteoarthritis include, but are not limited to, decreased drug administration frequency, extended release in situ, increased drug efficacy, decreased cartilage degeneration and/or decreased joint inflammation. The constructs have anti-catabolic and pro-anabolic chondrocyte metabolism, inhibit MMP activity, inhibis aggrecanase activity and/or other catabolic enzymatic activity. Furthermore, the constructs modify the PCM to modulate mechanobiological pathways.

[49] In certain embodiments, the administration of the novel BPG constructs of the invention improves the conditions of a subject suffering from at least one disease selected from the group consisting of; osteoarthritis, cancer, pain, chronic pain, neuropathic pain, postoperative pain, sports injuries, erosive arthritis, rheumatoid arthritis, psoriatic arthritis, Lyme arthritis, juvenile arthritis, ankylosing spondylosis, neuralgia, neuropathies, algesia, nerve injury, ischaemia, neurodegeneration, inflammatory diseases, cartilage degeneration, diseases affecting the larynx, trachea, auditory canal, intervertebral discs, ligaments, tendons, joint capsules or bone development, intervertebral disc degeneration, osteopenia, or periodontal diseases, acute joint injury, and/or a disease related to joint destruction.

[50] The novel BPG constructs described herein may be administered by standard routes. In general, the compositions and formulations may be administered by the topical, intraarticular, transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular, epidural) ophthalmic (including intravitreal or intracameral), nasal, topical (including buccal and sublingual), administration, route. In certain embodiments, osmotic minipumps may also be used to provide controlled delivery of therapeutic agents through cannulae to the site of interest. The biodegradable polymers and their use are described, for example, in detail in Brem et al., J. Neurosurg. 74:441-446 (1991), which is hereby incorporated by reference in its entirety.

[51] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. [52] Suitable dosages of the compositions and formulations disclosed herein will depend on the level of pain, condition or disease state being treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound. It is to be understood that the invention has application for both human and veterinary use. The methods of the invention contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time.

[53] Preferred unit dosage formulations are those containing a daily dose or unit, daily subdose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present invention may include other agents conventional in the art having regard to the type of formulation in question.

[54] The following examples are given to illustrate exemplary embodiments of the present disclosure. It should be understood, however, that the present disclosure is not to be limited to the specific conditions or details described in these examples. Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention.

EXAMPLES

Example 1

Cellular Uptake of Biomimetic Proteoglycan-Dexamethasone

[55] BPGs were surprisingly found to enter chondrocytes, the resident cell type of articular cartilage. This is a non-obvious and useful attribute of the constructs described and claimed herein. By themselves, small molecule drugs and steroids (<1 kDa), like dexamethasone, can easily diffuse across the cell membrane. Considering the molecular weight and size of dexamethasone is comparatively small to BPG (BPG —180 kDa, dexamethasone ~0.4 kDa), and the addition of dexamethasone to BPG adding very little bulk to BPG, BPG-Dex will enter chondrocytes in a similar manner to BPG alone. Therefore, as demonstrated herein, in at least one embodiment, BPG-Dex can be used as an intracellular drug delivery vehicle. [56] Prior research indicated BPG is retained within the cartilage, preferentially localizing in the pericellular matrix - the immediate niche surrounding the chondrocyte (Kahle 2022). The presumed mechanism of retention being molecular interactions with aggrecan, which are more heavily concentrated in the pericellular matrix (Kahle 2022). However, until now, BPGs were not assumed to enter chondrocytes.

[57] As described in detail below, two experimental techniques were employed to confirm cellular entry of BPGs within chondrocytes: confocal microscopy, for visualization, and flow cytometry, for quantification. Confocal imaging (Figure 1) shows fluorescently tagged BPGs (white) have entered chondrocytes (gray) in 24 hours. Z-stack imaging, a series of focal distances spanning the full thickness of the cell, indicating that BPGs are retained within the cell, do not span the surface of the cell membrane, and are retained within the cell. Flow cytometry (Figure 2) quantifies entry of fluorescently-tagged BPGs into cells by tracking the fluorescence intensity of the fluorescent marker, Cy5.5, in cells that have passed by the detector. Prior to flow cytometry, cells are trypsinized to cleave any remaining BPG from the plasma membrane and detach cells from their surface, allowing them to freely flow passed the detector while the fluorescence intensity of Cy5.5 is quantified for each cell. The mean fluorescence intensity shows a significant increase between cells treated with and without BPG. The graph shown (Figure 2) compares untreated and treated cells, confirming flow cytometry’s quantification with confocal microscopy results.

[58] With the confirmation of BPG uptake in chondrocytes, the present inventors can leverage BPG constructs such as BPG-Dex to deliver active agents such as dexamethasone into cells to halt osteoarthritis progression and alter metabolic activity. Within the field of biomaterials design and drug delivery, researchers have emphasized the difficulty of delivering therapeutics into cells and the present invention satisfies this long felt need. A 2024 journal article by Chan and Tsoukas highlights the challenges of delivering therapeutics to the surrounding region of cells and shuttling material inside cells to treat diseases. BPGs and BPG- Dex offers a new way to circumnavigate cellular entry obstacles, opening the door to a new and significant drug delivery strategy.

