Sulfated glycosaminoglycan biomaterials as proteoglycan mimetics

Abstract

Polymer conjugates are provided that can mimic the function of natural proteoglycans found in the extracellular matrix of connective tissue. The polymer conjugates of the present invention have utility in treating subjects suffering from soft tissue conditions. A simple and scalable chemical process for preparing the polymer conjugates of the present invention is also provided.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 395,805, filed Sep. 16, 2016, the entire teachings of which are incorporated herein by reference. [Background technology]

[0002] The extracellular matrix (ECM) forms the acellular scaffold for soft and connective tissues. It provides both biochemical and structural support required by resident cells and plays a crucial role in maintaining tissue shape and withstanding mechanical stress. Proteoglycans are natural macromolecules of the ECM that maintain tissue integrity and prevent ECM degradation. Proteoglycans are the protectors of healthy ECM through their strong osmotic hydration and ability to bind and regulate important growth factors. In response to aging, disease or injury, the ECM loses functionality. As proteoglycan content declines, collagen fibers and other matrix components also begin to degrade concomitantly. Such degradation is an underlying factor in numerous soft tissue diseases, disorders and / or conditions, including those of skin, spinal disc, cartilage and urethral tissues, to name just a few. Restoring proteoglycan functionality is one option to address the loss of ECM functionality. Summary of the Invention

[0003] The present disclosure describes medium to high molecular weight polymer conjugates that are soluble in aqueous biological solutions and are composed of sulfated glycosaminoglycan (GAG) chains. The provided polymer conjugates are biocompatible, can be easily injected using small-gauge needles, and can mimic certain proteoglycan functions in the ECM of soft tissues. In some cases, the provided polymer conjugates include sulfated GAG chains and biocompatible natural and synthetic polymers. In some embodiments, the GAG ​​chains are chemically modified with therapeutically useful groups. The present disclosure also provides methods for producing the polymer conjugates, including, inter alia, a convenient one-pot aqueous chemical process that can be scaled to produce commercially reasonable amounts of material. The present disclosure also provides methods for treating a subject suffering from a soft tissue degenerative condition. [Brief description of the drawings]

[0004] [Figure 1] FIG. 1 is a schematic diagram showing the formation of an extensive crosslinked network. [Diagram 2] FIG. 1 is a schematic showing the formation of soluble branched polymer chains. [Diagram 3] We show that sulfated GAGs are activated with a bifunctional linking agent, and the activated sulfated GAGs are then linked to additional sulfated GAG chains to form bottlebrush-like polymer conjugates. [Figure 4] It is shown that the core polymer chain is activated with a bifunctional linking agent and then the activated core polymer chain is reacted with an excess of sulfated GAG to form a hybrid bottlebrush-like polymer conjugate (i.e., in this case, the core polymer chain is not a sulfated GAG). [Diagram 5] A plot of [DVS] / OH versus polymer wt % for chondroitin sulfate, Mw=14 kDa, is shown, highlighting conditions under which gel formation and / or loss of solubility is observed. [Figure 6]A plot of [DVS] / OH versus polymer wt % for carboxymethyl cellulose, Mw=90 kDa, is shown, highlighting conditions where gel formation and / or loss of solubility is observed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0005] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS To address a long-standing need in the treatment of soft tissue diseases, disorders and conditions, it is desirable to produce proteoglycan mimetics that can mimic the morphology and physical properties of natural proteoglycans, which are composed of GAG chains that are highly negatively charged under physiological conditions due to the presence of sulfate and carboxylate groups.

[0006] Prior to this disclosure, polymer conjugates composed of sulfated GAG chains linked by multifunctional linking agents to form a significantly soluble product (i.e., not an extensively cross-linked network) have not been exemplified. The present invention encompasses the recognition that under certain conditions, sulfated GAGs can be reacted with linking agents in a controlled manner to produce high molecular weight, branched, sulfated GAG compositions that remain soluble in aqueous solutions. The molecular weight of such polymer conjugates can be characterized using standard methodologies for soluble polymers, i.e., gel permeation chromatography and dynamic light scattering. Such soluble, branched, high molecular weight sulfated GAGs are not currently available or have been previously described. Such soluble, branched, high molecular weight sulfated GAGs have utility as proteoglycan mimetics in treating numerous indications, such as those described above.

[0007] definition As used herein, headings and section headings are provided for organizational purposes and are not meant to be limiting, and thus, embodiments described in one section apply throughout this application, unless otherwise specified.

[0008] The term "approximately" or "about" when applied to one or more values ​​at issue indicates a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" indicates a range of values ​​that includes 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value, unless otherwise specified or otherwise clear from the context (except insofar as such number would exceed 100% of the possible values).

[0009] The term "administration" as used herein typically refers to administering a composition to a subject or system. Those skilled in the art will be aware of the various routes that may be utilized for administration to a subject (e.g., a human) in appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, transdermal (which may be or include, e.g., one or more of topical administration to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravesical, intravenous, intraventricular, inside a particular organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, intravitreal, etc. In some embodiments, administration may involve dosing that is intermittent dosing (e.g., multiple doses separated in time) and / or cyclical dosing (e.g., individual doses separated by regular periods of time). In some embodiments, administration may involve continuous dosing (eg, perfusion) for at least a selected period of time.

[0010] As used herein, "biocompatible" is intended to refer to materials that exert minimal disruptive or host response effects while in contact with body fluids or living cells or tissues. The term is also intended to mean such materials that result in minimal interaction with recognition proteins, such as naturally occurring antibodies, cellular proteins, cells and other components of biological systems, unless such interaction is specifically desired. Thus, materials and functional groups that are specifically intended to cause the above effects and that induce minimal and medically acceptable inflammation, foreign body reactions, immunotoxicity, chemical toxicity or other such adverse effects upon in vivo administration are considered to be biocompatible.

[0011] The term "biomolecule," as used herein, refers to molecules from various classes of chemical compounds commonly found in cells and tissues, whether naturally occurring or artificially created (e.g., by synthetic or recombinant methods), such as proteins, amino acids, peptides, polynucleotides, nucleotides, carbohydrates, sugars, lipids, nucleoproteins, glycoproteins, lipoproteins, steroids, etc. Exemplary types of biomolecules include, but are not limited to, peptides, enzymes, receptors, neurotransmitters, hormones, cytokines, cell response regulators (e.g., growth factors and chemotactic factors), antibodies, vaccines, interferons, ribozymes, antisense agents, plasmids, DNA, and RNA.

[0012] The term "treatment" (also "treat" or "treating"), as used herein, refers to any administration of a substance (e.g., a pharmaceutical composition) that partially or completely relieves, ameliorates, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the occurrence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. Such treatment may be treatment of subjects who do not exhibit symptoms of the relevant disease, disorder, and / or condition and / or who exhibit only early signs of the relevant disease, disorder, and / or condition. Alternatively, or in addition, such treatment may be treatment of subjects who exhibit one or more documented signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be treatment of subjects who have been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be treatment of subjects who are known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the relevant disease, disorder, and / or condition.

[0013] As used herein, "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, the subject is afflicted with the relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is a person having one or more characteristics characteristic of susceptibility to or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or who has been diagnosed and / or treated. In some embodiments, for any of the methods described herein, the subject is a mammal. In some embodiments, for any of the methods described herein, the subject is a human.

[0014] The terms "glycosaminoglycan" and "GAG", as used interchangeably herein, refer to a polysaccharide composed of repeating disaccharide units containing an amino sugar (such as N-acetylglucosamine or N-acetylgalactosamine) and a uronic sugar (such as glucuronic acid or iduronic acid) or galactose. GAGs for use in the present invention may vary in size and may be either sulfated or non-sulfated. GAGs that may be used in the methods of the present invention include, but are not limited to, hyaluronic acid, chondroitin, chondroitin sulfate (such as chondroitin 6-sulfate and chondroitin 4-sulfate), heparan, heparan sulfate, heparin, dermatan, dermatan sulfate, and keratan sulfate.

[0015] As used herein, the terms "improve," "increase," or "reduce," or their grammatical equivalents, refer to a value that is relative to a baseline measurement (e.g., a measurement in the same individual prior to initiating a treatment described herein, or a measurement in a control individual (or a number of control individuals) not receiving a treatment described herein, etc.).

[0016] As used herein, the term "modifier" refers to an organic, inorganic or bioorganic moiety that is covalently attached to the polymer conjugate. Modifiers can be small molecules or polymers and can belong to any chemical or pharmaceutical class, such as nucleotides, chemotherapeutic agents, antibacterial agents, antiviral agents, immunomodulators, hormones or analogs thereof, enzymes, inhibitors, alkaloids and therapeutic radionuclides, therapeutic radionuclides (e.g., alpha emitters, beta emitters or positron emitters). In certain embodiments, modifiers according to the present invention include, but are not limited to, biomolecules, small molecules, therapeutic agents, medicamentously useful groups or entities, macromolecules, diagnostic labels, chelators, hydrophilic moieties, dispersants, charge modifiers, viscosity modifiers, surfactants, coagulants and flocculants, to name a few. In some embodiments, modifiers are targeting peptides that have affinity for specific biomolecules or tissues and may enhance the delivery and / or efficacy of the polymer conjugate. Modulating agents can have one or more pharmaceutical functions (e.g., biological activity and pharmacokinetic modification). Pharmacokinetic modulating agents can include, for example, antibodies, antigens, receptor ligands, hydrophilic groups, hydrophobic groups, or charged groups. Biologically active modulating agents include, for example, therapeutic drugs and prodrugs, antigens, immunomodulators. Detectable modulating agents include diagnostic labels, such as radioactive, fluorescent, paramagnetic, superparamagnetic, ferromagnetic, x-ray modulating, x-ray opaque, ultrasound reflective, and other substances that are detectable by one of the available clinical or laboratory methods (e.g., scintigraphy, NMR spectroscopy, MRI, x-ray tomography, sonotomography, photoimaging, radioimmunoassay).

[0017] The term "prevent" or "prevention," as used herein, when used in reference to the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing a disease, disorder, and / or condition and / or delaying the onset of one or more characteristics or symptoms of a disease, disorder, or condition. Prevention may be considered complete when the onset of a disease, disorder, or condition has been delayed for a predetermined period of time.

[0018] The term "reference" as used herein refers to a standard or control to which a comparison is made. For example, in some embodiments, the agent, animal, individual, population, sample, sequence or value of interest is compared to the agent, animal, individual, population, sample, sequence or value that is the reference or control. In some embodiments, the reference or control is tested and / or measured substantially simultaneously with the test or measurement of interest. In some embodiments, the reference or control is a historical reference or control, embodied in a tangible medium if necessary. Typically, as will be understood by those skilled in the art, the reference or control is measured or characterized under conditions or circumstances equivalent to those under evaluation. Those skilled in the art will understand when there is sufficient similarity to justify reliance and / or comparison to a particular possible reference or control. In some embodiments, the reference is aggrecan. In some embodiments, the reference is a polymer starting material. In some embodiments, the reference is a reactant without conjugation. In some embodiments, the reference is a reaction without conjugation that is identical in all respects to the formation of a provided polymer conjugate, except for the omission of the linker agent.

[0019] The term "gel" refers to a viscoelastic material that is distinguished from solutions or solids by its rheological properties. A composition is considered to be a gel if it does not flow under steady state or low shear conditions, but exhibits some fluidity or flow when agitated. Gels consist of three-dimensional extensive networks that constitute a continuous solid phase in which a fluid phase (water in the case of hydrogels) is dispersed. Generally, the fluid phase is present in much greater amounts than the solid phase. The extensive crosslinked networks can be formed either through chemical covalent bonds or through physical associations in solution.

[0020] The term "molecular weight" refers to the weight average molecular weight or "M w ” (which is used interchangeably herein with “Mw”).

[0021] The term "soluble" refers to a chemical state in which a molecule (the solute) is completely dispersed at the molecular level in another molecule (the solvent) and no strong interactions exist between the solute molecules.

[0022] Proteoglycan Proteoglycans are glycoproteins found in the extracellular matrix (ECM) of all connective tissues of the body. A large number of proteoglycans and their tissue-specific expression have been identified. Despite the existence of a great deal of structural diversity, the common structural element of all proteoglycans is a protein core that is glycosylated with one or many sulfated glycosaminoglycan (GAG) chains. This protein core may contain several modular structural elements (e.g., IgG-like, EGF-like, HA-binding motifs, leucine-rich motifs, etc.) that are important for various biological functions. The covalently attached sulfated GAG chains are most typically chondroitin sulfate, dermatan sulfate, keratan sulfate, or heparan sulfate. They are often attached to the protein core as O-linked glycans that are attached to serine moieties in the core protein chain.

[0023] Hydration is crucial for ECM homeostasis. Water content determines tissue volume and resistance to compression. Hydration also provides the space required for cell migration, organization of the structural components of the ECM (e.g., collagen and elastin), and transport of biomolecules. The main structural function of proteoglycans in the ECM is to maintain hydration. This is particularly relevant for large, aggregating proteoglycans with numerous sulfated GAG chains. Proteoglycans in the hyalectan family, such as aggrecan and versican, contain numerous (e.g., about 10-100) GAG chains concentrated within specific subunits of the core protein. These unique biopolymer structures have a bottle-brush-like polymer structure and a very high density of anionic charges derived from the numerous sulfate and carboxylate moieties on the surface of the GAG ​​chains concentrated in a small volume. In addition to providing crucial hydration and structural support in the ECM, proteoglycans are known to play a key role in extracellular signaling. Proteoglycans are known to bind tightly to several growth factors, chemokines and cytokines and to influence signaling pathways for apoptosis, cell development, cell motility and cell adhesion.

[0024] A growing body of scientific evidence supports a significant role for proteoglycans in maintaining the integrity of connective tissue, protecting against tissue degradation, promoting healing after injury, and preventing disease. Due to the important role that proteoglycans play in determining the physical properties of connective tissues, and the understanding that age-related changes in connective tissues (such as the dermis) correlate with proteoglycan degradation, proteoglycan-based therapies (such as proteoglycan replacement therapy) are promising approaches for treating age-related changes and wound healing, and in addressing unmet medical needs in the areas of dermatology, urology, cardiovascular, and orthopedics.

[0025] Although proteoglycans are understood to be crucial biomolecules in the ECM of cartilage and soft tissues, they are present in most tissues in small amounts. Proteoglycans are difficult to isolate from natural sources and purify on a large scale. This is why biomolecules such as aggrecan are currently only available as research tools in small quantities. Using proteoglycans isolated from tissues as therapeutic agents is cost prohibitive and impractical. Moreover, proteoglycans extracted from xenobiotic tissues (bovine, porcine, marine) may be unsuitable for direct use in human medicine due to immunological host responses.

