Crosslinked polycarboxylated polysaccharides and methods of use thereof

Abstract

The present invention relates to crosslinked polycarboxylated polysaccharides and methods of use thereof. The present invention relates to polymers comprising a first hyaluronic acid (HA) chain and a second HA chain crosslinked by a linker comprising an unsaturated moiety or derivative thereof coupled to a tetrazine moiety or derivative thereof. In some embodiments, the polymers of the present invention are characterized by having a degree of crosslinking of 0.2 to 4%.

Description

Cross-linked polycarboxylated polysaccharides and their application methods

[0001] This application is a divisional application. The original application was filed on June 11, 2019, with application number 201980056276.2, and the invention title was "Cross-linked polycarboxylated polysaccharides and methods of using them," the entirety of which is incorporated herein by reference.

[0002] Cross-reference to related applications

[0003] This application claims the priority of U.S. Provisional Patent Application No. 62 / 691,035, filed June 28, 2018, entitled “CROSS-LINKED POLYCARBOXYLATED POLYSACCHARIDES AND METHODS OF USE THEREOF”, the entire contents of which are incorporated herein by reference. Technical Field

[0004] In some embodiments, the present invention relates to cross-linked hyaluronic acid. Background Technology

[0005] The cross-linking of hyaluronic acid in dermal fillers endows the material with desired mechanical properties, specifically enhancing (or filling) properties.

[0006] The need to use materials that are as close as possible to endogenous hyaluronic acid necessitates the production of materials with low levels of modification (e.g., % crosslinking). Both hyaluronic acid and crosslinked hyaluronic acid are known to be highly hygroscopic. Therefore, when these materials are injected, they tend to absorb water from tissues, leading to swelling and edema. The water absorption rate is positively correlated with the content of hyaluronic acid and crosslinked hyaluronic acid. Therefore, due to the need to reduce the risk of swelling and edema, it is desirable to produce dermal fillers with low contents of hyaluronic acid and crosslinked hyaluronic acid.

[0007] Materials currently on the market are known to contain high cross-linking (4 to 10%) or total hyaluronic acid content (equal to or greater than 20 mg / gR) to provide sufficient enhancing properties. Summary of the Invention

[0008] In some embodiments, the present invention relates to cross-linked hyaluronic acid.

[0009] In one aspect of the invention, a polymer is provided comprising a first hyaluronic acid (HA) chain and a second HA chain crosslinked via a linker, the linker comprising an unsaturated portion or a derivative thereof bound to a tetrazine moiety or a derivative thereof, and wherein the crosslinking is characterized by a degree of crosslinking of 0.2 to 4%.

[0010] In some embodiments, the unsaturated portion includes: unsaturated cycloalkyl, unsaturated alkylaryl, unsaturated alkyl, or combinations thereof.

[0011] In some embodiments, one or more linkers comprise a compound represented by the following formula:

[0012] Formula (A)

[0013]

[0014] Or formula (B):

[0015]

[0016] or combinations thereof; wherein:

[0017] --- indicates a single or double bond;

[0018] R 1 R 2 Or both are selected from: bond, alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocycloalicyclic, C(O)-NH-alkyl-NH and alkyl-NZ, wherein Z is bond, aryl or heteroaryl;

[0019] A is selected from: bond, alkyl and aryl;

[0020] Q 1 Q 2 Either indicates hydrogen or its absence;

[0021] And R 3 Selected from: hydrogen, substituted or unsubstituted alkyl, aryl and heteroaryl.

[0022] In some embodiments, the unsaturated portion comprises a styrene portion or a derivative thereof.

[0023] In some embodiments, the styrene portion or its derivatives are represented by formulas IA-D:

[0024]

[0025] In some embodiments, one or more linkers comprise a compound represented by the following formula:

[0026] Formula (C)

[0027]

[0028] Or formula (D):

[0029]

[0030] or combinations thereof; wherein:

[0031] --- indicates a single or double bond;

[0032] R 1 R 2 Or both are selected from: bond, alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocycloalicyclic, C(O)-NH-alkyl-NH and alkyl-NZ, wherein Z is bond, aryl or heteroaryl;

[0033] Q 1 Q 2 Either indicates hydrogen or its absence;

[0034] And R 3 It is selected from hydrogen, substituted or unsubstituted alkyl, aryl and heteroaryl.

[0035] In some implementations, one or more connectors include:

[0036] The first compound is derived from the norbornene moiety, which is selected from formulas IIA-G:

[0037]

[0038] b) A second compound derived from the tetrazine moiety selected from formulas IIIA-F:

[0039]

[0040] In some embodiments, the first compound and the second compound are covalently bonded.

[0041] In some embodiments, the norbornene moiety and the tetrazine moiety are covalently bonded to the first HA chain and the second HA chain.

[0042] In some embodiments, the covalent bond is selected from amides, amines, esters, ethers, ureas, thioureas, and carbamates.

[0043] In some embodiments, the average molecular weight Mw of the first HA chain and the second HA chain is from 100,000 to 4,000,000 Daltons (Da).