[59] The experimental design below explains how BPG-Dex is tested to determine whether dexamethasone conjugation hinders cell entry. Additionally, other cell types are also tested to evaluate the expansiveness of BPG as a drug delivery vehicle to other tissues and for the treatment of different diseases.

Experimental Design for Testing Fluorescently Labeled BPG-Dex Cellular Uptake

[60] Using the two techniques stated above, confocal imaging and flow cytometry, BPG- Dex are labeled with the fluorescent marker, for example Cy5.5. The cyan color produced by this marker can be detected with both confocal imaging and flow cytometry. After growing cells at various concentrations, cells are treated with the fluorescent marker at different concentrations. Following different time points of treatment with the fluorescently labeled BPG-Dex, the present inventors perform confocal imaging to visualize fluorescent BPG-Dex entry into cells and flow cytometry to determine the uptake of fluorescent BPG-Dex, in addition to the percentage of cells that BPG-Dex enters. We will compare the mean fluorescence intensity and percent uptake of fluorescently labeled BPG-Dex with BPGCy5.5 alone to understand the effects of dexamethasone’s conjugation on cellular uptake. These experiments provide qualitative and quantitative evidence of the uptake of BPG-Dex into cells.

CITED REFERENCES

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2. Sarkar S, Lightfoot- Vidal SE, Schauer CL, Vresilovic E, Marcolongo M. Terminal-end functionalization of chondroitin sulfate for the synthesis of biomimetic proteoglycans. Carbohydr Polym 2012;90:431-440.

3. Sarkar S, Moorehead C, Prudnikova K, Schauer CL, Penn LS, Marcolongo M. Synthesis of macromolecular mimics of small leucine-rich proteoglycans with a poly(ethylene glycol) core and chondroitin sulphate bristles. Carbohydr Polym 2017;166:338-347.

4. Prudnikova K, Yucha RW, Patel P, Kriete AS, Han L, Penn LS, Marcolongo MSJB. Biomimetic proteoglycans mimic macromolecular architecture and water uptake of natural proteoglycans. 2017;18:1713-1723.

5. Prudnikova K, Lightfoot Vidal SE, Sarkar S, Yu T, Yucha RW, Ganesh N, Penn LS, Han L, Schauer CL, Vresilovic EJ, Marcolongo MS. Aggrecan-like biomimetic proteoglycans (BPGs) composed of natural chondroitin sulfate bristles grafted onto a poly(acrylic acid) core for molecular engineering of the extracellular matrix. Acta Biomater 2018;75:93-104.

6. Phillips ER, Haislup BD, Bertha N, Lefchak M, Sincavage J, Prudnikova K, Shallop B, Mulcahey MK, Marcolongo MSJJoBMRPA. Biomimetic proteoglycans diffuse throughout articular cartilage and localize within the pericellular matrix. 2019.

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Claims

1. A method for intracellular delivery of an active agent, comprising the use of a biomimetic proteoglycan (BPG).

2. The method of Claim 2, wherein BPG is combined with an active agent creating a construct.

3. The method of Claim 2, wherein the construct effectively diffuses across a cellular membrane.

4. The method of Claim 3, wherein the construct is comprised of ester-linked glycine-active agent which is conjugated to BPG via an amide bond.

5. The method of Claim 4, wherein the BPG comprises BPGIO, BPG250, BPG0.5 or Small Leucine-Rich Proteoglycans (SLRP) mimics.

6. The method of Claim 4, wherein the active agent comprises pharmaceutical agent(s), therapeutic agent(s), immunomodulatory agent(s), cosmeceutical agent(s), cytotoxic agent(s), enzymes, proteins, antimicrobial agent(s), interferons, vitamins, nucleic acids, steroids, aminoglycosides, antioxidants, stem cells, anesthetic agents, analgesics, anti-inflammatories or hormonal agents, disease modifying osteoarthritic drugs (DMOAD), tumor necrosis factor, endostatin, angiostatin, thalidomide, taxol, melphalan, paclitaxel, vinblastin, vincristine, doxorubicin, acyclovir, cisplatin, tacrine, 5-fluorouracil, mitaxantrone, VM-16, etoposide, VM- 26, teniposide, or taxanes.

7. The method of Claim 6, wherein the DMOAD comprise bone active drugs glucocorticoids (including, but not limited to, dexamethasone, cortisol, cortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamicinolone, deflazacort, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, beclometasone) strontium ranelate, Zoledronic acid, Risedronate, anticytokine therapy, antibodies, statins (atorvastatin, pravastatin, fluvastatin, Tanezumab, nerve growth factor antibody, AMG 108, interleukin- 1 (IL-1) receptor type 1 antibody, adalimumab, tumor necrosis factor alpha antibody, bispecific antibodies, Lutikizumab, bispecific antibody for IL-la and IL-1|3), Canakinumab, receptor antagonists, Anakinra, a IL-1 receptor antagonist)), enzyme inhibitors (including but not limted to, nanobodies, M6495 (Anti- ADAMTS5), small molecules inhibitors, doxycycline, Cindunistat (SD-6010)), growth factors (including, but not limited to, transforming growth factor beta family, bone morphogenetic protein 7, fibroblast growth factor (FGF) family, FGF-18 (sprifermin), nucleic acids (gene therapy), antisense oligonucleotides, peptides, Calcitonin, SM04690 (a Wnt inhibitor), UBX101 (Senolitic), Transient receptor potential vanilloid 4, Neural EGFL-like 1, TPCA-1 (a KB kinase inhibitor) and tofacitinib (a Janus kinase inhibitor), Lorecivivint, and Quercitrin.