[0026] Proteoglycan-mimicking materials Several studies have attempted to design compositions capable of mimicking important structural and / or biological functions of naturally occurring proteoglycans (PGs) in connective tissues. These efforts fall into several categories.

[0027] Sulfation of synthetic polymers or natural polysaccharides. For example, one of the most convenient approaches to synthesize PG mimics is the sulfation of carbohydrates (e.g., dextran, etc.) [D Papy-Garcia et al., Macromolecules, 2005, 38:4647-4654]. Sulfation of synthetic polymers (e.g., aromatic polyphenols, etc.) has also been reported to produce molecules with GAG or PG bioactivity [UR Desai, Future Med.Chem., 2013, 5:1363-1366].

[0028] b. Attachment of sulfated GAGs to surfaces or particles. For example, chondroitin sulfate was conjugated to the surface of carbon nanotubes to provide GAG-functional nanoparticles as PG mimics in hydrogel constructs for cartilage replacement [J Wei et al., Materials Chemistry and Physics, 2015, 166:66-72]. Chondroitin sulfate was attached to the surface of agarose gel after activation of the gel with a reactive cyanate ester that can react with the serine moiety at the reducing end of chondroitin sulfate [KJ Mattern et al., Carbohydrate Research, 2007, 342:2192-2201]. Chondroitin sulfate was attached to poly(ethylene terephthalate) fiber surfaces and chitosan-coated PET fiber surfaces [CH Jou et al., Polym. Adv. Technol., 2005, 16:821-826].

[0029] c. Creation of insoluble particles by complexing anionic sulfated GAGs with cationic polymers. The formation of complexes between highly anionic GAGs and polycations, such as chitosan, has been described as a method to obtain nanoparticles capable of binding FGF-2 [S Boddohi et al., Biomacromolecules, 2009, 10:1402-1409][LW Place et al., Biomacromolecules, 2014, 15:3772-3780]. Heparan has been complexed with various reactive polymers to form insoluble coatings that are applied to the surfaces of medical devices [US Patent Publication No. 2005 / 0281857].

[0030] d. Conjugation of certain bioactive peptides with sulfated GAGs to provide well-defined soluble peptidoglycan derivatives. For example, conjugation of dermatan sulfate with peptides capable of binding either type II collagen or hyaluronic acid has been extensively reviewed and described [S Sharma et al., Acta Biomaterialia, 2013, 9:4618-4625][JC Bernhard et al., Acta Biomaterialia, 2012, 8:1543-1550][U.S. Patent No. 9,200,039].

[0031] e. Polymerization of monomers bearing sulfated di- or oligosaccharides. For example, polymeric mimics of chondroitin sulfate have been made via synthesis of ROMP polymerizable monomers substituted with simple chondroitin sulfate disaccharide units [SG Lee et al., Chem. Sci., 2010, 1:322-325].

[0032] f. Synthesis of polyvalent oligosaccharide glycans. Specific di- and tetrasaccharides representing single-entity heparan sulfate (HS) structural motifs have been prepared and conjugated to four-arm dendritic linking molecules. These heparan sulfate mimics were found to be able to mimic the ability of long-chain native HS in their interactions with certain therapeutic proteins [PC Tyler et al., Angew. Chem. Int. Ed., 2015, 54:2718-2723].

[0033] g. Conjugating GAGs with other polymers. For example, some small and oligosaccharides have been conjugated to synthetic polymers by reacting their reducing ends with complementary functional groups on the synthetic polymer core [K Godula et al., J. Am. Chem. Soc., 2010, 132:9963-9965]. Related efforts have reported aggrecan-like bottle brush compositions using hyaluronic acid derivatives as polymer cores that can react with the reducing ends of full-length native heparan or chondroitin sulfate chains as setae [LW Place et al., Biomacromolecules, 2014, 15:3772-3780]. In another approach to forming bottlebrush-type structures, chondroitin sulfate with an O-linked serine glycan at the reducing end of the chain has been used as a monotelechelic amine in several reaction contexts, including amide-forming reactions with poly(acrylic acid) as the core [US Patent Application Publication No. 20130052155 (A1)].

[0034] A different research field, which has some relevance to the area of ​​proteoglycan mimetics, has focused on crosslinked GAG hydrogels. In these cases, extensively crosslinked networks have been obtained, rather than soluble polymeric compounds. The properties of the crosslinked networks most fundamentally stem from their crosslink density and particle size. In contrast, soluble polymers are characterized by their molecular weight and degree of branching. In general, crosslinked gels have a high modulus and can be difficult to administer by injection.

[0035] Water-swelling hydrogel particles prepared by crosslinking GAG materials provide a GAG-rich surface in biological environments. However, after being injected into tissues, these crosslinked gels behave as individual particles in the ECM, and therefore cannot function as proteoglycan-mimetic materials. These crosslinked gels cannot integrate into soft tissues and interact with other components of the ECM, as proteoglycans (such as versican) are known to integrate and interact in the dermis.

[0036] The majority of research on crosslinked GAG networks has focused on hyaluronic acid (HA) because of its large-scale production from bacterial cultures as well as its natural source and commercial availability. Furthermore, HA is generally available in very high molecular weight forms, usually exceeding 500,000 Da and extending into the millions of Da. The large molecular weight favors the formation of extensive hydrogel structures. For this reason, significant research has been conducted on the synthesis and use of crosslinked hydrogels based on HA, with hyaluronic acid being by far the most widely used GAG in the development of biopharmaceutical and medical device products. Various HA gels are widely known as dermal fillers, viscosupplements, and cosmetics.

[0037] In contrast to HA, sulfated GAGs (e.g., chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate) are currently only available from natural sources, and generally in much smaller quantities. Commercial sources of high-quality GMP material are limited. In addition, when extracted from natural tissues, these sulfated GAGs are found to have much lower molecular weights than HA. For example, chondroitin sulfate of bovine origin is generally found to have molecular weights less than 50,000 Da, and most typically less than 25,000 Da. The low molecular weight of these biopolymers, as well as the difficulty in sourcing high-purity material, have limited their use in the development of biopharmaceutical and medical device products.

[0038] Research reports and patents on crosslinked HA hydrogels indicate that other GAGs may be used in place of HA. However, given the significant differences between sulfated GAGs and HA, most notably the vast difference in molecular weight, it cannot be assumed that existing synthetic methods for forming gels with HA are applicable to sulfated GAGs, nor can it be assumed that the properties of crosslinked hydrogels obtained from sulfated GAGs will resemble those of HA crosslinked hydrogels.

[0039] For the formation of crosslinked GAG hydrogels, several one-step direct linking agents have been described in the literature and have been found to result in biocompatible hydrogels. These crosslinked HA hydrogels are utilized in various commercial products, such as dermal fillers (e.g., HYLAFORM®, PREVELLE®, RESTYLANE®, JUVEDERM®) and viscosupplements (e.g., SYNVISC®, SYNVISC-ONE®, SUPARTZ®, EUFLEXXA®, JONEXA®, MONOVISC®, ORTHOVISC®) and adhesion barriers (e.g., INCERT®, INCERT-S®, HYALOBARRIER®). Non-limiting examples of direct linking agents include divinylsulfone (DVS), epichlorohydrin (epi), butanediol diglycidyl ether (BDDE), diepoxyoctane, ethylene glycol diglycidyl ether, phenylene-bis(ethylcarbodiimide), 1,1'carbonyldiimidazole (CDI).

[0040] The reaction of various direct crosslinkers with sulfated GAGs is known to form strong hydrogels. However, applicants recognize that such gel formation is sensitive to reaction conditions and may result in unexpected outcomes. For example, the reaction of DVS with chondroitin sulfate may result in several outcomes. In some cases, a strong, transparent gel is obtained. In some cases, a viscous, transparent fluid is obtained. In some cases, a cloudy suspension of insoluble modified chondroitin sulfate is obtained. In some cases, a cloudy gel is obtained. Applicants disclose herein methods to control and manage these various outcomes to produce soluble polymer conjugates.

[0041] Despite several attempts aimed at developing proteoglycan mimetic materials, there are currently no known polymer conjugates that effectively impart the beneficial physical and biological functions of natural proteoglycans, that are known or predicted to be biocompatible, that are soluble and capable of being integrated into soft tissues by diffusion, that are easy to inject or administer, that are retained in soft tissues for extended periods of time, and that can be made using efficient and simple chemical processes that are scalable to commercial quantities.

[0042] Polymer Conjugates The present invention relates to the use of several different strategies to create proteoglycan mimics. The present invention encompasses the recognition that sulfated GAGs (and other polymers) contain numerous functional moieties that can be reacted with appropriate linking agents to form soluble, higher-order polymer conjugates, including polymer conjugates with branched and bottle-brush-like structures. Such functional moieties may be reacted with linking agents to "activate" the polymer chain for conjugation with one or more other polymer chains. Previous efforts in this area have produced GAG compositions that are gels, but the present invention provides polymer conjugates that are not gels and remain soluble in aqueous solutions. In some embodiments, the soluble polymer conjugates of the present invention are produced by controlling the stoichiometry of the linking agent and sulfated GAG, the concentration of sulfated GAG, the molecular weight of sulfated GAG, and / or the reaction time.

[0043] Functional moieties on GAGs or other polymers that can be utilized in the linking chemistry described herein include, but are not limited to, hydroxyl groups, amines, thiols, and carboxyl groups. In some embodiments, the functional moieties are or include one or more hydroxyl groups along the GAG ​​polymer backbone. In some embodiments, the functional moieties are or include one or more carboxyl groups along the GAG ​​polymer backbone.

[0044] In some embodiments, the polymer conjugate of the present invention comprises a plurality of sulfated GAG polymer chains linked via a linking agent. In some embodiments, the polymer conjugate of the present invention comprises a plurality of sulfated GAG polymer chains linked via a linking agent and at least one additional polymer. In some embodiments, the polymer conjugate of the present invention comprises a plurality of sulfated GAG polymer chains linked via a linking agent and at least two additional polymers. In some embodiments, the polymer conjugate of the present invention comprises a plurality of sulfated GAG polymer chains linked via a linking agent and at least three additional polymers. In some embodiments, the sulfated GAG is chondroitin sulfate. In some embodiments, the additional polymer is a sulfated GAG other than chondroitin sulfate. In some embodiments, the additional polymer is a non-sulfated GAG. In some embodiments, the additional polymer is hyaluronic acid (HA) or carboxymethylcellulose (CMC).

[0045] It will be understood that the polymer conjugates of the invention will generally have a higher (e.g., increased) molecular weight compared to the individual GAG polymer chains, but will not form gels with extensive crosslinked networks. In some embodiments, the polymer conjugates of the invention have a molecular weight in a particular range compared to the unlinked sulfated GAGs used as starting materials (e.g., the polymer conjugates have a molecular weight 3-100 times that of the individual unlinked sulfated GAGs). In some embodiments, the polymer conjugates of the invention are branched multi-chain conjugates having a molecular weight in a particular range (e.g., having a molecular weight 3-100 times that of the individual unlinked sulfated GAGs). In some embodiments, the polymer conjugates of the invention are bottlebrush multi-chain conjugates having a molecular weight in a particular range (e.g., having a molecular weight 3-100 times that of the individual unlinked sulfated GAGs). In some embodiments, the polymer conjugates of the invention have a molecular weight in the range of between about 3-100, 3-75, 3-50, 3-25, 5-100, 5-75, 5-50, and 5-25 times the molecular weight of the individual unlinked sulfated GAGs. In some embodiments, the polymer conjugates of the invention have a molecular weight in the range of 5-25 times the molecular weight of the individual unlinked sulfated GAGs.

[0046] In some embodiments, the polymer conjugates of the invention are soluble in aqueous solution. In some embodiments, the polymer conjugates of the invention comprise a plurality of sulfated glycosaminoglycan (GAG) polymer chains, where each sulfated GAG is linked to one or more sulfated GAG polymer chains via a linker agent, and the polymer conjugate is soluble in aqueous solution and has a molecular weight that is 3-100 times the molecular weight of the individual unlinked sulfated GAGs.

[0047] Without wishing to be bound by any particular theory, variations in the polymer conjugates include, but are not limited to, varying length, sulfation patterns, molecular weight, and chemical composition, etc. These variations may be controlled using the methods provided herein, while affecting the conformation, molecular weight, hydration, mechanical function, and cell signaling function of the polymer conjugates.

[0048] Linker Agent Those skilled in the art will be familiar with the various types of direct linker agents that are suitable for linking GAG polymers and other polymers used in accordance with the present invention. It will be understood that the terms "linking agent" and "linker" are interchangeable, with the understanding that the linker is the part of the conjugate that results from reaction with the linker agent.

[0049] In some embodiments, the linker is bifunctional. In some embodiments, the linker is not polymeric. In some embodiments, the linker is only a polymer with 10 or fewer repeats of the monomer unit. In some embodiments, the linker is only a polymer with 5 or fewer repeats of the monomer unit. In some embodiments, the linker has a molecular weight of less than about 150 Da, less than 200 Da, less than 250 Da, less than 300 Da, less than 350 Da, less than 400 Da, less than 450 Da, less than 500 Da, less than 600 Da, less than 700 Da, less than 800 Da, less than 900 Da, or less than 1000 Da. In some embodiments, the linker is non-polymeric and is less than about 150 Da, less than 200 Da, less than 250 Da, less than 300 Da, less than 350 Da, less than 400 Da, less than 450 Da, less than 500 Da, less than 600 Da, less than 700 Da, less than 800 Da, less than 900 Da, or less than 1000 Da. In some embodiments, the linker is non-polymeric and is less than about 1000 Da. In some embodiments, the linker is non-polymeric and is less than about 500 Da. In some embodiments, the linker is non-polymeric and is less than about 250 Da. In some embodiments, the linker is non-polymeric and is less than about 200 Da. In some embodiments, the linker is non-polymeric and is less than about 150 Da. In some embodiments, the linker agent is not polymeric and is less than 150 Da, less than 200 Da, less than 250 Da, less than 300 Da, less than 350 Da, less than 400 Da, less than 450 Da, less than 500 Da, less than 600 Da, less than 700 Da, less than 800 Da, less than 900 Da, or less than 1000 Da.

[0050] In some embodiments, the linker agent is selected from the group consisting of divinylsulfone (DVS), diepoxide, epichlorohydrin (Epi), butanediol diglycidyl ether (BDDE), and combinations thereof. In some embodiments, the linker agent is epichlorohydrin (Epi). In some embodiments, the linker agent is butanediol diglycidyl ether (BDDE). In some embodiments, the linker agent is a biscarbodiimide. In some embodiments, the linker agent is phenylene-bis(ethylcarbodiimide). In some embodiments, the linker agent is 1,1'-carbonyldiimidazole. In some embodiments, the linker agent is divinylsulfone (DVS). In some embodiments, the linker agent is bromoacetic NHS ester, 6-(iodoacetamido)caproic acid NHS ester, maleimidoacetic acid NHS ester, maleimidobenzoic acid NHS ester, or MMCCH (4-(maleimidomethyl)cyclohexane-1-carboxylhydrazide).