[0044] In some embodiments, the polymer has a phase angle (δ) of 0.1 to 10°.

[0045] In some embodiments, the elastic modulus (G') of the polymer is 10 to 1,000 Pa.

[0046] In some embodiments, the polymer includes an HA content of 1 mg / gR to 20 mg / gR.

[0047] In some implementations, the degree of crosslinking is determined by... 1 HNMR confirmed.

[0048] According to another aspect, a composition is provided comprising the polymer of the present invention and a pharmaceutically acceptable carrier.

[0049] In some embodiments, the composition comprises one or more of the following:

[0050] 1) A polymer comprising a linker represented by formula (A);

[0051] 2) Polymers including the linkers represented by formula (B);

[0052] 3) Polymers including linkers represented by formula (C);

[0053] 4) Polymers including the linkers represented by formula (D).

[0054] In some embodiments, the composition further includes 0.1-30% (w / w) of non-crosslinked HA in the total HA content of the composition.

[0055] In some embodiments, the composition further includes one or more compounds selected from the group consisting of amino acids, minerals, vitamins, antioxidants, nucleic acids, coenzymes, enzymes, growth factors, proteins, antitumor drugs, steroids, nonsteroidal anti-inflammatory drugs, antibiotics, anesthetics, antimicrobial drugs, or any combination thereof.

[0056] According to another aspect, a method for filling or filling tissue in an object in need is provided, comprising giving the tissue the composition of the present invention or the polymer of the present invention, thereby filling or filling the tissue in the object in need.

[0057] In some implementations, the tissue is selected from: skin, gums, cartilage and ophthalmic tissues, muscle and subcutaneous tissue.

[0058] According to another aspect, a method for preparing a polymer is provided in its embodiments, the method comprising: mixing a first hyaluronic acid (HA) chain or a derivative thereof with a second HA chain or a derivative thereof, wherein the first HA chain comprises an unsaturated portion or a derivative thereof, and the second HA chain comprises a tetrazine portion or a derivative thereof; wherein the molar ratio of the unsaturated portion or a derivative thereof to the tetrazine portion or a derivative thereof is 3:1 to 1:3; thereby crosslinking the first HA chain or a derivative thereof with the second HA chain or a derivative thereof.

[0059] In some embodiments, the unsaturated portion includes norbornene or a derivative thereof.

[0060] In some embodiments, the first HA chain or a derivative thereof and the second HA chain or a derivative thereof have an average molecular weight Mw of 100,000 to 4,000,000 Daltons (Da).

[0061] In some embodiments, crosslinking includes forming a covalent bond between the norbornene moiety or a derivative thereof and the tetrazine moiety or a derivative thereof.

[0062] According to another aspect, a kit is provided comprising a first HA chain or a derivative thereof coupled to an unsaturated moiety or a derivative thereof, and a second HA chain or a derivative thereof coupled to a tetrazine moiety or a derivative thereof.

[0063] In some embodiments, the unsaturated portion includes norbornene or a derivative thereof.

[0064] In some implementations, the kit further includes a pharmaceutically acceptable carrier.

[0065] In some implementations, the kit also includes an injection device.

[0066] In some embodiments, the kit also includes components selected from the following: free HA chains, amino acids, minerals, vitamins, or anesthetics.

[0067] In some implementations, the kit includes instructions regarding the following:

[0068] a. Mixing a first HA chain or a derivative thereof coupled to norbornene moiety or a derivative thereof and a second HA chain or a derivative thereof coupled to tetrazine moiety or a derivative thereof in a ratio of 3:1 to 1:3; and

[0069] b. Applying a composition formed by mixing a first HA chain or a derivative thereof coupled to a norbornene moiety or a derivative thereof and a second HA chain or a derivative thereof coupled to a tetrazine moiety or a derivative thereof to a filling or filling method.

[0070] In some embodiments, the kit also includes instructions on mixing the composition with free HA chains, amino acids, minerals, vitamins, anesthetics, or any combination thereof.

[0071] In some implementations, the mixing is carried out in an injection device.

[0072] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials described herein may be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are exemplary only and are not intended to be necessarily limiting.

[0073] Other embodiments and the full scope of the invention will become apparent from the detailed description given below. However, it should be understood that while the detailed description and specific examples indicate preferred embodiments of the invention, they are given by way of example only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art based on this detailed description. Attached Figure Description

[0074] Figure 1: Hyaluronic acid modified with tetrazine (HA-tetrazine) 1 HNMR spectra show aromatic peaks of tetrazine and aliphatic peaks of N-acetylglucosamine residues.

[0075] Figure 2: Hyaluronic acid modified with norbornene (HA-norbornene) 1 HNMR spectra show olefinic peaks of norbornene and aliphatic peaks of N-acetylglucosamine residues.

[0076] Figure 3: Hyaluronic acid modified with styrene (HA-styrene) 1 HNMR spectra show olefin peaks for styrene and aliphatic peaks for N-acetylglucosamine residues.

[0077] Figure 4: A diagram depicting the onset point in the linear viscoelastic region (LVR) of a polymer.