8. The method of Claim 7, wherein the construct comprises a BPGlO-dexamethasone construct, comprised of ester-linked glycine-dexamethasone (GD) conjugated to BPGIO via an amide bond (BGD).

9. The method of Claim 8, wherein the construct is delivered to the subject via injection into intra-articular joint space and wherein the construct diffuses into cartilage and into chondrocytes, and wherein the targeted delivery results in the decrease of symptoms of osteoarthritis, wherein the symptoms of osteoarthritis comprise pain, stiffness, tenderness, loss of flexibility, swelling, grating sensation, or bone spurs.

10. The method Claim 9, wherein the drug from the construct is released over an extended period of time, over 1-7 days, 1-20 weeks, 1-12 weeks, 1-10 weeks, 1-8 weeks, 1-6 weeks, 1-4 weeks and/or 1-2 weeks.

11. The method of Claim 9, wherein the drug construct is provided in a dose of BPG10 at lOmg/mL, dexamethasone at 25mM and BGD at lOmg/mL.

12. The method of Claim 11, wherein delivery of construct results in mechanobiological support of PCM, and arrests cartilage degeneration.

13. A construct comprising BPG and an ester-linked glycine-therapeutic agent conjugated to BPG via an amide bond.

14. The construct of Claim 13, wherein: the BPG comprises BPG10, BPG250, BPG0.5 or Small Leucine-Rich Proteoglycans (SLRP) mimics; the therapeutic agent comprises pharmaceutical agents, immunomodulatory agents, cosmeceutical agent(s), cytotoxic agents, enzymes, proteins, antimicrobial agents, interferons, vitamins, nucleic acids, steroids, aminoglycosides, antioxidants, stem cells, anesthetic agents, analgesics, anti-inflammatories or hormonal agents, disease modifying osteoarthritic drugs (DMOAD), tumor necrosis factor, endostatin, angiostatin, thalidomide, taxol, melphalan, paclitaxel, vinblastin, vincristine, doxorubicin, acyclovir, cisplatin, tacrine, 5-fluorouracil, mitaxantrone, VM-16, etoposide, VM-26, teniposide, or taxanes; and wherein the DMOAD comprises bone active drugs glucocorticoids (including, but not limited to, dexamethasone, cortisol, cortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamicinolone, deflazacort, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, beclometasone) strontium ranelate, Zoledronic acid, Risedronate, anticytokine therapy, antibodies, statins (atorvastatin, pravastatin, fluvastatin, Tanezumab, nerve growth factor antibody, AMG 108, interleukin- 1 (IL-1) receptor type 1 antibody, adalimumab, tumor necrosis factor alpha antibody, bispecific antibodies, Lutikizumab, bispecific antibody for IL-la and IL-ip), Canakinumab, receptor antagonists, Anakinra, a IL-1 receptor antagonist)), enzyme inhibitors (including but not limted to, nanobodies, M6495 (Anti-ADAMTS5), small molecules inhibitors, doxycycline, Cindunistat (SD-6010)), growth factors (including, but not limited to, transforming growth factor beta family, bone morphogenetic protein 7, fibroblast growth factor (FGF) family, FGF-18 (sprifermin), nucleic acids (gene therapy), antisense oligonucleotides, peptides, Calcitonin, SM04690 (a Wnt inhibitor), UBX101 (Senolitic), Transient receptor potential vanilloid 4, Neural EGFL-like 1, TPCA-1 (a KB kinase inhibitor) and tofacitinib (a Janus kinase inhibitor), Lorecivivint, and Quercitrin.

15. The construct of Claim 14, wherein delivery of the construct results in extended release of the therapeutic agent.

16. The construct of Claim 14, wherein construct diffuses across a cellular membrane enabling intracellular drug delivery.

17. The construct of Claim 14, wherein the BPG consists of BPG10, wherein the DMOAD consists of dexamethasone; and wherein administration of the construct results in site-specific and targeted delivery of dexamethasone to chondrocytes.

18. The construct of Claim 17, wherein BPGIO and DMOAD are linked via an ester-linked glycine.

19. The construct of Claim 17, wherein the DMOAD is released over an extended period of time.

20. The construct of Claim 14, wherein the disease modifying therapeutic effect comprises mechanical and mechanobiological support for the extracelluar matrix (ECM) of cartilage, the pericellular matrix (PCM) of cartilage, rescue of chondrocyte catabolism, and/or arrested cartilage degeneration.