[0051] In some embodiments, the linker is a peptidic fragment containing from 2 to about 20 aminoacyl residues, a straight or branched chain alkyl or aryl carboxylate ester, or a C 1~20 wherein one or more methylene units of the linker are optionally and independently replaced by cyclopropylene, -CHOH-, -NR-, -N(R)C(O)-, -C(O)N(R)-, -N(R)SO2-, -SON(R)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -S-, -SO-, -SO2-, -C(=S)- or -C(=NR)-.

[0052] In some embodiments, the linker or linker agent contains a short poly(alkylene oxide) chain, hi some embodiments, the linker or linker agent is a short poly(ethylene oxide) chain terminated with an epoxide group, such as poly(ethylene glycol) diglycidyl ether.

[0053] Sulfated GAGs In some embodiments, the sulfated GAG for use according to the present invention is selected from the group consisting of chondroitin sulfate, heparan sulfate, dermatan sulfate, keratan sulfate, heparin, and combinations thereof.In some embodiments, the sulfated GAG is chondroitin sulfate.Chondroitin sulfate is composed of repeating disaccharide units of N-acetylgalactosamine (GalN) and glucuronic acid (GlcN).In some embodiments, chondroitin sulfate can have more than 100 sugars, each of which can be sulfated at different positions and amounts (e.g., chondroitin sulfate A, C, D, and E). In some embodiments, the molecular weight of the sulfated GAGs may be greater than about 1,000 Da, 5,000 Da, 10,000 Da, 15,000 Da, 20,000 Da, 30,000 Da, 40,000 Da, 50,000 Da, 100,000 Da, or a range inclusive of any two of these numbers. In some embodiments, the molecular weight of the chondroitin sulfates may be greater than about 1,000 Da, 5,000 Da, 10,000 Da, 15,000 Da, 20,000 Da, 30,000 Da, 40,000 Da, 50,000 Da, 100,000 Da, 200,000 Da, or a range inclusive of any two of these numbers.

[0054] Qualified GAG In some embodiments, the polymer conjugates of the present invention can be prepared by using modified GAGs in which at least one modulating agent is introduced into at least one polymer GAG chain. As described above, GAGs have multiple hydroxyl and carboxyl functional groups along the chain. In addition, the reducing end of GAGs provides a unique and unique chemical functional group. To expand and enhance the therapeutic benefits of the novel compositions described in the present invention, the present invention embraces the recognition that modulating agents can be introduced onto the GAG ​​chain prior to reaction with the linking agent. By carrying out the methods of the present invention with chemically modified GAGs, i.e., GAG glycoconjugates, a high molecular weight proteoglycan mimic will be provided with the additional benefits provided by the modulating agent. For example, sulfated GAGs bearing peptides with affinity for other ECM components or cell surface proteins, including but not limited to collagen I, collagen II, other collagen isoforms, elastin, integrin receptors, or various galectins, will allow more specific binding of the proteoglycan mimic to the target biomolecule. The literature describes several examples of covalent modifications of GAGs and suitable chemistries for such modifications are known to those skilled in the art.

[0055] In some embodiments, sulfated GAGs may be modified along the GAG ​​polymer chain. In some embodiments, modulating agents may be introduced into sulfated GAGs before linking the GAG ​​chain backbone with a linking agent by various methods known to those skilled in the art. In some embodiments, modulating agents may be introduced into sulfated GAGs at their reducing end using reducing end chemistry (e.g., reductive amination) that is well known to those skilled in the art.

[0056] In some embodiments, sulfated GAGs are modified via carboxyl groups along the GAG ​​polymer chain. In some embodiments, the carboxyl groups are subjected to peptide coupling conditions to form amide bonds, thereby introducing the modulating agent. Suitable peptide coupling conditions are well known in the art and include those detailed in Han et al., Tetrahedron, 60, 2447-67 (2004) and VR Pattabiraman et al., Nature, 480, 471-479 (2011), which are hereby incorporated by reference in their entirety. In some embodiments, suitable peptide coupling conditions include a peptide coupling reagent selected from a carbodiimide-based or triazole-based activation reagent in the presence of a base, such as DIEA or other bases familiar to those skilled in the art. In certain embodiments, the peptide coupling conditions include the addition of HOBt, HOAt, DMAP, BOP, HBTU, HATU, BOMI, DCC, EDC, IBCF, or a combination thereof. In some embodiments, the peptide coupling agent is selected from triazine-based activating agents, such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM).

[0057] In some embodiments, soluble, high molecular weight sulfated GAG compositions may be prepared using polymers that are chemically substituted with various groups to enhance their capabilities in their intended applications. In some embodiments, such substitution with modulating agents is random along the GAG ​​polysaccharide chain or only at the reducing end of the chain. In some embodiments, the provided polymer conjugates include sulfated GAGs (e.g., chondroitin sulfate (ChS) and the like) that are substituted with peptide modulating agents known to have strong affinity for a given component of the ECM (e.g., collagen, elastin). In other embodiments, the provided polymer conjugates include sulfated GAGs that are substituted with antioxidant modulating agents or other molecules to enhance their therapeutic benefits. Peptide conjugation is widely known in the art as a means of adding biological recognition and function to synthetic polymers and biomaterials. Many short peptide motifs have been identified and utilized in biomaterial applications that may be useful in forming GAG conjugates for the present invention. Many of these peptides are derived from natural proteins that have the desired affinity for a given target biomolecule.

[0058] In some embodiments, the provided polymer conjugates comprise sulfated GAGs substituted with integrin binding modulating agents. The most widely known are peptide motifs for binding to cell surface integrins, which are derived from fibronectin: GRGDS (SEQ ID NO: 1), PHSRN (SEQ ID NO: 2), REDV (SEQ ID NO: 3) and LVD. These peptides and their derivatives have affinity for cell surface integrins and have been covalently attached to biomaterial matrices to immobilize cells. Integrin binding peptides derived from laminin have also been used to attract cells into biomaterials: YIGSR (SEQ ID NO: 4), GIIFFL (SEQ ID NO: 5), IKVAV (SEQ ID NO: 6) and their derivatives, etc.

[0059] In some embodiments, the provided polymer conjugates include sulfated GAGs substituted with collagen binders. There are several peptides known to bind to collagen surfaces. Some are derived from decorin: SYIRIADTNITGC (SEQ ID NO: 7) (known as dc-13), LRELHLNNN (SEQ ID NO: 8) (IS-6) and LHERHLNNN (SEQ ID NO: 9). Another widely known collagen-binding peptide is [GPO]7, which is a heptameric repeat of the collagen motif of glycine-proline-hydroxyproline and has helicogenic affinity for fibrillar collagen. The peptide GLRSKSKKFRRPDIQYPDA (SEQ ID NO: 10) was described in US Patent No. 9,133,246 (B2), where it was used as part of a collagen-targeting fusion protein. US Patent No. 9,200,039 (B2) describes collagen-binding peptides RRANAALKAGELYKSILYGC (SEQ ID NO: 11) (known as SILY) and WYRGRLGC (SEQ ID NO: 12), as well as several other examples. In addition, US Patent No. 8,846,003 (B2) describes peptides with specificity for binding to the surface of type III collagen, such as KELNLVYTGC (SEQ ID NO: 13) and GSITTIDVPWNVGC (SEQ ID NO: 14). Several cyclic peptides with affinity for collagen are described in US Patent No. 8,034,898 (B2), including WHCYTYFPHHYCVYG (SEQ ID NO: 15), GWHCYTYFPHHYCTYG (SEQ ID NO: 16), AWHCYTYFPHHYCVYG (SEQ ID NO: 17), LWHCYTYFPHHYCVYG (SEQ ID NO: 18), and YWHCYTYFPHHYCVYG (SEQ ID NO: 19).

[0060] In some embodiments, the provided polymer conjugates comprise sulfated GAGs substituted with hyaluronan-binding modulating agents. Peptides having affinity for binding to hyaluronan in the ECM are described in U.S. Patent No. 9,200,039 (B2). These include GAHWQFNALTVRGGGC (SEQ ID NO: 20) (also known as GAH) and other examples.

[0061] Preferably, the polymer conjugate according to the present invention comprises at least one sulfated GAG polymer chain substituted with at least one glycan ligand for a galectin, e.g., a sulfated GAG polymer chain comprising at least one β-galactose residue (e.g., β-galactoside).

[0062] In some embodiments, the provided polymer conjugates comprise any of the above peptides or glycans as modulating agents.

[0063] Further Polymers In some embodiments of the provided polymer conjugates, sulfated GAGs are directly conjugated with other polymers and biomolecules. In some embodiments, hyaluronic acid (HA) or carboxymethylcellulose (CMC) are incorporated to form hybrid high molecular weight soluble polymer compositions. In some embodiments, sulfated GAGs may be directly conjugated with other polymers and biopolymers having molecular weights of more than about 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 1,000 kDa, or any two of these numbers inclusive. Exemplary such conjugates are described in more detail below.

[0064] Methods for preparing GAG polymer conjugates As described above, the polymer conjugate of the present invention is synthesized by appropriate selection of synthetic reagents and synthetic methods.The following discussion is provided to illustrate some of the various methods that are available for use in assembling the polymer conjugate of the present invention.However, the following discussion is not intended to limit the scope of reactions or reaction sequences that are useful in preparing the compounds of the present invention.

[0065] In Scheme A, as illustrated below, a sulfated GAG (eg, chondroitin sulfate) and various positions to which a linker agent (eg, DVS) may be attached are shown.

[0066] Scheme A [ka]

[0067] In Scheme B, as illustrated below, an example of a sulfated GAG is shown that is reacted with a second sulfated GAG to which a linker agent (DVS) is attached to form a polymer conjugate.

[0068] Scheme B [ka]

[0069] Applicants have found that under conditions where sulfated GAGs are present at high concentrations, strong, transparent gels can be rapidly formed. For example, when using a commercially available bovine chondroitin sulfate material with Mw=14,000 Da, when the chondroitin sulfate is at a concentration of greater than 8 wt% (8 g of polymer in 100 g of solution) and sufficient DVS is used, hydrogels can be formed within 1-2 hours of adding DVS in 0.1 N NaOH solution.

[0070] Figures 5 and 6 illustrate various combinations of DVS / OH ratio and polymer concentration, as expressed as weight percent of polymer in solution. The figures show that some combinations form gels and others do not. In addition, the figures include the approximate time required for a gel to form, and it is possible to stop such reactions before a gel forms. Figures 5 and 6 also show conditions under which the reaction mixture remains clear and conditions under which the reaction becomes cloudy or opaque due to the formation of insoluble highly modified polymer derivatives.

[0071] The present invention provides, inter alia, a method for preparing a polymer conjugate, wherein the primary product is a sulfated GAG polymer conjugate that is soluble in aqueous solution. According to one aspect of the present invention, a sulfated GAG is used in the methods provided herein at a concentration selected to avoid the formation of a gel.

[0072] In some embodiments, the sulfated GAGs have a concentration of greater than about 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, or a range inclusive of any two of these numbers. In some embodiments, the sulfated GAGs are present in a concentration ranging from 2 wt% to 20 wt%, between 2 wt% to 18 wt%, between 2 wt% to 16 wt%, between 2 wt% to 14 wt%, between 2 wt% to 12 wt%, between 2 wt% to 10 wt%, between 4 wt% to 20 wt%, between 6 wt% to 20 wt%, between 8 wt% to 20 wt%, between 10 wt% to 20 wt%, between 5 wt% to 15 wt%, between 5 wt% to 10 wt%, between 8 wt% to 16 wt%, or between 8 wt% to 12 wt%. In some embodiments, chondroitin sulfate (ChS) is used at a concentration of greater than about 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, or a range inclusive of any two of these numbers.

[0073] In some embodiments, experiments conducted with ChS in the range between about 8 wt% and 16 wt% reveal that the rate of gel formation increases with both the concentration of ChS and the amount of DVS used. For example, when the molar ratio of DVS / hydroxyl equivalents available to the biopolymer is less than 0.1, gels do not form after 90 minutes, even for more concentrated solutions of ChS (10-12 wt%).

[0074] In some embodiments, it was found that the gel formation rate accelerated when these reactions were carried out under conditions in which the DVS / hydroxyl ratio was systematically increased. Moreover, it was found that some reactions became cloudy or even formed a white solid precipitate when the DVS / hydroxyl level was high (close to or above 1.0). Characterization of the insoluble product by NMR spectroscopy revealed that the insoluble product was a chondroitin sulfate derivative highly substituted with vinyl sulfone groups.

[0075] "Branched" Polymer Conjugates In some embodiments, the polymer conjugates of the invention have a branched structure. See, for example, FIG. 2. In some embodiments, sulfated GAGs are reacted with linking agents under conditions where the GAG ​​concentration and the molar ratio of linking agent to GAG are selected to provide a soluble branched polymer rather than an extensively crosslinked network. In some embodiments, the linking agent is DVS and the DVS / hydroxyl ratio is between about 0.01-0.6. In some embodiments, the DVS / hydroxyl ratio is 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, or a range inclusive of any two of these numbers.

[0076] In certain embodiments, the invention provides a method of preparing a polymer conjugate comprising the steps of: i) providing an aqueous solution of a sulfated GAG at a concentration of about 2 wt% to 20 wt%; and ii) contacting the sulfated GAG with a linking agent to form a soluble branched polymer, wherein the molar ratio of GAG hydroxyl groups to linking agent is less than the molar ratio required for gel formation. In some embodiments, the sulfated GAG in step i has a molecular weight of 10,000 Da to 100,000 Da. In some embodiments, the molar ratio of GAG hydroxyl groups to linking agent (e.g., DVS / hydroxyl ratio) is 0.01 to 0.6.

[0077] In some embodiments, sulfated GAGs are reacted with direct linking agents under conditions that allow the reaction to terminate before extensive crosslinking networks are formed. In these cases, the linking reaction is easily stopped by adding an acid (e.g., HCl, etc.) to reduce the pH to neutral values. Again, a soluble branched polymer is obtained, rather than an extensive crosslinking network. In some embodiments, the reaction is allowed to proceed for a period of time before the reaction is terminated. In some embodiments, the reaction is allowed to proceed for about 1 minute to 120 minutes. In some embodiments, the reaction is allowed to proceed for about 25 minutes to 40 minutes. In some embodiments, the reaction is allowed to proceed for about 40 minutes. In some embodiments, the reaction is allowed to proceed for about 90 minutes.