[0078] Figure 5: A diagram depicting the dimensions of the subcutaneous gel material administered in vivo to the product vs. the polymer of the present invention over a period of 95 days. Detailed Implementation

[0079] In some embodiments, the present invention relates to a polymer comprising a first hyaluronic acid (HA) chain and a second HA chain crosslinked by a linker comprising an unsaturated portion or a derivative thereof bonded to a tetrazine moiety or a derivative thereof. In some embodiments, the polymer of the present invention is characterized by having a crosslinking degree of 0.2 to 4%.

[0080] In some embodiments, the unsaturated portion includes: unsaturated cycloalkyl, unsaturated alkylaryl, unsaturated alkyl, or combinations thereof. In some embodiments, the unsaturated portion includes cyclic or acyclic alkynes. In some embodiments, the unsaturated portion includes cyclic or acyclic olefins. In some embodiments, the unsaturated portion includes optionally fused cyclic olefins.

[0081] In some embodiments, the unsaturated portion is selected from: substituted or unsubstituted styrene, cyclohexene, cyclopentene, cyclohexadiene, cyclopentadiene, norbornadiene, fused norbornadiene, norbornene, and fused norbornene. In an exemplary embodiment, the unsaturated portion is norbornene.

[0082] polymer

[0083] In some embodiments, the present invention relates to polymers comprising hyaluronic acid (HA) chains or derivatives thereof. As used herein, HA chains or derivatives thereof include D-glucuronic acid and N-acetyl-glucosamine.

[0084] As used herein, derivatives of the HA chain relate to chemically modified HA. In some embodiments, the chemically modified HA includes side-chain modifications (e.g., acetylation of hydroxyl groups, decarboxylation, esterification, or amidation). In some embodiments, the chemically modified HA includes one or more of the aforementioned side-chain modifications. In some embodiments, the modifications are the same. In some embodiments, the modifications are different. In some embodiments, the chemically modified HA includes a combination of modified side chains.

[0085] In some embodiments, the HA chain of the present invention has the following molecular weights: 50,000-200,000 Da, 100,000-200,000 Da, 150,000-400,000 Da, 150,000-1,000,000 Da, 250,000-1,500,000 Da, 350,000-5,000,000 Da, 750,000-4,000,000 Da. 0 Da, 50,000-5,000,000 Da, 1,000,000-7,500,000 Da, 2,000,000-10,000,000 Da, 400,000-5,000,000 Da, 650,000-8,000,000 Da, 4,000,000-10,000,000 Da, or 7,500,000-15,000,000 Da. Each possibility represents a single embodiment of the invention.

[0086] As used herein, the term "molecular weight" includes any of the following average weight values ​​selected from: Mn (number-average molar mass), NAMW (number-average molecular weight), Mw (mass-average molar mass), WAMW (weight-average molecular weight), Mz (Z-average molar mass), Mv (viscosity-average molar mass), and MWCO (molecular weight cutoff). Unless otherwise stated, the term refers to Mw.

[0087] In one embodiment, the present invention relates to a polymer comprising a polycarboxylated polysaccharide or a derivative thereof with a molecular weight in the above range.

[0088] In some embodiments, the polymer of the present invention comprises one or more HA chains. In some embodiments, "one or more" means two. In some embodiments, the two HA chains of the present invention are cross-linked. In one embodiment, the cross-linking is mutual cross-linking. As defined herein, the term "mutual" refers to bonding between two portions located in two different chains, as opposed to the formation of an "internal" bond between two residues located in the same chain. In some embodiments, the cross-linking of the two HA chains is carried out by a linker.

[0089] As defined herein, a "linker" refers to a molecule or macromolecule used to connect different portions or functional groups of one or more polycarboxylated polysaccharides. In one embodiment, linkers may also facilitate other functions, including but not limited to preserving biological activity, maintaining interactions, and others.

[0090] In some embodiments, the polymer of the present invention includes a first HA chain connected to a second HA chain via one or more linkers, said linkers comprising compounds represented by the following formula:

[0091] Formula (A)

[0092]

[0093] Or formula (B)

[0094]

[0095] or combinations thereof; wherein:

[0096] Indicates a single bond or a double bond.

[0097] In some embodiments, the polymer of the present invention includes a first HA chain crosslinked to a second HA chain via one or more linkers, the linkers comprising compounds represented by formula (A) and / or (B).

[0098] In some implementations, R 1 R2 Or both are selected from: bond, alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocycloalicyclic, C(O)-NH-alkyl-NH and alkyl-NZ, where Z is bond, aryl or heteroaryl.

[0099] In some embodiments, A is selected from: bond, alkyl and aryl.

[0100] In some implementations, Q 1 Q 2 "Or both" indicates that hydrogen or it is not present.

[0101] In some implementations, R 3 Selected from: hydrogen, substituted or unsubstituted alkyl, aryl and heteroaryl.

[0102] In some embodiments, the polymer of the present invention comprises a first HA chain connected to a second HA chain via one or more linkers, the linkers comprising compounds represented by the following formula:

[0103] Formula (C)

[0104]

[0105] Or (D)

[0106]

[0107] or combinations thereof;

[0108] in Indicates a single bond or a double bond.

[0109] In some embodiments, the polymer of the present invention includes a first HA chain crosslinked with a second HA chain via a linker, the linker comprising a compound represented by formula (C) or (D).

[0110] In some embodiments, the linker comprises a first compound derived from the unsaturated portion and a second compound derived from the tetrazine portion, as described below. In some embodiments, the first and second compounds are covalently bonded. In some embodiments, the first and second compounds are covalently bonded to form a compound represented by any one of formulas (A) to (D).

[0111] In some implementations, R 1 R 2 and R 3Substituents selected from the following: alkyl, cycloalkyl, aryl, heterocycloalicyclic, heteroaryl, alkoxy, hydroxyl, phosphonic acid, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halogen, trihalomethyl, cyano, amide, amine, alkylamine, carboxyl, sulfonyl, sulfoxy, sulfinyl, and sulfonamide.

[0112] In some implementations, Q 1 Q 2 "Or both" indicates that hydrogen or it is not present.

[0113] In some embodiments, the polymer of the present invention includes a first HA chain connected to a second HA chain via a linker, the linker comprising compounds selected from:

[0114]

[0115] In some implementations, R 1 Selected from: -C0-C6 alkyl-NZ-, -C0-C6 alkyl-O- and C0-C3 alkyl-C(O)-.

[0116] In some embodiments, Z is selected from: bond, aryl or heteroaryl, wherein the aryl and heteroaryl groups are optionally substituted with halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, (C1-C6 alkyl)amino and di(C1-C6 alkyl)amino.

[0117] In some implementations, R 2 Selected from: C0-C6 alkyl-NZ, -C0-C6 alkyl-O- and C0-C3 alkyl-C(O)-.

[0118] In some implementations, R 3 Selected from: hydrogen, C1-C6 alkyl, aryl or heteroaryl, wherein the aryl and heteroaryl groups are optionally substituted with halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, (C1-C6 alkyl)amino and di(C1-C6 alkyl)amino.

[0119] In one implementation method Refers to a single bond, or in some implementations to a double bond—where feasible.

[0120] In some implementations, R 1 R 2 Or both are selected from: -NZ-, -C1-C6 alkyl-NZ-, -O-, -C1-C6 alkyl-O-, -C(O)- or -C1-C3 alkyl-C(O)-; -methyl-O, -pentyl-O-; -C(O)-; and -methyl-C(O)-.

[0121] In some embodiments, Z is a bond. In some embodiments, Z is selected from: aryl and heteroaryl, phenyl; and pyridyl, pyrimidinyl and pyrazinyl; each group may optionally be substituted.

[0122] Another embodiment provides a linker comprising a compound of formula (C) or (D) according to any of the foregoing embodiments, wherein R 1 R 2 Or both selected from: -NZ-, -C1-C6 alkyl-NZ-, -O-, -C1-C6 alkyl-O-, -C(O)- or -C1-C3 alkyl-C(O)-; -C1-C6 alkyl-NZ-; C1-C3 alkyl-NZ-; -methyl-NH- or -pentyl-NH-; -C1-C6 alkyl-O-; -C1-C3 alkyl-O-; -methyl-O or -pentyl-O-; -C0-C3 alkyl-C(O)-; C(O)-; and -methyl-C(O)-.

[0123] In some implementations, R 3 It is hydrogen.

[0124] In some implementations, R 3 Selected from: C1-C6 alkyl, aryl or heteroaryl, wherein the aryl or heteroaryl may optionally be substituted; aryl or heteroaryl, wherein the aryl and heteroaryl are optionally substituted; phenyl; pyridyl, pyrimidinyl or pyrazinyl.

[0125] In some embodiments, R in the linker of a compound of formula (C) or (D) 3 Selected from: C1-C6 alkyl, C1-C3 alkyl and methyl.

[0126] In some embodiments, the polymer includes a variety of links comprising compounds of formula (C) or (D).

[0127] In some embodiments, the present invention relates to polymers comprising a first HA chain and a second HA chain interconnected by linkers, resulting in cyclization, such as but not bound by any particular mechanism, including the reverse electron-demanding Diels-AldeR cyclization of an unsaturated moiety (e.g., norbornene moiety) or a derivative thereof, and a tetrazine moiety or a derivative thereof.

[0128] In some embodiments, the first HA chain of the present invention includes an unsaturated portion, as described above. In some embodiments, the first HA chain of the present invention includes a styrene portion or a derivative thereof.

[0129] In some embodiments, the styrene portion is represented by formulas IA-D:

[0130]

[0131] In some embodiments, the styrene moiety derivative includes a styrene moiety of formula IA-ID that is covalently bonded to the first HA chain.

[0132] Non-limiting examples of covalent bonds include, but are not limited to: amides, amines, esters, ethers, ureas, thioureas, and carbamates.