[0078] In some embodiments, the branched polymer conjugate has a molecular weight greater than about 15,000 Da, 20,000 Da, 30,000 Da, 40,000 Da, 50,000 Da, 100,000 Da, 200,000 Da, 300,000 Da, 400,000 Da, 500,000 Da, 1,000,000 Da, or a range inclusive of any two of these numbers.

[0079] "Bottlebrush-like" polymer conjugates In some embodiments, the polymer conjugate of the present invention has a bottle brush-like structure. See, for example, FIG. 3. In some embodiments, sulfated GAG is reacted with a linking agent under conditions in which the reactants are introduced sequentially. In some embodiments, this stepwise addition of reactants greatly affects the molecular structure and properties of the product. For example, in a one-pot procedure, a small amount of sulfated GAG can be activated with a linking agent in a dilute solution to form a multivalent reactive core polymer intermediate. Subsequent addition of an excess of the same sulfated GAG or a different sulfated GAG results in the formation of a soluble, high molecular weight sulfated GAG composition with a bottle brush-like structure.

[0080] Thus, in some embodiments, the present invention provides a method for preparing a polymer conjugate by sequential introduction of sulfated GAG in a single reaction, comprising the steps of: i) providing a sulfated GAG; ii) reacting the sulfated GAG with a linking agent under conditions such that a small amount of the sulfated GAG is reacted with the entire amount of the linking agent; and iii) adding the remaining portion of the sulfated GAG to form a soluble conjugate having a bottlebrush-like structure.

[0081] In some embodiments, a high molecular weight core polymer capable of direct reaction with a linking agent (e.g., CMC, HA) is reacted in the first step of a two-step synthetic procedure. A sulfated GAG may then be introduced to react with the modified core polymer to form a bottle brush-like polymer composition in a one-pot procedure.

[0082] In some embodiments, the present invention provides a method for preparing a polymer conjugate, comprising: i) activating a core polymer with a linking agent in a dilute solution to form a multifunctional reactive core polymer intermediate; and ii) adding an excess of sulfated GAG to form a soluble bottle brush-like polymer. In certain embodiments, step i comprises activating the core polymer with a linking agent under conditions where a small amount of the core polymer is reacted with the entire amount of linking agent. In certain embodiments, step i comprises activating a substoichiometric amount of the core polymer (i.e., an excess of linking agent over the hydroxyl groups of the polymer) with a linking agent in a dilute solution to form a multifunctional reactive core polymer intermediate. In some embodiments, the core polymer in step i is a sulfated GAG that is the same as the sulfated GAG added in step ii. In some embodiments, the core polymer in step i is a sulfated GAG that is different from the sulfated GAG added in step ii. In some embodiments, the core polymer in step i is not a sulfated GAG. In certain embodiments, the core polymer in step i is carboxymethylcellulose. In certain embodiments, the core polymer in step i is hyaluronic acid.

[0083] In some embodiments, the provided polymer conjugates are prepared in a two-step reaction in which a core polymer is first functionalized with a linking agent in dilute solution and then isolated by precipitation or other means. The linking agent-modified core polymer intermediate can be characterized and / or purified. Subsequent reaction of this core polymer intermediate in a second reaction with sulfated GAG in a concentrated solution results in a soluble bottle brush-like polymer composition.

[0084] Thus, in some embodiments, the present invention provides a method of preparing a polymer conjugate comprising the steps of: i) functionalizing a core polymer with a linking agent in dilute solution to form a core polymer intermediate; ii) isolating the core polymer intermediate; and iii) reacting the core polymer intermediate with a sulfated GAG in concentrated solution to form a soluble bottle brush-like polymer.

[0085] In some embodiments, the bottle brush-like polymer conjugate has a molecular weight greater than about 15,000 Da, 20,000 Da, 30,000 Da, 40,000 Da, 50,000 Da, 100,000 Da, 200,000 Da, 300,000 Da, 400,000 Da, 500,000 Da, 1,000,000 Da, 2,000,000 Da, or a range inclusive of any two of these numbers.

[0086] Characterization techniques As described above, in some embodiments, the polymer conjugates of the present invention are soluble in aqueous solutions. Such conjugates are in contrast to known GAG polymer conjugates, which are gels with extensive crosslinked networks. One skilled in the art can distinguish between materials that are gels and those that are not gels, but for the avoidance of doubt, it is noted that for polymerization in homogeneous solutions, the formation of an extensive crosslinked network will be characterized by the loss of solution properties. For example, the reaction mixture will no longer flow, and when the gel is added to a large amount of water, it may swell, but it will not dissolve. Such gels exhibit the properties of a solid or viscoelastic material. In addition, such gels have viscoelastic properties that can be quantified using rheometry. For example, many strong gels have a storage modulus (G') that is greater than their loss modulus (G'').

[0087] In some embodiments, the provided polymer conjugates will maintain solution flow properties when dissolved in water. In some embodiments, the provided polymer conjugates will have a molecular weight distribution and degree of branching that will be characteristic of the synthesis method and will be reproducible from batch to batch. In some embodiments, the provided polymer conjugates are characterized in that a clear viscous fluid, rather than a gel, is observed during the manufacture of the provided polymer conjugates. In some embodiments, the polymer conjugates are clear viscous fluids in aqueous solution.

[0088] Characterization of the provided polymer conjugates may be provided by gel permeation chromatography (GPC) and dynamic light scattering (DLS). In some embodiments, parameters related to flow (such as, for example, viscosity or elastic modulus) may be determined by viscometry and rheology.

[0089] Hydrodynamic radius (Rh) is determined by DLS and is directly related to molecular weight and molecular structure (type / degree of branching). In some embodiments, an enhancement or increase in Rh over that of the starting material will be achieved. In some embodiments, the polymer conjugate of the present invention will have an increased hydrodynamic radius compared to the hydrodynamic radius of the reference. In some embodiments, aggrecan may be the reference used to model the upper limit for both molecular weight and Rh. In some embodiments, the starting material (e.g., unlinked sulfated GAG) may be used as the reference.

[0090] DLS is a convenient method to directly determine the size (Rh) of a polymer in solution, however, it does not directly measure the molecular weight. By knowing the hydrodynamic radius, it is possible to estimate the molecular weight. In DYMANICS® software (Wyatt technologies), a shape model is used to estimate Mw from Rh. This calculation can be done after inputting common polymer structure models (spherical, coiled, branched).

[0091] Purification of polymer conjugates In some embodiments, the polymer conjugate may be purified by various methods known to those skilled in the art. In some embodiments, the polymer conjugate may be purified by dialysis. In some embodiments, the polymer conjugate may be purified by tangential flow filtration. In some embodiments, the polymer conjugate may be precipitated from the crude reaction product. In some cases, the polymer conjugate may be precipitated from the reaction mixture, collected, redissolved in water, and precipitated again. Several cycles of redissolution / precipitation may be performed.

[0092] How to use Injuries to soft tissues (e.g., vascular, dermal or musculoskeletal tissues) are quite common. Surgical approaches to correct soft tissue defects and / or damage in the body generally involve the implantation of structures made of biocompatible, inert materials that attempt to replace or serve as substitutes for the defective function. The implantation of non-biodegradable materials results in permanent structures that remain in the body as foreign bodies. Various implants made of absorbable materials have been proposed for use as temporary substitutes, where the purpose is to allow healing processes to replace the absorbable material. However, these approaches have had limited success in the long-term correction of structures in the body.

[0093] As people age, facial rhytides and folds appear in response to loss of facial fat and decreased skin elasticity. Skin loses shape, acute wounds require longer periods to heal, and scar more easily. Over the years, physicians have tried various methods and materials to address facial volume loss in facial soft tissue. Scientists and physicians are constantly searching for the ideal dermal filler.

[0094] Soft tissue conditions further include, for example, skin conditions (e.g., scar revision or treatment of traumatic wounds, severe burns, skin ulcers (e.g., decubitus (pressure) ulcers, venous ulcers, and diabetic ulcers), and surgical wounds (e.g., surgical wounds associated with the removal of skin cancer), vascular conditions (e.g., vascular diseases (e.g., peripheral arterial disease, abdominal aortic aneurysm, carotid artery disease, and venous disease); vascular injuries; improper vascular development), conditions affecting the vocal cords, cosmetic conditions (e.g., conditions involving repair, augmentation, or beautification), muscle diseases, connective tissue (e.g., tendons and ligaments, including, but not limited to, the periodontal ligament and anterior cruciate ligament), and conditions of organs and / or fascia (e.g., bladder, bowel, pelvic floor).

[0095] Degenerated and damaged soft tissues of the musculoskeletal system are the cause of and increase the risk of various medical complications that result in severe pain and restricted movement.For example, degenerated and damaged soft tissues of the spine are the main source of back pain for millions of people worldwide.Soft tissue degeneration of ligaments and intervertebral discs also increases the risk of damage to and back pain from local spinal joints, including zygapophysical (facet joints), costovertebral joints, sacroiliac joints, sacral joints and atlantoaxial joints.

[0096] In some embodiments, the polymer conjugate of the present invention is for use in medical treatment. In some embodiments, the polymer conjugate of the present invention is for use in treating diseases, disorders or conditions associated with soft tissue in mammals. In some embodiments, the polymer conjugate of the present invention is for use in treating diseases, disorders or conditions associated with soft tissue loss and / or damage, where administering the conjugate of the present invention to a soft tissue site results in full or partial functional recovery of the soft tissue.

[0097] In some embodiments, the soft tissue treated according to the present invention is selected from the group consisting of intervertebral disc, skin, heart valve, articular cartilage, cartilage, meniscus, adipose tissue, craniofacial, ocular, tendon, ligament, fascia, fibrous tissue, synovium, muscle, nerve, blood vessel, and any combination thereof. In some embodiments, the polymer conjugates of the present invention are for use in skin repair, orthopedic repair, urology, wound repair, and topical cosmetics.

[0098] In some embodiments, the polymer conjugates of the invention are for use in treating a disease, disorder, or condition associated with ECM degradation in a mammal, hi some embodiments, the polymer conjugates of the invention are for use in treating a disease, disorder, or condition associated with ECM deficiency and / or impairment, where administration of the conjugates of the invention to the ECM results in, in whole or in part, functional restoration of the ECM.

[0099] In some embodiments, the polymer conjugates of the invention provide a method of slowing the onset of soft tissue loss (e.g., preventing soft tissue loss). In some embodiments, the polymer conjugates of the invention provide a method for augmenting soft tissue. In some embodiments, the polymer conjugates of the invention provide a method for cosmetic augmentation. In some embodiments, the polymer conjugates of the invention provide a method of treating a subject suffering from age-related degeneration of connective tissue or a disease associated with degeneration of connective tissue.

[0100] In some embodiments, the polymer conjugates of the invention are for use in acute wound healing, hi some embodiments, the polymer conjugates of the invention are for use in regenerative medicine.

[0101] Interstitial cystitis (IC) or painful bladder syndrome (BPS) is a chronic disease that affects 4 to 12 million people in the United States, mostly women. IC / BPS is characterized by frequent urination, increased urgency, and pain associated with bladder filling. Thus, the polymer conjugates of the present invention are preferably for use in treating damaged urothelium of the bladder found in patients suffering from painful bladder syndrome or interstitial cystitis. In some embodiments, the polymer is preferably administered to the bladder by intravesical instillation.

[0102] Despite the unknown etiology, and without being limited to any particular theory, one leading theory proposes that bladder pain symptoms result from a loss of a dense impermeable barrier at the bladder luminal surface that triggers activation of visceral afferent fibers that innervate the urothelium. The "umbrella cells" that make up the luminal cell layer responsible for bladder impermeability may be absent or may not be fully differentiated, the normal layer of glycosaminoglycans (GAGs) at the surface is compromised, and the expression of tight junction component proteins is altered. Parsons found that IC patients exhibit significantly greater absorption of urea that flows into the bladder than controls. Hurst unambiguously demonstrated using MRI that the urothelium of IC / BPS patients is significantly more permeable than normal controls. What is unclear is how the bladder loses its impermeability. Evidence suggests that it may arise both endogenously, via neural connections possibly regulated by inflammatory cells, and from substances in the urine or from loss of cation sequestrants.

[0103] Treatment options for IC / BPS are limited, despite a wide variety of drugs that have been tried. Some success has been achieved through restoration of urothelial impermeability by GAG replacement therapy (30-32). GAG replacement involves intravesicular administration of chondroitin sulfate and hyaluronan (either alone or together), heparin, or pentosan polysulfate (ELMIRON®). Unfortunately, response rates rarely exceed 50%-60%. The limited efficacy of current GAG replacement therapies may be explained by the inability of these agents to recapitulate the native GAG ​​layer of the urothelium. The GAG ​​layer of the urothelium is composed of proteoglycans (PGs), mainly biglycan and perlecan. PGs are glycoproteins that are usually replaced by clusters of sulfated GAG chains, resulting in regions of significant anionic charge that increase the interaction of these sulfated GAGs with other biomolecules. The resulting osmotic pressure ensures highly effective hydration for PG-rich tissues and interfaces. Current efforts for GAG supplementation in IC / BPS only deliver linear, single-chain GAGs, such as hyaluronic acid (which is not sulfated), or sulfated GAGs with low MW (<50 kDa), such as chondroitin sulfate. These single-chain GAGs cannot mimic the clustered sulfated GAG environment provided by PGs on the surface of native urothelium. PGs are complex biomolecules that are difficult to isolate and purify from tissues, and thus PGs themselves are not practical therapeutic agents.

[0104] However, the proteoglycan-mimetic conjugated polymers of the present invention mimic the PG structure by displaying a multivalent array of sulfated GAG chains to bind to biological surfaces in a way that is not possible for a single linear GAG chain. In order to restore bladder impermeability in IC / BPS, binding to the bladder endothelium is crucial, and therefore this multivalent display of sulfated GAG chains provided by the proteoglycan-mimetic conjugated polymers of the present invention represents a meaningful innovation. Preferably, the proteoglycan mimics of the present invention provide targeted treatment of IC / BPS, for example, by further functionalization with glycan ligands for galectins, such as ligands containing β-galactosides. For example, such polymer conjugates "decorated" with β-galactosides will target galectins present in the bladder epithelium. Thus, the present invention provides a method of treating interstitial cystitis (IC) in a patient, comprising administering to the patient a polymer conjugate of the present invention, preferably a polymer conjugate of the present invention, in which at least one sulfated GAG polymer chain comprises at least one glycan ligand for a galectin (e.g., a β-galactoside).

[0105] In some embodiments, the polymer conjugate of the present invention is for use in treating degenerated intervertebral discs. In some embodiments, the polymer conjugate of the present invention is for use in a method of administering the polymer conjugate into the nucleus of a degenerated intervertebral disc to increase the osmotic potential of the disc. By administering the material of the polymer conjugate into the nucleus of a degenerated disc, normal disc height and function can be restored. Preferably, the polymer conjugate of the present invention is administered by direct injection into the intervertebral disc. Such administration can result in full or partial restoration of the load-bearing and viscoelastic properties of the defective intervertebral disc.

[0106] In some embodiments, the polymer conjugate of the present invention is for use in osteoarthritis OA of knee joints and other joints. OA, also known as degenerative joint disease, is the most common form of arthritis and results from the slow breakdown of cartilage associated with aging. Typically, OA occurs following chronic joint damage caused by trauma or some other type of arthritis (such as rheumatoid arthritis). Alternatively, OA may result from overuse of a particular joint. OA most commonly involves the joints of the elbow, finger, hip, knee, shoulder, wrist, spine and toes. Clinically, OA is characterized by joint pain, tenderness, limited movement, crepitus and relentlessly progressive disability. OA may be present in only one of these joints or in all of these joints. Although most body tissues are capable of repairing themselves after injury, it is believed that cartilage repair is hindered by limited blood supply and lack of effective mechanisms for cartilage regrowth. Preferably, the present invention provides a method for administering the polymer conjugate of the present invention to a patient suffering from OA.Preferably, the polymer conjugate of the present invention may be administered to a patient by direct injection into affected joint.Preferably, the polymer conjugate of the present invention may be administered to a patient by direct injection into affected joint in combination with additional viscosupplementation therapy, including (but not limited to) hyaluronic acid (HA)-containing viscosupplements, such as EUFLEXXA®, HYALGAN®, ORTHOVISC®, SUPARTZ® and SYNVISC®.

[0107] The polymer conjugates of the present invention can be used in conjunction with any known or heretofore unknown method of treating a disease or condition of the intervertebral disc in a mammal. Preferably, the subject is a human.

[0108] Administration In some embodiments, the polymer conjugates of the present invention may be formulated with one or more excipients, buffers, carriers, stabilizers, preservatives and / or bulking agents. In some embodiments, the polymer conjugates of the present invention may be formulated using excipients that are completely biocompatible (i.e., non-toxic). In some embodiments, the polymer conjugates of the present invention may be formulated using excipients and buffered at physiological pH with salts (e.g., sodium phosphate salts). The polymer conjugates of the present invention may be administered to a subject at a soft tissue site for functional restoration thereof using various methods and in various formulations known in the art. The method of administration is selected depending on the condition and pharmaceutical composition being treated. The administration of the polymer conjugates of the present invention may be performed in various ways, including, but not limited to, dermal, subcutaneous, intravenous, oral, topical, transdermal, intraperitoneal, intramuscular and intravesical. For example, microparticle, microsphere and microcapsule formulations are useful for oral, intramuscular or subcutaneous administration. In addition, liposome and nanoparticle are suitable for intravenous administration.The administration of the polymer conjugate of the present invention may be via only one route or may be via several routes simultaneously.For example, oral administration may involve intravenous injection or parenteral injection.

[0109] Preferably, the subject compositions are administered by intravesical instillation. This procedure generally involves inserting a catheter into the urinary tract and filling the bladder with a suitable diluent containing the subject compositions. Filling may be performed by manual infusion or by kidney pump. Powered drug administration can further assist intravesical drug delivery (see, e.g., Riedl, CR et al., J. Endourol., 12:269-72 (1998); which is incorporated by reference).

[0110] Preferably, the conjugates of the present invention are administered by direct injection into the dermis using a small-bore needle or microneedle or microneedle array. As branched biopolymers, the polymer conjugates of the present invention have the advantage of being of low viscosity when in solution, which facilitates injection through a small-bore needle.

[0111] In some embodiments, it is preferred that the polymer conjugate of the present invention does not degrade appreciably after administration.In some embodiments, it is preferred that the composition of the present invention degrades in tissues, either quickly or slowly.Thus, when administered to the body, the polymer conjugate may be permanent, may be enzymatically degraded, or may be degraded in the presence of a solvent (such as water).

[0112] The method of the present invention includes determining the optimal dose of the compound and pharmaceutical composition for treating IC symptoms, and the optimal dose may be determined by considering the results of animal experiments.More specific doses obviously vary depending on the administration method, the condition of the subject, such as age, weight, sex, sensitivity, food, administration interval, co-administered drugs, and the severity and degree of IC.The optimal dose and administration frequency under given conditions must be determined by appropriate dosage tests by medical professionals based on these guidelines, and do not require undue experimentation by those skilled in the art.

[0113] The polymer conjugates of the present invention may also be administered using sustained release or long-term delivery methods well known to those skilled in the art. By "sustained release" or "long-term release" as used herein, it is meant that the delivery system administers a pharma- ceutical amount of the polymer conjugate for more than one day, preferably for more than one week, and most preferably for at least about 30 to 60 days or more. Long-term release systems may include implantable solids or gels (e.g., biodegradable polymers, etc.) that contain the polymer conjugate.

[0114] The polymer conjugates of the present invention may be administered in combination with one or more other drugs (or as any combination thereof). The polymer conjugates of the present invention may be usefully combined with another pharmacologically active compound, or with two or more other pharmacologically active compounds, to treat pain and / or lower urinary tract symptoms (LUTS) associated with IC and / or painful bladder syndrome and / or painful bladder syndrome. For example, the polymer conjugates of the present invention may be administered simultaneously, sequentially, or separately in combination with one or more drugs selected from the following drugs: Opioid analgesics, such as morphine, heroin, hydromorphone, oxymorphone, levorphanol, levallorphan, methadone, meperidine, fentanyl, cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine or pentazocine; Nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, nimesulide, nitroflurbiprofen, olsalazine, oxaprozin, phenylbutazone, piroxicam, sulfasalazine, sulindac, tolmetin or zomepirac; Barbiturates such as amobarbital, aprobarbital, butabarbital, butabital, mephobarbital, metharbital, methohexital, pentobarbital, phenobarbital, secobarbital, talbutal, theamylal or thiopental; benzodiazepines with sedative properties, such as chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam or triazolam; Sedating H1 antagonists, such as diphenhydramine, pyrilamine, promethazine, chlorpheniramine or chlorcyclizine; Sedatives, such as glutethimide, meprobamate, methaqualone, or dichloralphenazone; Skeletal muscle relaxants such as baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol or orphrenadine; NMDA receptor antagonists, such as dextromethorphan ((+)-3-hydroxy-N-methylmorphinan) or its metabolite dextrorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine, pyrroloquinoline quinine, cis-4-(phosphonomethyl)-2-piperidine carboxylic acid, budipine, EN-3231 (MorphiDex®, a combination formulation of morphine and dextromethorphan), topiramate, neramexane or perzinfotel, as well as NR2B antagonists, such as ifenprodil, traxoprodil or (−)-(R)-6-{2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl-3,4-dihydro-2(1H)-quinolinone; Alpha-adrenergic agents, such as doxazosin, tamsulosin, clonidine, guanfacine, dexmetatomidine, modafinil, terazosin, indoramin, alfuzosin, silodosin or 4-amino-6,7-dimethoxy-2-(5-methane-sulfonamido-1,2,3,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl)quinazoline; prazosin; Tricyclic antidepressants, such as desipramine, imipramine, amitriptyline or nortriptyline; Anticonvulsants, such as carbamazepine, lamotrigine, topiratomate or valproate; Tachykinin (NK) antagonists, in particular NK-3 antagonists, NK-2 antagonists or NK-1 antagonists, such as (αR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]-naphthyridine-6-13-dione (TAK-637), 5-[[(2R, 3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorophenyl)-4-morpholinyl]-methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one (MK-869), aprepitant, lanepitant, dapitant or 3-[[2-methoxy-5-(trifluoromethoxy)phenyl]-methylamino]-2-phenylpiperidine (2S,3S); Muscarinic antagonists, such as oxybutynin, tolterodine, fesoterodine, 5-hydroxymethyltolterodine, propiverine, trospium chloride, darifenacin, solifenacin, temiverine and ipratropium; COX-2 selective inhibitors, such as celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib or lumiracoxib; Coal tar pain relievers, especially acetaminophen / paracetamol; Neuroleptics, such as droperidol, chlorpromazine, haloperidol, perphenazine, thioridazine, mesoridazine, trifluoperazine, fluphenazine, clozapine, olanzapine, risperidone, ziprasidone, quetiapine, sertindole, aripiprazole, sonepiprazole, blonanserin, iloperidone, perospirone, raclopride, zotepine, bifeprunox, asenapine, lurasidone, amisulpride, balaperidone, palindore, eplivanserin, osanetant, rimonabant, meclinertant, MIRAXION® or sarizotan; Vanilloid receptor agonists (e.g., resinferatoxin) or antagonists (e.g., capsazepine); Beta-adrenergic agents, such as propranolol; Local anesthetics, such as mexiletine; Corticosteroids, such as dexamethasone; 5-HT receptor agonists or antagonists (e.g., pizotifen), especially 5-HT 1B / 1D Agonists, such as eletriptan, sumatriptan, naratriptan, zolmitriptan or rizatriptan; 5-HT 2A Receptor antagonists, such as R(+)-alpha-(2,3-dimethoxy-phenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (MDL-100907); Cholinergic (nicotinic) analgesics, such as ispronicline (TC-1734), (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine (RJR-2403), (R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594) or nicotine; Tramadol(trademark); PDE-5 inhibitors, such as 5-[2-ethoxy-5-(4-methyl-1-piperazinyl-sulfonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (sildenafil), (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazino[2'1',1:6,1]pyrido[3,4-b]ine Dole-1,4-dione (IC-351 or tadalafil), 2-[2-ethoxy-5-(4-ethyl-piperazin-1-yl-1-sulfonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one (vardenafil), 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3 -d]pyrimidin-7-one, 5-(5-acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-azetidinyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, 5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulfonyl)pyridin-3-yl]-3-ethyl-2-[2-methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one ion, 4-[(3-chloro-4-methoxybenzyl)amino]-2-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-N-(pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide, 3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamide, etc.; Alpha-2-delta ligands, such as gabapentin, pregabalin, 3-methylgabapentin, (1α,3α,5α)(3-amino-methyl-bicyclo[3.2.0]hept-3-yl)-acetic acid, (3S,5R)-3-aminomethyl-5-methyl-heptanoic acid, (3S,5R)-3-amino-5-methyl-heptanoic acid, (3S,5R)-3-amino-5-methyl-octanoic acid, (2S,4S)-4-(3-chlorophenoxy)proline, (2S,4S)-4-(3-fluorobenzyl)proline, [(1R,5R,6S)-6-(aminomethyl)bicyclo[3.2.0]hept-3-yl]-acetic acid, (3S,5R)-3-aminomethyl-5-methyl-heptanoic acid, (3S,5R)-3-amino-5-methyl-octanoic acid, but-6-yl]acetic acid, 3-(1-aminomethyl-cyclohexylmethyl)-4H-[1,2,4]oxadiazol-5-one, C-[1-(1H-tetrazol-5-ylmethyl)-cycloheptyl]methylamine, (3S,4S)-(1-aminomethyl-3,4-dimethyl-cyclopentyl)-acetic acid, (3S,5R)-3-amino-5-methyl-nonanoic acid, (3R,4R,5R)-3-amino-4,5-dimethyl-heptanoic acid, and (3R,4R,5R)-3-amino-4,5-dimethyl-octanoic acid; (3S,5R)-3-aminomethyl-5-methyloctanoic acid; Cannabinoids; metabotropic glutamate subtype 1 receptor (mGluR1) antagonists; Serotonin reuptake inhibitors, such as sertraline, the sertraline metabolite desmethylsertraline, fluoxetine, norfluoxetine (the desmethyl metabolite of fluoxetine), fluvoxamine, paroxetine, citalopram, the citalopram metabolite desmethylcitalopram, escitalopram, d,l-fenfluramine, femoxetine, ifoxetine, cyanodothiepin, litoxetine, dapoxetine, nefazodone, cericlamine, and trazodone; noradrenaline (norepinephrine) reuptake inhibitors, such as maprotiline, lofepramine, mirtazepine, oxaprotiline, fezolamine, tomoxetine, mianserin, buproprion, the buproprion metabolite hydroxybuproprion, nomifensine and viloxazine (VIVALAN®), especially selective noradrenaline reuptake inhibitors, such as reboxetine (especially (S,S)-reboxetine); Dual serotonin-norepinephrine reuptake inhibitors, such as venlafaxine, the venlafaxine metabolite O-desmethylvenlafaxine, clomipramine, the clomipramine metabolite desmethylclomipramine, duloxetine, milnacipran, and imipramine; Inhibitors of inducible nitric oxide synthase (iNOS), such as S-[2-[(1-iminoethyl)amino]ethyl]-L-homocysteine, S-[2-[(1-iminoethyl)-amino]ethyl]-4,4-dioxo-L-cysteine, S-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine, (2S,5Z)-2-amino-2-methyl-7-[(1-iminoethyl)amino]-5-heptenoic acid, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-5-chloro-3-pyridinecarbonitrile; 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5 -thiazolyl)butyl]thio]-4-chlorobenzonitrile, (2S,4R)-2-amino-4-[[2-chloro-5-(trifluoromethyl)phenyl]thio]-5-thiazolebutanol, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-6-(trifluoromethyl)-3-pyridinecarbonitrile, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-5-chlorobenzonitrile, N-[4-[2-(3-chlorobenzylamino)ethyl]phenyl]thiophene-2-carboxamidine, or guanidinoethyl disulfide; Acetylcholinesterase inhibitors, such as donepezil; Prostaglandin E2 subtype 4 (EP4) antagonists, such as N-[({2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c)pyridin-1-yl)phenyl]ethyl}amino)-carbonyl]-4-methylbenzenesulfonamide or 4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]benzoic acid; Leukotriene B4 antagonists, such as 1-(3-biphenyl-4-ylmethyl-4-hydroxy-chroman-7-yl)-cyclopentanecarboxylic acid (CP-105696), 5-[2-(2-carboxyethyl)-3-[6-4-methoxyphenyl)-5E-hexenyl]oxyphenoxy]-valeric acid (ONO-4057) or DPC-11870; 5-lipoxygenase inhibitors, such as zileuton, 6-[(3-fluoro-5-[4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4-yl)phenoxy-methyl]-1-methyl-2-quinolone (ZD-2138), or 2,3,5-trimethyl-6-(3-pyridylmethyl),1,4-benzoquinone (CV-6504); Sodium channel blockers, such as lidocaine; or bupivicaine 5-HT3 antagonists, such as ondansetron; Glycosaminoglycan layer substitutes and anti-inflammatory agents, such as pentosan polysulfate (Elmiron-trademark); beta-3 agonists, such as YM-178 (mirabegron or 2-amino-N-[4-[2-[[(2R)-2-hydroxy-2-phenylethyl]amino]ethyl]phenyl]-4-thiazoleacetamide), solabegron, KUC-7483 (ritobegron or 2-[4-[2-[[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-2,5-dimethylphenoxy]-acetic acid) or AK-134; Antihistamines, such as hydroxyzine; H2-antagonists, such as cimetidine; or ranitidine silver nitrate; steroid; Doxorubicin; Chondroitin sulfate; Disodium chromoglycate; Oxychlorosene (Clorpactin-brand); and Immunosuppressants such as cyclosporine.