[0133] In some embodiments, the amine of the styrene moiety is covalently bonded to the first HA chain. In some embodiments, the styrene moiety is bonded to the carboxyl group of the first HA chain. In some embodiments, a derivative of the styrene moiety refers to a styrene moiety bonded to the first HA chain via an amide bond (e.g., a compound of formula IA), as shown in formula IE:

[0134]

[0135] In some embodiments, the first HA chain of the present invention comprises a norbornene moiety or a derivative thereof. In some embodiments, the number of norbornene moiety or derivative thereof in the first HA chain of the present invention can be any integer between 1 and 100,000. In some embodiments, the second HA chain of the present invention comprises a tetrazine moiety or a derivative thereof. In some embodiments, the number of tetrazine moiety or derivative thereof attached to the second HA chain of the present invention can be any integer between 1 and 100,000. In some embodiments, each first HA chain comprises 1-10,000, 1-5,000, 1-1,000, 5,000-50,000, 5,000-10,000, 1,000-10,000, 1,000-5,000, 500-5,000, 500-1000, or 1-500 norbornene moiety or derivative thereof. In some embodiments, each second HA chain comprises 1-100,000, 1-50,000, 1-10,000, 1-5,000, 1-1,000, 5,000-50,000, 5,000-10,000, 1,000-10,000, 1,000-5,000, 500-5,000, 500-1,000, or 1-500 tetrazine moieties or derivatives thereof. Each possibility represents a single embodiment of the invention.

[0136] In some embodiments, the norbornene moiety is an internal, external, or mixed conformation. Non-limiting examples of the norbornene moiety include, but are not limited to, compounds of formulas IIA-IIG:

[0137]

[0138] Non-limiting examples of the tetrazine moiety include, but are not limited to, compounds of formulas IIIA-IIIF:

[0139]

[0140]

[0141] In some embodiments, the norbornene moiety is a compound of formula IIA:

[0142]

[0143] In some embodiments, the derivative of the norbornene moiety includes a norbornene moiety represented by formula IIA-IIG that is covalently bonded to the first HA chain.

[0144] In some embodiments, the amine of the norbornene moiety is bonded to the first HA chain. In some embodiments, the norbornene moiety is bonded to the carboxyl group of the first HA chain. In some embodiments, derivatives of the norbornene moiety refer to the norbornene moiety bonded to the first HA chain via an amide bond (e.g., compounds of formula IIG), as shown in formula IF:

[0145]

[0146] In some embodiments, the norbornene moiety is bonded to the first HA chain by reacting the first HA chain and the norbornene moiety (e.g., norbornene methylamine, norbornene methanol) with a suitable coupling agent.

[0147] Non-limiting examples of coupling agents include, but are not limited to, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), carbonyl diimidazole, N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM), and N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)urea hexafluorophosphate (HBTU). In one embodiment, as defined above, the tetrazine moiety derivative relates to the tetrazine moiety and is covalently bonded to the second HA chain. In some embodiments, the covalent bond is as defined above.

[0148] In some embodiments, the tetrazine moiety derivative comprises a tetrazine moiety represented by formulas IIIA-IIIF that is covalently bonded to the second HA chain. In some embodiments, the amine of the tetrazine moiety is bonded to the second HA chain. In some embodiments, the tetrazine moiety is bonded to the carboxyl group of the second HA chain. In some embodiments, the tetrazine moiety derivative refers to a tetrazine moiety bonded to the second HA chain via an amide bond (e.g., compounds of formula IIIB), as shown in formula IG:

[0149]

[0150] In some embodiments, the tetrazine moiety is bound to the second HA chain by reacting the second HA chain and the tetrazine moiety (e.g., benzylamine tetrazine) with a suitable coupling agent.

[0151] In some embodiments, a first HA chain coupled to the norbornene moiety or a derivative thereof and a second HA chain coupled to the tetrazine moiety or a derivative thereof are present in the polymer of the invention in a ratio of 3:1, 3:2, 2:1, 1:1, 1:2, 2:3, or 1:3, including any values ​​and ranges therebetween. Each possibility represents a single embodiment of the invention. As defined herein, ratios are any of the following: moles, weights, or concentrations.

[0152] As defined herein, the term "degree of crosslinking" refers to the molar ratio between the linkers forming the polymers of the present invention and the repeating polycarboxylated polysaccharides. In some embodiments, the degree of crosslinking refers to the molar ratio between the linkers comprising unreacted norbornene and tetrazine and the repeating polycarboxylated polysaccharides forming the polymers of the present invention. In some embodiments, the degree of crosslinking of the polymers of the present invention is at most 0.1%, at most 0.2%, at most 0.5%, at most 0.7%, at most 0.9%, at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10%, or at most 12%. In some embodiments, the degree of crosslinking of the polymer of the present invention is 0.01-0.1%, 0.01-0.5%, 0.05-0.1%, 0.1-0.3%, 0.1-0.5%, 0.1-0.75%, 0.1-1%, 1-1.75%, 1-2%, 1-2.5%, 2-2.5%, 2.25-3%, 2.5-3.25%, 3-3.75%, 3.6-4.2%, 4-5.25%, 5-6.5%, 6-7.5%, 7-8.5%, 8-9.25%, 9-10.5%, or 10-12.5%. Each possibility represents a single embodiment of the invention. In one embodiment, the degree of crosslinking of the polymer refers to the calculated average of the degree of crosslinking of the plurality of HA chains within the polymer of the present invention.

[0153] In some embodiments, the polymers of the present invention are elastic. As used herein, the elasticity of a polymer is characterized by its elastic modulus (G'). In some embodiments, the term "elastic modulus" refers to the elastic modulus as determined below.

[0154] In some embodiments, the polymer of the present invention has an elastic modulus of 10-500 Pa, 20-1,000 Pa, 30-600 Pa, 40-1,000 Pa, 40-5,000 Pa, 50-10,000 Pa, 500-50,000 Pa, 500-10,000 Pa, 500-5,000 Pa, 500-1,000 Pa, 1,000-50,000 Pa, 1,000-10,000 Pa, 1,000-5,000 Pa, 50-20,000 Pa, 500-20,000 Pa, or 1,000-20,000 Pa. In some embodiments, the polymer of the present invention has an elastic modulus of up to 10,000 Pa, up to 20,000 Pa, up to 30,000 Pa, up to 40,000 Pa, or up to 50,000 Pa. Each possibility represents a single embodiment of the invention.

[0155] As defined herein, the term "phase angle" or "δ" refers to the degree of viscoelasticity of a material. It will be apparent to those skilled in the art that δ can be calculated using the following equation:

[0156] Where G” is the viscosity modulus and G' is the elastic modulus. In some embodiments, G' and G” are obtained by oscillatory rheology and measured in a viscoelastic domain with low oscillations in stress or amplitude.

[0157] In some embodiments, the polymers of the present invention have phase angles (d) of 0.1-25°, 0.1-0.5°, 0.1-0.9°, 0.5-1°, 0.7-1.5°, 1-2.5°, 2-4.5°, 3-4.75°, 4.7-5.5°, 5-7.5°, 6-8°, 7-8.5°, 8.25-9.5°, 9-10.5°, and 9.5-12°. In some embodiments, the polymer of the present invention has a phase angle (d) of up to 0.1°, up to 0.5°, up to 0.7°, up to 0.9°, up to 1°, up to 1.5°, up to 2°, up to 2.7°, up to 3.2°, up to 4°, up to 4.5°, up to 6°, up to 7.5°, up to 8°, up to 9°, up to 10°, up to 11°, or up to 13°. Each possibility represents a single embodiment of the invention.

[0158] In some embodiments, the present invention relates to a method for preparing the polymer of the present invention, the method comprising the steps of: (i) attaching an unsaturated portion (e.g., a norbornene portion) (or a derivative thereof) to a first hyaluronic acid (HA) chain (or a derivative thereof) and attaching a tetrazine portion (or a derivative thereof) to a second HA chain (or a derivative thereof); (ii) mixing the first (HA) chain (or a derivative thereof) comprising the norbornene portion (or a derivative thereof) and the second HA chain (or a derivative thereof) comprising the tetrazine portion (or a derivative thereof); and (iii) crosslinking the first HA chain with the second HA chain.

[0159] In some embodiments, step (i) further includes purifying the HA chain linked to the unsaturated or tetrazine moiety.

[0160] After the unsaturated or tetrazine moiety is attached to the HA chain, the resulting product is purified from the unreacted starting material and inorganic salt. Purification can be carried out by any method known in the art, such as dialysis, precipitation, ultrafiltration, or tangential flow filtration.

[0161] In some instances, the crosslinking reaction occurs within the range of temperatures and conditions available for polymer formation without the input of external energy. In one embodiment, the crosslinking reaction is heated to increase the reaction efficiency.

[0162] In some embodiments, the method for preparing the polymer of the present invention includes a spontaneous crosslinking reaction. In some embodiments, "spontaneous chemical reaction" means a process without the assistance of, for example, light, heat, or free radicals. In some embodiments, the crosslinking reaction can occur in water, in an aqueous buffer, or in a cell culture medium. Non-limiting examples of culture media include, but are not limited to, phosphate-buffered saline, Hank balanced salt solution, DulbeCCo modified Eagle medium, etc. In some embodiments, crosslinking can occur in an organic solvent. Non-limiting examples of organic solvents include, but are not limited to, methanol, ethanol, dichloromethane, dimethylformamide, etc.

[0163] In some embodiments, the crosslinking reaction can occur within a wide temperature range of at least -80°C, at least -50°C, at least -20°C, at least 0°C, at least 4°C, at least 22°C, at least 37°C, or at least 45°C, but not exceeding 60°C. In some embodiments, the crosslinking reaction can occur within a wide temperature range of (-80)-(-50)°C, (-60)-(-15)°C, (-20)-(-4)°C, (-5)-0°C, (-2)-4°C, 2-8°C, 5-20°C, 15-30°C, 25-40°C, or 35-55°C.

[0164] In some embodiments, steps (ii) and (iii) are performed in situ, thereby forming a crosslinked polymer by mixing the first (HA) chain and the second (HA) chain. In some embodiments, the preparation method does not involve post-processing steps such as sieving or homogenization.