[0115] example The following examples are meant to illustrate certain embodiments of the invention and are not meant to limit the scope of the invention.

[0116] material and method Chondroitin sulfate was obtained from Bioiberica: EP injection grade (GPC data from the supplier: Mn=11,400, Mw=13,700 Da, PDI=1.21). The equivalent weight of the structural repeat unit of disodium chondroitin sulfate A was 503.35 g / equivalent (C 14 H 19 O 14 SNa2) with a hydroxyl equivalent weight of 503.35 / 3=167.78 g / OH equivalent. Divinyl sulfone (99%) was purchased from ACROS Organics. Carboxymethyl cellulose (MW=250 kDa and 90 kDa, degree of substitution=0.80, 226.16 g / eq, 113.08 g / OH equivalent) was purchased from Sigma Aldrich.

[0117] Protocol for DLS Dynamic light scattering analysis was performed on a DynaPro Nanostar instrument (Wyatt Technology) using Wyatt cyclic olefin copolymer disposable microcuvettes. Data were collected at 25°C with 10 s acquisition time and hydrodynamic radii were averaged over 20 acquisitions. Data fitting was performed using DYNAMICS software (version 7.5; Wyatt Technology) to obtain hydrodynamic radii and estimate molar masses.

[0118] Example 1. Synthesis of soluble high MW chondroitin sulfate compositions by direct reaction with DVS under conditions where no gel is formed. Sodium chondroitin sulfate (0.34 g, 2.0 mmol equivalent hydroxyl groups) was dissolved in 2.25 mL of DI water in an 8 mL reaction vessel. A clear, colorless solution resulted. DVS (0.034 g, 30 uL, 0.28 mmol) was added volumetrically via microliter pipette. After gentle mixing, the solution was clear and colorless. The reaction was initiated by adding 0.25 mL of 1.0 N NaOH using a microliter pipette. Upon addition of NaOH, the solution immediately turned pale yellow in color while remaining clear. The reaction is 12 wt% chondroitin sulfate and 0.1 M NaOH (approximately pH 13). The reaction was gently mixed on a rotisserie. The reaction remained fluid and clear over time. Under these reaction conditions (12 wt% polymer, DVS / hydroxyl=0.14), no gel formation was observed for periods of more than 1 hour. The reaction was stopped 40 minutes after initiation by adding 0.25 mL of 1.0 N HCl. The pH after neutralization was found to be approximately 5.0. The reaction was diluted with PBS to a total volume of 25 mL.

[0119] The diluted reactions were analyzed using dynamic light scattering and compared to a control sample obtained from a null reaction that was identical in all respects except that DVS was omitted. The following hydrodynamic radius and Mw values ​​were found (Table 1): [Table 1]

[0120] Example 2. Synthesis of soluble high MW chondroitin sulfate compositions by direct reaction with DVS with quenching prior to gel formation. Sodium chondroitin sulfate (0.34 g, 2.0 mmol equivalent hydroxyl groups) was dissolved in 2.25 mL of DI water in an 8 mL reaction vessel. A clear, colorless solution was obtained. DVS (0.067 g, 60 uL, 0.57 mmol) was added volumetrically via microliter pipette. After gentle mixing, the solution was clear and colorless. The reaction was started by adding 0.25 mL of 1.0 N NaOH using a microliter pipette. Upon addition of NaOH, the solution immediately turned pale yellow in color while remaining clear. The reaction is 12 wt% chondroitin sulfate, DVS / hydroxyl=0.28, and 0.1 M NaOH (pH 13). The reaction was gently mixed on a rotisserie. The reaction became more viscous over time while remaining clear. Forty minutes after the start of the reaction, a rapid viscosity increase was observed and the reaction was stopped just before gelation by adding 0.25 mL of 1.0 N HCl. The pH after neutralization was found to be approximately 5.0. The viscous reaction solution was diluted with PBS to a total volume of 25 mL.

[0121] The diluted reactions were analyzed using dynamic light scattering and compared to a control sample derived from a null reaction that was identical in all respects except that DVS was omitted. The following hydrodynamic radius and Mw values ​​were found (Table 2): [Table 2]

[0122] Example 3. Synthesis of soluble high MW chondroitin sulfate compositions modified with type II collagen binding peptides by direct reaction with DVS with quenching prior to gel formation. Peptide-modified chondroitin sulfate can be prepared using methods described in the literature [Caravan, U.S. Patent No. 9,386,938 (B2)] [Panitch, U.S. Patent No. 9,200,039 (B2)]. The peptide-modified chondroitin sulfate (2.0 mmol equivalent of hydroxyl groups) is dissolved in 2.25 mL of DI water in an 8 mL reaction vessel to obtain a clear, colorless solution. DVS (0.067 g, 60 uL, 0.57 mmol) is volumetrically added with a microliter pipette and gently mixed to obtain a clear, colorless solution. The reaction is started by adding 0.25 mL of 1.0 N NaOH using a microliter pipette. The reaction is greater than 12 wt% peptide-modified chondroitin sulfate and 0.1 M NaOH (pH 13). The reaction is gently mixed on a rotisserie. The reaction becomes slightly more viscous over time, but remains clear. A rapid viscosity increase may be observed 20-40 minutes after the start of the reaction, at which point the reaction is stopped just before gelation by adding 0.25 mL of 1.0 N HCl. The pH after neutralization is approximately 5.0. The viscous reaction solution can be diluted with PBS to a total volume of 25 mL.

[0123] The diluted reactions can be analyzed using dynamic light scattering and compared to a control sample obtained from a null reaction that is identical in all respects except that DVS was omitted. The product of Example 3 will have a significantly higher hydrodynamic radius and Mw relative to the null reaction comparator.

[0124] Example 4. Synthesis of a soluble chondroitin sulfate / carboxymethylcellulose composition by direct reaction with DVS. Carboxymethylcellulose (0.050 g, 0.44 mmol equivalents of hydroxyl groups) and chondroitin sulfate (0.600 g, 3.58 mmol equivalents of hydroxyl groups) are dissolved in 4.5 mL of DI water in an 8 mL reaction vessel. A clear, colorless solution is obtained. DVS (0.100 g, 85 uL, 0.85 mmol) is added volumetrically with a microliter pipette. After gentle mixing, the solution is clear and colorless. The reaction is started by adding 0.5 mL of 1.0 N NaOH using a microliter pipette. Upon addition of NaOH, the solution immediately turns pale yellow in color while the solution remains clear. The reaction has a CMC of 0.88 wt%, chondroitin sulfate of 10.62 wt%, and NaOH of 0.1 M (approximate pH 13). The reaction is gently mixed on a rotisserie. The reaction solution becomes more viscous over time while remaining clear. A few minutes after the start of the reaction, a rapid increase in viscosity is observed and the reaction is stopped by adding 0.25 mL of 1.0 N HCl. The neutralized pH is found to be approximately 5.0. The viscous reaction solution is diluted with PBS to a total volume of 25 ml.

[0125] The diluted reactions are analyzed using dynamic light scattering and compared to control samples obtained from null reactions that are identical in all respects except that DVS was omitted. The following hydrodynamic radius and Mw values ​​are found (Table 3): [Table 3]

[0126] Example 5. Synthesis of soluble high MW chondroitin sulfate with bottlebrush-like structure by stepwise addition of reactants in one pot. Sodium chondroitin sulfate (0.11 g, 0.66 mmol equivalents of hydroxyl groups) was dissolved in 4.5 mL of DI water in an 8 mL reaction vessel. A clear, colorless solution was obtained. DVS (0.097 g, 82 uL, 0.82 mmol) was added volumetrically via a microliter pipette. After gentle mixing, the solution was clear and colorless. The reaction was started by adding 0.5 mL of 1.0 N NaOH using a microliter pipette. Upon addition of NaOH, the solution immediately turned pale yellow in color while remaining clear. The reaction is 2.15 wt% chondroitin sulfate and 0.1 M NaOH (pH 13). The reaction was mixed gently on a rotisserie. After 30 minutes, additional sodium chondroitin sulfate was added (0.572 g, 3.41 mmol equivalents of hydroxyl groups) and the reaction mixture was stirred on a rotisserie. The reaction became more viscous but remained clear. Five hours after initiation, the reaction had not formed a gel. The reaction remained a pale yellow, clear, viscous solution, and the reaction was stopped by adding 0.5 mL of 1.0 N HCl. The pH after neutralization was found to be approximately 5.0.

[0127] A comparative reaction was run in parallel (Table 4). In the comparative reaction, all of the chondroitin sulfate (0.682 g, 4.1 mmol equivalents of hydroxyl groups) was added at once at the start of the reaction. This reaction mixture was also clear, pale yellow, and viscous. The reaction mixture became increasingly viscous and gelled after 52 minutes. [Table 4]

[0128] Example 6. Synthesis of soluble high MW proteoglycan-mimetic polymers with bottlebrush-like structures by stepwise addition of CMC followed by chondroitin sulfate in a one-pot process. Sodium carboxymethylcellulose (250 kDa, 0.050 g, 0.44 mmol equivalent hydroxyl groups) is dissolved in 4.5 mL of DI water in an 8 mL reaction vessel. A clear, colorless solution results. DVS (0.100 g, 85 uL, 0.85 mmol) is added volumetrically via a microliter pipette. After gentle mixing, the solution is clear and colorless. The reaction is started by adding 0.5 mL of 1.0 N NaOH using a microliter pipette. Upon addition of NaOH, the solution immediately turns pale yellow in color while the solution remains clear. The reaction has a CMC of 0.88 wt% and NaOH of 0.1 M (pH 13). The reaction is gently mixed on a rotisserie. After 30 minutes, sodium chondroitin sulfate is added (0.600 g, 3.58 mmol equivalent hydroxyl groups). The chondroitin sulfate is completely dissolved after approximately 5 minutes of stirring on a rotisserie. The reaction becomes viscous but remains clear. 90 minutes after addition of the chondroitin sulfate, the clear viscous reaction is quenched by adding 0.5 mL of 1.0 N HCl. The pH after neutralization is found to be approximately 5.0.

[0129] The diluted reactions are analyzed using dynamic light scattering and compared to a control sample obtained from a null reaction that is identical in all respects except that DVS was omitted. The product of Example 6 is found to have a much larger hydrodynamic radius and Mw relative to the null reaction counterpart.

[0130] Example 7. Synthesis of soluble high MW chondroitin sulfate with bottlebrush-like structure in a two-step reaction sequence. 7A. Synthesis and Characterization of Vinyl Sulfone-Modified Chondroitin Sulfate Sodium chondroitin sulfate (0.11 g, 0.66 mmol equivalents of hydroxyl groups) was dissolved in 4.5 mL of DI water in an 8 mL reaction vessel. A clear, colorless solution was obtained. DVS (0.097 g, 82 uL, 0.82 mmol) was added volumetrically via a microliter pipette. After gentle mixing, the solution was clear and colorless. The reaction was started by adding 0.5 mL of 1.0 N NaOH using a microliter pipette. Upon addition of NaOH, the solution immediately turned light yellow in color while remaining clear. The reaction was 2.15 wt% chondroitin sulfate and 0.1 M NaOH (pH 13). The reaction was mixed gently on a rotisserie. After 30 minutes, the clear reaction was quenched by adding 0.5 mL of 1.0 N HCl. The reaction mixture was then dripped into a 50 mL conical centrifuge tube containing 25 ml of ethanol. A white solid immediately formed. The solid was collected by centrifugation followed by removal of the supernatant. The solid was suspended in another 25 mL volume of ethanol in the same tube, shaken, and then centrifuged again. The solid was collected after removal of the supernatant and placed under high vacuum to remove residual ethanol and water.

[0131] DVS-functionalized chondroitin sulfate can be characterized by H-NMR and the extent of DVS functionalization can be quantified by integration of the pendant vinyl groups.

[0132] 7B. Formation of a soluble, high MW, proteoglycan-mimetic polymer with a bottlebrush-like structure by reacting chondroitin sulfate with vinylsulfone-modified chondroitin sulfate.

[0133] The white solid formed in reaction 7A is dissolved in 4.5 mL of DI water in an 8 mL reaction vessel. A clear, colorless solution is obtained. Sodium chondroitin sulfate is added (0.572 g, 3.41 mmol equivalent of hydroxyl groups) and the reaction mixture is stirred on a rotisserie for several minutes until all the polymer is dissolved. The reaction is started by adding 0.5 mL of 1.0 N NaOH using a microliter pipette. The reaction is mixed gently on a rotisserie. After 2 hours the reaction becomes viscous but remains clear. The reaction is stopped by adding 0.5 mL of 1.0 N HCl. The pH after neutralization is found to be approximately 5.0. The viscous reaction is diluted with PBS to a total volume of 25 mL.

[0134] The diluted reaction solution can be analyzed using dynamic light scattering and compared to a control sample of the starting chondroitin sulfate material. The product of Example 7B will have a much larger hydrodynamic radius and Mw relative to the starting material comparator.

[0135] Example 8. Synthesis of a soluble, high MW CMC / chondroitin sulfate composition with bottlebrush-like structure in a two-step reaction sequence. 8A. Synthesis and Characterization of Vinyl Sulfone-Modified CMC Sodium carboxymethylcellulose (250 kDa, 0.050 g, 0.44 mmol equivalents of hydroxyl groups) was dissolved in 4.5 mL of DI water in an 8 mL reaction vessel. A clear, colorless solution was obtained. DVS (0.100 g, 85 uL, 0.85 mmol) was added volumetrically via a microliter pipette. After gentle mixing, the solution was clear and colorless. The reaction was initiated by adding 0.5 mL of 1.0 N NaOH using a microliter pipette. Upon addition of NaOH, the solution immediately turned pale yellow in color while the solution remained clear. The reaction had a CMC of 0.88 wt% and 0.1 M NaOH (pH 13). The reaction was mixed gently on a rotisserie. After 30 minutes, the clear reaction was quenched by adding 0.5 mL of 1.0 N HCl. The reaction mixture was then dripped into a 50 mL conical centrifuge tube containing 25 mL of ethanol. A white solid formed immediately. The solid was collected by centrifugation followed by removal of the supernatant. This solid was suspended in another 25 mL volume of ethanol in the same tube, shaken, and then centrifuged again. The solid was collected after removal of the supernatant and placed under high vacuum to remove residual ethanol and water.

[0136] DVS-functionalized CMC can be characterized by H-NMR and the extent of DVS functionalization can be quantified by integration of the pendant vinyl groups.

[0137] 8B. Formation of a soluble, high MW, proteoglycan-mimetic polymer with a bottlebrush-like structure by reacting chondroitin sulfate with vinyl sulfone-modified CMC.

[0138] The white solid formed in reaction 8A is dissolved in 4.5 mL of DI water in an 8 mL reaction vessel. A clear, colorless solution is obtained. Sodium chondroitin sulfate is added (0.572 g, 3.41 mmol equivalent of hydroxyl groups) and the reaction mixture is stirred for several minutes until all the polymer has dissolved. The reaction is started by adding 0.5 mL of 1.0 N NaOH using a microliter pipette. The reaction is mixed gently on a rotisserie. After 2 hours the reaction becomes viscous but remains clear. The reaction is stopped by adding 0.5 mL of 1.0 N HCl. The pH after neutralization is found to be approximately 5.0. The viscous reaction is diluted with PBS to a total volume of 25 mL.

[0139] The diluted reaction solution can be analyzed using dynamic light scattering and compared to a control sample of the starting chondroitin sulfate material. The diluted reaction solution can also be compared to a solution of the CMC starting material. The product of Example 8B will have a significantly higher hydrodynamic radius and Mw relative to the comparison of these two starting materials.

[0140] Example 9. Synthesis of a soluble, high MW, branched chondroitin sulfate composition with a bottlebrush-like structure by stepwise addition of reactants in one pot, purification of the product using tangential flow filtration, and SEC analysis of the purified isolated product. 9A. Stepwise addition reactions Sodium chondroitin sulfate (0.153 g, 0.913 mmol equivalents of hydroxyl groups) was dissolved in 4.865 g of DI water in an 8 mL reaction vessel. A clear, colorless solution was obtained. DVS (0.127 g, 108 uL, 1.07 mmol) was added volumetrically via a microliter pipette. After gentle mixing, the solution was clear and colorless. The reaction was started by adding 0.51 mL of 1.0 N NaOH using a microliter pipette. Upon addition of NaOH, the solution immediately turned pale yellow in color while remaining clear. The reaction is 2.69 wt% chondroitin sulfate and 0.1 M NaOH (pH 13). The reaction was gently mixed on a rotisserie. After 10 minutes, additional sodium chondroitin sulfate was added (0.454 g, 3.62 mmol equivalents of hydroxyl groups) and the reaction mixture was stirred on a rotisserie. The reaction became more viscous but remained clear and mobile. Two hours after initiation with NaOH, the reaction was stopped by adding 0.51 mL of 1.0 N HCl using a microliter pipette. The clear, mobile reaction mixture was added to a vial containing 30 g of PBS and the total weight was brought to 50 g by addition of PBS.

[0141] 9B. Purification Using Tangential Flow Filtration A Spectrum Labs KR2i TFF system was equipped with a small volume feed reservoir (50 ml) and a 20 cm hollow fiber filter module containing modified polyethersulfone filter fibers (1 mm diameter, 100 kDa MWCO, 75 cm 2A sample of the diluted product of Example 9A (46 g) was placed in a small volume feed reservoir. Tangential flow filtration was started at a flow rate of 100 ml / min, which was increased (increments of 50 ml / min) to 300 ml / min while keeping the inlet pressure below 20 psig. TFF was performed in dialysis mode where the volume of solution lost to the permeate was continuously replenished with fresh deionized water. In this way, the volume of the retentate remained constant for the duration of the filtration procedure during which 6 volumes (270 ml) of permeate were obtained. The make-up of deionized water was then discontinued and filtration was performed in concentration mode to reduce the retentate volume to approximately 30 mL. TFF was then stopped and the system was flushed (10 ml DI water) to recover the retentate. The purified retentate (approximately 40 mL) was then dried by lyophilization for 72 hours, which gave the purified product (0.251 g, 43% of the starting chondroitin sulfate weight) as a white fluffy solid.

[0142] 9C. Size Exclusion Chromatography Analysis SEC analysis was performed on an Agilent 1100 HPLC system equipped with an Agilent G1312 binary pump, G1322A micro degasser, G1367A well plate autosampler, G1316 column compartment, Wyatt Dawn EOS multi-angle light scattering detector, and OptiLab rEx refractive index detector. A dn / dc value of 0.15 was used. The column chosen for separation was a TSK6000 in addition to a guard column. Samples were diluted to a concentration of 5 mg / mL in PBS (pH=7.4), filtered through a 0.45 micron PVDF syringe filter, and analyzed using the conditions in Table 5 below. [Table 5]

[0143] The SEC analysis of the product was compared to that of the starting chondroitin sulfate material, and the following results were obtained (Table 6): [Table 6]

[0144] Example 9 demonstrates that the stepwise addition reaction protocol results in a soluble polymer that is filterable through a 0.45 um membrane and can be purified by tangential flow filtration using a 100 kDa MWCO filter. The purified polymer is obtained in good yield, has a significantly higher molecular weight than the starting material, and has a branched conformation.

[0145] Example 10. Synthesis of a high MW soluble chondroitin sulfate composition with bottlebrush-like structure by stepwise addition of reactants in a one-pot with sodium chloride present in the second step, purification of the product using tangential flow filtration, and SEC analysis of the purified isolated product. 10A. Stepwise addition reaction with salt present in the second step Sodium chondroitin sulfate (0.154 g, 0.920 mmol equivalents of hydroxyl groups) was dissolved in 4.865 g of DI water in an 8 mL reaction vessel. A clear, colorless solution was obtained. DVS (0.127 g, 108 uL, 1.07 mmol) was added volumetrically via a microliter pipette. After gentle mixing, the solution was clear and colorless. The reaction was started by adding 0.51 mL of 1.0 N NaOH using a microliter pipette. Upon addition of NaOH, the solution immediately turned pale yellow in color while remaining clear. The reaction is 3.07 wt% chondroitin sulfate and 0.1 M NaOH (pH 13). The reaction was gently mixed on a rotisserie. After 20 minutes, additional sodium chondroitin sulfate was added (0.454 g, 3.63 mmol equivalents of hydroxyl groups). Sodium chloride was also added (47.6 mg, 0.81 mmol) and the reaction mixture was stirred on a rotisserie. The reaction became increasingly viscous but remained clear and mobile. Two hours after initiation with NaOH, the reaction was stopped by adding 0.51 mL of 1.0 N HCl using a microliter pipette. The clear, mobile reaction mixture was added to a vial containing 30 g of PBS and the total weight was brought to 50 g by addition of PBS. The diluted reaction mixture was easily filtered through a 0.45 um PVDF syringe filter.

[0146] 10B. Purification using Tangential Flow Filtration The Spectrum Lab KR2i TFF system was equipped with a small volume feed reservoir (50 ml) and a 20 cm hollow fiber filter module containing modified polyethersulfone filter fibers (1 mm diameter, 100 kDa MWCO, 75 cm 2 A 100 ml / min total surface area was used with a 100 ml / min filter (part # D02-E100-10-N). A sample of the diluted product of Example 10A was placed in a small volume feed reservoir. Tangential flow filtration was started at a flow rate of 100 ml / min and the flow rate was increased (50 ml / min increments) to 300 ml / min while keeping the inlet pressure below 25 psig. TFF was performed in dialysis mode where the volume of solution lost to the permeate was continuously replenished with fresh deionized water. In this way, the volume of the retentate remained constant for the duration of the filtration procedure during which more than 6 volumes (>270 ml) of permeate were obtained. The make-up of deionized water was then discontinued and filtration was performed in concentration mode to reduce the retentate volume to approximately 30 mL. TFF was then stopped and the system was flushed (10 ml DI water) to recover the retentate. The purified retentate (approximately 40 mL) was then dried by lyophilization for 72 hours, which gave the purified product as a white fluffy solid.

[0147] 10C. Size Exclusion Chromatography Analysis SEC analysis was performed exactly as in Example 9C, and the following results were obtained (Table 7): [Table 7]

[0148] Example 10 demonstrates that the product of the stepwise addition reaction protocol was a soluble polymer that was filterable through a 0.45um membrane and purified by tangential flow filtration using a 100kDa MWCO filter. The soluble polymer was obtained in good yield after TFF purification, had a significantly higher molecular weight than the starting material, and had a branched conformation. The addition of salt in the second step of the reaction resulted in a higher molecular weight relative to Example 9.

[0149] Example 11. Synthesis of a soluble, high MW chondroitin sulfate composition with bottlebrush-like structure by stepwise addition of reactants in one pot in the presence of sodium chloride (in steps 1 and 2). Product purification using an optimized tangential flow filtration protocol. 11A. Stepwise addition reactions Sodium chondroitin sulfate (0.306 g, 1.823 mmol equivalent of hydroxyl groups) and sodium chloride (85.2 mg, 1.46 mmol) were dissolved in 9.746 g DI water in a 20 mL reaction vessel. A clear, colorless solution was obtained. DVS (0.254 g, 216 uL, 2.15 mmol) was added volumetrically via a microliter pipette. After gentle mixing, the solution was clear and colorless. The reaction was initiated by adding 1.03 mL of 1.0 N NaOH using a microliter pipette. Upon addition of NaOH, the solution immediately turned pale yellow in color while remaining clear. The reaction is 3.04 wt% chondroitin sulfate and 0.1 M NaOH (pH 13). The reaction was gently mixed on a rotisserie. After 15 minutes, additional sodium chondroitin sulfate was added (0.909 g, 7.25 mmol equivalents of hydroxyl groups) and the reaction mixture was stirred on a rotisserie. The reaction became more viscous but remained clear and mobile. Two hours after initiation with NaOH, the reaction was stopped by adding 1.03 mL of 1.0 N HCl using a microliter pipette. The clear mobile reaction mixture was added to a vial containing 50 g of PBS and the total weight was brought to 80 g by addition of PBS. The diluted reaction mixture was easily filtered through a 0.45 um PVDF syringe filter.

[0150] 11B. Purification using Tangential Flow Filtration The Spectrum Lab KR2i TFF system was equipped with a 250 ml feed reservoir and a 20 cm hollow fiber filter module containing modified polyethersulfone filter fibers (1 mm diameter, 100 kDa MWCO, 75 cm 2 A total of 80 g of diluted product from Example 11A was placed in the feed reservoir. Tangential flow filtration was started at a flow rate of 200 ml / min, which was increased to 300 ml / min while keeping the inlet pressure below 25 psig. TFF was performed in dialysis mode where the volume of solution lost to the permeate was continuously replenished with additional PBS. In this way, the volume of the retentate remained constant for the duration of the filtration procedure during which 5 volumes (400 ml) of permeate were obtained. TFF was then continued in desalting mode by refilling the feed reservoir with DI water (instead of PBS) and continuing the filtration until 5 more volumes of permeate (400 ml) were obtained. The DI water make-up was then discontinued and the filtration was performed in concentration mode to reduce the retentate volume to approximately 50 mL. TFF was then stopped and the system was flushed (10 ml DI water) to recover the retentate. The purified retentate was then dried by lyophilization for 72 hours, which gave the purified product (0.598 g, 45% yield based on the starting chondroitin sulfate weight) as a white fluffy solid.

[0151] Example 11 demonstrates that the stepwise addition reaction protocol in the presence of 0.15M sodium chloride results in a soluble polymer that is filterable through a 0.45um membrane and purified by tangential flow filtration using a 100kDa MWCO filter. The soluble polymer was obtained in good yield after TFF purification. The soluble polymer is expected to have a significantly higher molecular weight and branched conformation than the starting material. Furthermore, the addition of salt is expected to result in a higher molecular weight relative to Examples 9 and 10.

[0152] Example 12. Synthesis of a soluble, high MW chondroitin sulfate composition with bottlebrush-like structures containing galactosyl epitopes. 12A. Reactions by stepwise addition Sodium chondroitin sulfate (0.306 g, 1.823 mmol equivalent of hydroxyl groups) and sodium chloride (85.2 mg, 1.46 mmol) are dissolved in 9.746 g of DI water in a 20 mL reaction vessel. A clear, colorless solution results. DVS (0.254 g, 216 uL, 2.15 mmol) is added volumetrically via microliter pipette. After gentle mixing, the solution is clear and colorless. The reaction is started by adding 1.03 mL of 1.0 N NaOH using a microliter pipette. Upon addition of NaOH, the solution immediately turns pale yellow in color, but remains clear. The reaction is 3.04 wt% chondroitin sulfate and 0.1 M NaOH (pH 13). The reaction is gently mixed on a rotisserie. After 15 minutes, additional sodium chondroitin sulfate (0.909 g, 7.25 mmol equivalent of hydroxyl groups) and lactosylamine (36.5 mg, 0.107 mmol) are added. The reaction mixture is stirred on a rotisserie. The reaction becomes slightly more viscous, but remains clear and mobile. Two hours after initiation with NaOH, the reaction is stopped by adding 1.03 mL of 1.0 N HCl using a microliter pipette. The clear, mobile reaction mixture is added to a vial containing 50 g of PBS, and the total weight is brought to 80 g by addition of PBS. The diluted reaction mixture is easily filtered through a 0.45 um PVDF syringe filter.

[0153] 12B. Purification using Tangential Flow Filtration The Spectrum Lab KR2i TFF system was equipped with a 250 ml feed reservoir and a 20 cm hollow fiber filter module containing modified polyethersulfone filter fibers (1 mm diameter, 100 kDa MWCO, 75 cm 2(total surface area of ​​100 ml, part #D02-E100-10-N). A total of 80 g of the diluted product of Example 12A is placed in the feed reservoir. Tangential flow filtration is started at a flow rate of 200 ml / min, which is increased to 300 ml / min while keeping the inlet pressure below 25 psig. TFF is performed in dialysis mode where the volume of solution lost to the permeate is continuously replenished with additional PBS. In this way, the volume of the retentate remains constant for the duration of the filtration procedure during which 5 volumes (400 ml) of permeate are obtained. TFF is then continued in desalting mode by refilling the feed reservoir with DI water and continuing the filtration until 5 more volumes of permeate (400 ml) are obtained. The make-up of deionized water is then discontinued and the filtration is performed in concentration mode to reduce the retentate volume to approximately 50 mL. TFF is then stopped and the system is flushed (10 ml DI water) to recover the retentate. The purified retentate is then dried by lyophilization for 72 hours, which gives the purified product as a white fluffy solid in good yield.