[0165] In some embodiments of the preparation method, after the crosslinking step, unreacted norbornene or its derivatives and / or tetrazine or its derivatives may remain attached to the HA chain. As defined herein, the term "unreacted" refers to a norbornene moiety or its derivative and / or tetrazine moiety or its derivative that is not bound to another HA chain or moiety.

[0166] In some embodiments, norbornene or its derivatives and / or tetrazine or its derivatives may be modified. In some embodiments, post-crosslinking modification includes the binding of one or more molecules to unbound norbornene or its derivatives and / or unbound tetrazine or its derivatives on the crosslinked polymer.

[0167] Non-limiting examples of the one or more molecules include, but are not limited to, amino acids, anesthetics, minerals, vitamins, etc. The amount of unreacted norbornene or its derivatives and / or tetrazine or its derivatives on the HA chain can be adjusted by changing the ratio of the first HA chain to the second HA chain, or vice versa, during the cross-linking reaction.

[0168] Composition

[0169] In some embodiments, the present invention relates to compositions comprising the polymers of the present invention.

[0170] In some embodiments, the compositions of the present invention comprise cross-linked and non-cross-linked HA. In some embodiments, the total HA polymer content (cross-linked and non-cross-linked HA) of the composition is present at concentrations of up to 1 mg / gr, up to 5 mg / gr, up to 7 mg / gr, up to 8.5 mg / gr, up to 10 mg / gr, up to 12 mg / gr, up to 15 mg / gr, up to 17 mg / gr, up to 18.5 mg / gr, up to 20 mg / gr, up to 22 mg / gr, or up to 25 mg / gr. In some embodiments, the total HA polymer content of the composition is 1-2.5 mg / gr, 3-5 mg / gr, 4-7 mg / gr, 6-9 mg / gr, 8-12 mg / gr, 10-13 mg / gr, 12-15 mg / gr, 14-17 mg / gr, 16-19 mg / gr, 18-22 mg / gr, or 20-25 mg / gr. Each possibility represents a single embodiment of the invention.

[0171] According to another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the polymer of the present invention, and a pharmaceutically acceptable carrier and / or diluent. In some embodiments, the pharmaceutical composition may facilitate the delivery of the polymer to a living organism.

[0172] In some embodiments, the compositions of the present invention further include amino acids. In some embodiments, the amino acids include any amino acid, naturally occurring or non-naturally occurring. Non-limiting examples of non-naturally occurring amino acids include, but are not limited to, D-amino acids, hydroxylysine, 4-hydroxyproline, N-Cb2-protected aminovaleric acid (Nva), ornithine (O), aminooctanoic acid (AoC), 2,4-diaminobutyric acid (Abu), homoarginine, ortholeucine (Nle), N-methylaminobutyric acid (MeB), 2-naphthylalanine (2Np), aminoheptanoic acid (Ahp), phenylglycine, β-phenylproline, tert-leucine, 4-aminocyclohexylalanine (Cha), N-methyl-ortholeucine, 3,4-dehydroproline, N,N-dimethylaminoglycine, N-methylaminoglycine, 4-aminopiperidin-4-carboxylic acid, 6-aminohexanoic acid, and trans-4-(aminomethyl)-cyclohexyl Hexanecarboxylic acid, 2-, 3-, and 4-(aminomethyl)benzoic acid, 1-aminocyclopentanecarboxylic acid, 1-aminocyclopropanecarboxylic acid, cyanopropionic acid, 2-benzyl-5-aminovaleric acid, n-valine (Nva), 4-O-methylthreonine (TMe), 5-O-methyl-homoserine (hSM), tert-butylalanine (tBu), cyclopentylalanine (Cpa), 2-aminoisobutyric acid (Aib), N-methylglycine (MeG), N-methylalanine (MeA), N-methylphenylalanine (MeF), 2-thienylalanine (2Th), 3-thienylalanine (3Th), O-methyltyrosine (YMe), 3-benzothienylalanine (Bzt), and D-alanine (DAl). In some embodiments, the amino acids are amino acid oligomers or dimers linked by peptide bonds. In some embodiments, the oligomer is a trimer, tetramer, pentamer, hexamer, heptametamer, octamer, nonametamer, or decamer, or in some embodiments, a polymer having more than 11 amino acids linked together by peptide bonds. In some embodiments, the amino acids are included in the composition in the form of peptides, polypeptides, or proteins. In some embodiments, the peptides, polypeptides, or proteins included in the compositions of the present invention are in forms selected from, but not limited to, the following: natural, denatured, neutralized, digested, cross-linked, unfolded, reduced, oxidized, or inactivated forms.

[0173] In some embodiments, the compositions of the present invention further include one or more minerals. Non-limiting examples of minerals include, but are not limited to: potassium, chloride, sodium, calcium, phosphorus, magnesium, iron, zinc, manganese, copper, iodine, chromium, molybdenum, selenium, or cobalt.