[0154] 12C. Analysis of high molecular weight conjugates of chondroitin sulfate and lactosylamine The purified product of Example 12 can be analyzed by SEC-MALLS as described for Examples 9 and 10. The lactosylamine incorporation level in the purified product can be determined by exhaustive hydrolysis followed by analysis of the resulting mixture of monosaccharides by high-performance anion exchange chromatography using pulsed amperometric detection. For example, the sample can be hydrolyzed in 2M trifluoroacetic acid at 100°C for 8 hours and the hydrolysate dried in a speed-vac. The resulting residue can be dissolved in water and analyzed on a Carbo Pac PA-1 column using 16 mM sodium hydroxide eluent (isocratic).

[0155] Example 12 illustrates how a high molecular weight chondroitin sulfate composition bearing a galactose epitope can be prepared, purified by TFF, and characterized. As illustrated in this example, the chemical process of the present invention for producing high molecular weight branched sulfated GAGs can be carried out in the presence of other biological epitopes in a second step that can react with the DVS-modified intermediate prepared in the first step.

[0156] Example 13. Synthesis of soluble high MW chondroitin sulfate compositions modified with type II collagen binding peptides. 13A. Reactions by stepwise addition Sodium chondroitin sulfate (0.306 g, 1.823 mmol equivalents of hydroxyl groups) and sodium chloride (85.2 mg, 1.46 mmol) are dissolved in 9.746 g of DI water in a 20 mL reaction vessel. A clear, colorless solution is obtained. DVS (0.254 g, 216 uL, 2.15 mmol) is added volumetrically with a microliter pipette. After gentle mixing, the solution is clear and colorless. The reaction is started by adding 1.03 mL of 1.0 N NaOH using a microliter pipette. Upon addition of NaOH, the solution immediately turns pale yellow in color, but remains clear. The reaction is 3.04 wt% chondroitin sulfate and 0.1 M NaOH (pH 13). The reaction is gently mixed on a rotisserie. After 15 minutes, peptide-modified chondroitin sulfate (7.25 mmol equivalents of hydroxyl groups) is added. The reaction mixture is stirred on a rotisserie. The reaction becomes slightly more viscous, but remains clear and fluid. Two hours after initiation with NaOH, the reaction is stopped by adding 1.03 mL of 1.0 N HCl using a microliter pipette. The clear, fluid reaction mixture is added to a vial containing 50 g of PBS, and the total weight is brought to 80 g by addition of PBS. The diluted reaction mixture is easily filtered through a 0.45 um PVDF syringe filter.

[0157] 13B. Purification using Tangential Flow Filtration The Spectrum Lab KR2i TFF system was equipped with a 250 ml feed reservoir and a 20 cm hollow fiber filter module containing modified polyethersulfone filter fibers (1 mm diameter, 100 kDa MWCO, 75 cm 2 (total surface area of ​​100 ml, part #D02-E100-10-N). A total of 80 g of the diluted product of Example 12A is placed in the feed reservoir. Tangential flow filtration is started at a flow rate of 200 ml / min, which is increased to 300 ml / min while keeping the inlet pressure below 25 psig. TFF is performed in dialysis mode where the volume of solution lost to the permeate is continuously replenished with additional PBS. In this way, the volume of the retentate remains constant for the duration of the filtration procedure during which 5 volumes (400 ml) of permeate are obtained. TFF is then continued in desalting mode by refilling the feed reservoir with DI water and continuing the filtration until 5 more volumes of permeate (400 ml) are obtained. The make-up of deionized water is then discontinued and the filtration is performed in concentration mode to reduce the retentate volume to approximately 50 mL. TFF is then stopped and the system is flushed (10 ml DI water) to recover the retentate. The purified retentate is then dried by lyophilization for 72 hours, which gives the purified product as a white fluffy solid in good yield.

[0158] Example 13 illustrates how to prepare high molecular weight chondroitin sulfate composition with type II collagen binding peptide epitope and purify by TFF.Various substances of peptide-modified chondroitin sulfate can be prepared using the methods described in the literature [Caravan, U.S. Patent No. 9,386,938 (B2)] [Panitch, U.S. Patent No. 9,200,039 (B2)].As illustrated in this example, the chemical process of the present invention for producing high molecular weight branched sulfated GAG can be carried out with chemically modified GAG with various peptide moieties or other biological epitopes.

[0159] Example 14. Evaluation of a high MW chondroitin sulfate composition having a bottlebrush-like structure in a rat model of interstitial cystitis. A rat model is used to reproduce the pathology of leaky bladder, which is understood to be a major factor in the development of interstitial cystitis (IC). Female ovariectomized (OVX) Sprague-Dawley rats (250g-300g) are purchased from Charles River Laboratories. Rats are housed two per cage under controlled temperature and humidity. OVX rats are used to avoid any influence of hormonal circulation and male rats are not able to catheterize through the urethra. All animals have free access to food and water and are acclimatized to the facility housing for at least one week prior to the experiment. Experimental protocols are approved by the relevant Institutional Animal Care and Use Committee.

[0160] Transurethral protamine sulfate (PS) treatment Seven-week-old OVX female SAS Sprague Dawley® rats weighing 250-300 grams are treated with protamine sulfate (PS) to induce leaky bladder as described in the literature [Towner et al., Journal of Urology, 2015, Vol. 193, pp. 1394-1400]. Rats are anesthetized with isoflurane (3%) for approximately 10 minutes with a constant supply of oxygen, and the bladder is emptied after catheterization using a lubricated 18-gauge intravenous catheter (Surflo, Terumo, Elkton, MD) and a custom-made guidewire. Care is taken not to injure the bladder by stopping the catheter immediately after it passes the pubic bone and not allowing the catheter to "bottom out." Animals are monitored for hematuria as an indicator of bladder trauma, and any animals with blood in the urine or solution are not used. PS (1 mg / ml in 400 μl saline) is slowly infused into the bladder through the catheter. After 15 minutes, the bladder is emptied by applying lower abdominal pressure. The bladder is then rinsed with saline (400 μl, 3 times). The transurethral catheter is then removed and the animal is returned to its home cage.

[0161] MRI imaging of the bladder and colon Bladder permeability is assessed by magnetic resonance imaging (MRI). Rats are anesthetized with isoflurane (1.5%-3.0%) with 800-1,000 ml O2 for MRI experiments. MRI is performed on a 7 Tesla, 30 cm internal diameter BioSpec® MRI system. For bladder imaging, in vivo diagnostic CE-MRI specifically uses Gd-DTPA (0.2 mmol Gd / kg diluted in 800 ml saline) administered via an intravesical catheter to visualize permeable bladder urothelial loss with bladder contrast images. Bladder contrast images are acquired every 3 min 43 s for a total of 20 min. For contrast images of the colon, Gd-DTPA (0.2 mmol Gd / kg diluted to 200 ml in saline) is administered intravenously via a 24-gauge 0.75 inch BD Insyte™ Autoguard™ shielded intravenous tail vein catheter. Images are acquired over a 30 minute period. All MRI images are acquired using a T1-weighted RARE (rapid acquisition with relaxation enhancement) MRI pulse sequence with certain parameters including a 1,200 ms repetition time, 9 ms echo time, a RARE factor of 4, 4 averages, a 1 mm image slice thickness, a 256x256 matrix with motion and fat suppression, and a 6.5x6.5 cm2 field of view.

[0162] Biopolymer Treatment Chondroitin sulfate-based biopolymers are instilled into the leaky bladder 24 hours after PS treatment. There are three treatment groups with 10 rats (n=10) per group: Group A, high molecular weight chondroitin sulfate with bottle brush structure obtained from Example 11; Group B, high molecular weight chondroitin sulfate with bottle brush structure with galactosyl epitope obtained from Example 12; Group C, saline-treated control. Biopolymers are administered by transurethral catheterization 24 hours after PS exposure. Biopolymers are dissolved in saline (20 mg / ml) and sterile filtered (0.2 μm PVDF syringe filter) before administration. Biopolymer administration is performed under the anesthesia protocol described for MRI imaging.

[0163] MRI is performed 24 hours after PS exposure and immediately after polymer treatment. MRI is performed again 5 days after PS exposure and 4 days after biopolymer treatment.

[0164] Data Analysis and Statistics MRI signal intensity was measured from regions of interest (ROIs) in the images. Four or five ROIs were used in the high-intensity areas in the bladder periphery, colonic mucosa, fat pad surrounding the bladder, surrounding colonic tissue, and medial thigh muscle, as well as corresponding areas in the control data set. These data were displayed using ParaVision™ (version 5.0). Statistical analysis was performed using ANOVA with post-Tukey multiple comparison test to evaluate the differences in treatment groups using InStat (Graph-Pad®). Signal intensity differences between groups with p<0.05, p<0.01, or p<0.001 were considered statistically significant.

[0165] Example 14 demonstrates how the high molecular weight chondroitin sulfate compositions of the invention having a bottlebrush structure with and without galactose epitopes restore impermeability to a leaky bladder in a rat protamine sulfate model. Animals treated with the materials of the invention prepared in Examples 11 and 12 will show significantly greater bladder impermeability (less leakage) compared to animals treated with saline.

[0166] Example 15. Varying the chondroitin sulfate / DVS molar ratio: observational study. A set of reaction vials was filled with various amounts of sodium chondroitin sulfate and DI water, respectively, to obtain solutions with 4 wt%, 6 wt%, 8 wt%, and 10 wt% chondroitin sulfate. The vials were gently agitated in a rotisserie mixer, which resulted in a set of clear, colorless solutions. Various amounts of DVS were then volumetrically added to each vial using a microliter pipette, so that for each concentration of chondroitin sulfate, the molar ratio of DVS to available chondroitin sulfate hydroxyl groups was 0.98, 0.65, and 0.32. After gentle mixing, all solutions were clear and colorless. The reactions were initiated by adding 1.0 N NaOH using a microliter pipette. Upon addition of NaOH, the solution immediately turned pale yellow in color while remaining clear. Each reaction was at 0.1 M NaOH (approximately pH 13). This set of 12 reactions was gently mixed on a rotisserie and observed over time for gelation and loss of transparency. Loss of transparency was determined by visually determining the occurrence of haze or opacity. Gelling was determined by visually determining loss of fluidity. When the reaction formed a non-fluid gel, the gel time was recorded. Figure 5 illustrates the composition of these 12 reactions and the observations made regarding homogeneity and fluidity for each.

[0167] Example 16 Varying the Carboxymethylcellulose / DVS Molar Ratio: Observational Study. A set of reaction vials were filled with various amounts of CMC and DI water, respectively, to obtain solutions at 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, and 6 wt% CMC. The vials were gently agitated in a rotisserie mixer, which resulted in a set of clear, colorless solutions. Various amounts of DVS were then volumetrically added to each vial using a microliter pipette, such that for each concentration of CMC, the molar ratio of DVS to available CMC hydroxyl groups was 1.2, 0.6, and 0.3. After gentle mixing, all solutions were clear and colorless. The reactions were initiated by adding 1.0 N NaOH using a microliter pipette. Upon addition of NaOH, the solutions immediately turned pale yellow in color while remaining clear. Each reaction was at 0.1 M in NaOH (approximately pH 13). This set of 18 reactions was gently mixed on a rotisserie and observed over time for gelling and loss of transparency. Loss of transparency was determined by visually determining the occurrence of haze or opacity. Gelling was determined by visually determining loss of flow. When the reaction formed a non-flowing gel, the gel time was recorded. Figure 6 illustrates the composition of these 18 reactions and the observations made regarding homogeneity and flow for each.

[0168] Equivalent While the present disclosure has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to illustrate, and not to limit, the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0169] The patents and scientific literature referenced herein reveal knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference. [Sequence List Free Text]

[0170] Sequence Listing 1-20 <223> Writing artificial sequences: synthetic peptides

Claims

1. A polymer conjugate comprising a plurality of sulfated glycosaminoglycan (GAG) polymer chains, each of which is linked to one or more sulfated GAG polymer chains via a linker derived from divinyl sulfone (DVS), the linker being linked to the sulfated glycosaminoglycan (GAG) polymer chain via a hydroxyl group of the GAG ​​polymer chain, the polymer conjugate being soluble in aqueous solution and having a molecular weight that is 3 to 100 times the molecular weight of an individual unlinked sulfated GAG.

2. 2. The polymer conjugate of claim 1, wherein the sulfated GAG is selected from the group consisting of chondroitin sulfate, heparan sulfate, dermatan sulfate, keratan sulfate, and combinations thereof.

3. The polymer conjugate of claim 1 or 2, wherein the linkers are randomly linked along the GAG ​​polymer chain.

4. The polymer conjugate of any one of claims 1 to 3, which has a branched structure.

5. 13. A method for preparing the polymer conjugate of claim 1, comprising: i) providing an aqueous solution of sulfated GAGs at a concentration of 2 wt % to 20 wt %, and ii) contacting the sulfated GAG with divinyl sulfone (DVS) to form a soluble branched polymer, wherein the molar ratio of DVS to GAG hydroxyl groups (DVS / OH) is less than the molar ratio required for gel formation. The method includes:

6. The method of claim 5, wherein the soluble branched polymer has a molecular weight of from 15,000 Da to 1,000,000 Da.

7. The method according to claim 5 or 6, wherein the molar ratio of DVS to GAG hydroxyl groups (DVS / OH) is 0.01 to 0.

6.

8. 13. A method for preparing the conjugate of claim 1 by sequential introduction of the sulfated GAGs in a single reaction, comprising: i) providing sulfated GAGs; ii) reacting the sulfated GAG with a linking agent under conditions such that a small amount of the sulfated GAG is reacted with the entire amount of divinyl sulfone (DVS); and iii) adding the remaining portion of the sulfated GAG to form a conjugate having a branched structure. The method includes:

9. The conjugate of claim 1 , wherein at least one of the GAG ​​polymer chains comprises at least one modifying agent capable of reacting with a carboxyl group of the GAG ​​polymer chain and attaching to the GAG ​​polymer chain.

10. The conjugate of claim 9, wherein the modifying agent is a collagen-binding peptide.

11. A pharmaceutical composition for treating a disease, disorder or condition associated with soft tissue in a subject, comprising a conjugate according to any one of claims 1 to 4, 9 and 10.

12. 12. The pharmaceutical composition according to claim 11 for treating interstitial cystitis (IC) in a patient.

13. The pharmaceutical composition of claim 12 , wherein the conjugate is functionalized with at least one glycan ligand for a galectin.

14. 14. The pharmaceutical composition of claim 12 or 13, which is administered by intravesical instillation.

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