[0174] In some embodiments, the compositions of the present invention further include vitamins. Non-limiting examples of vitamins include, but are not limited to: vitamin A (retinol, retinaldehyde, and four carotenoids, including β-carotene), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin, nicotinamide, nicotinamide nucleoside), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxamine, pyridoxal), vitamin B7 (biotin), vitamin B9 (folate (ester)), and vitamin B6. 12 (Cyanocobalamin, Hydroxycobalamin, Methylcobalamin, Adenosylcobalamin), Vitamin C (Ascorbic Acid), Vitamin D (Cholecalciferol (D3), Ergocalciferol (D2)), Vitamin E (Tocopherol, Tocotrienol) or Vitamin K (Phosphopone, Methylnaphthoquinone).

[0175] In some embodiments, the compositions of the present invention further include an anesthetic agent. As used herein, the term "anesthetic agent" refers to any molecule or substance that prevents pain (such as during surgery) or completely blocks any sensation. In one embodiment, the anesthetic agent is a general anesthetic. In one embodiment, the anesthetic agent is a local anesthetic. In some embodiments, a local anesthetic agent causes reversible loss of sensation limited to a specific area of ​​the body while maintaining consciousness.

[0176] Non-limiting examples of anesthetics include, but are not limited to, articaine, procaine, amethocaine, lidocaine, bupivacaine, levobupivacaine, ropivacaine, mepivacaine, dibucaine, and cocaine. In some embodiments, the compositions of the present invention comprise 0.01-0.1% (w / w), 0.05-0.15% (w / w), 0.1-0.3% (w / w), 0.2-0.5% (w / w), 0.4-0.7% (w / w), 0.6-0.85% (w / w), 0.8-1.25% (w / w), 1-1.5% (w / w), 1.4-2% (w / w), 1.75-3% (w / w), 2.5-3.75% (w / w), 3.5-4.5% (w / w), or 4.25-5.25% (w / w) of an anesthetic. In some embodiments, the compositions of the present invention comprise up to 0.01% (w / w), up to 0.05% (w / w), up to 0.75% (w / w), up to 1% (w / w), up to 1.5% (w / w), up to 2% (w / w), up to 3% (w / w), up to 4% (w / w), or up to 5.5% (w / w) of an anesthetic. Each possibility represents a single embodiment of the invention.

[0177] In some embodiments, the compositions of the present invention further include non-crosslinked HA. In some embodiments, non-crosslinked HA refers to any HA polymer that is not interconnected with another polymer (e.g., another HA polymer). In some embodiments, the term "interconnected" refers to a covalent bond formed between an unsaturated portion (e.g., a norbornene portion) or a derivative thereof and a tetrazine portion or a derivative thereof. In some embodiments, as defined herein, the percentage of non-crosslinked HA is a calculated proportion of non-crosslinked HA in the total HA content of the final product (i.e., the composition). In some embodiments, the composition comprises a total HA content of 1 to 6 mg / gr, 2 to 7 mg / gr, 3 to 8 mg / gr, 4 to 9 mg / gr, 5 to 10 mg / gr, 6 to 11 mg / gr, 7 to 12 mg / gr, 8 to 13 mg / gr, 9 to 14 mg / gr, 10 to 15 mg / gr, 11 to 16 mg / gr, 12 to 17 mg / gr, 13 to 18 mg / gr, 14 to 19 mg / gr, 15 to 20 mg / gr, 16 to 21 mg / gr, 17 to 22 mg / gr, 18 to 25 mg / gr, or any range thereof. In some embodiments, the non-crosslinked HA content in the compositions of the present invention is: up to 5%, up to 7%, up to 10%, up to 15%, up to 20%, up to 25%, up to 27%, up to 30%, or 35%. In some embodiments, non-crosslinked HA is present in the composition at concentrations of 1-5%, 2.5-7%, 4-9%, 8-12%, 10-16%, 15-20%, 18-25%, 22-28%, or 27-35%. Each possibility represents a single embodiment of the invention.

[0178] In another embodiment, the pharmaceutical composition of the present invention can be formulated as a pharmaceutically acceptable salt of the polymer of the present invention. In another embodiment, the pharmaceutically acceptable salt includes salts derived from non-toxic inorganic or organic acids (such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.). In some embodiments, the salt is formed with a free carboxyl group, such as salts derived from non-toxic inorganic or organic bases (such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.).

[0179] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or medium administered with the compounds disclosed herein. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil, etc., polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents. Water is a preferred carrier when the drug composition is administered intravenously. Saline solutions, as well as dextran solutions and aqueous solutions of glycerin, can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol, etc. If desired, the composition may also include small amounts of wetting agents or emulsifiers, or pH buffers such as acetates, citrates, or phosphates. Antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; and agents for regulating tension, such as sodium chloride or dextran, are also considered. The carrier may comprise, in total, from about 0.1% to about 99.9% by weight of the pharmaceutical composition presented herein.

[0180] As used herein, the term "pharmaceutical acceptable" means suitable for administration to an object, such as a human being. For example, the term "pharmaceutical acceptable" may mean approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals and, more specifically, in humans.

[0181] In another embodiment, the compositions of the present invention are in the form of solutions, suspensions, emulsions, tablets, powders, gels, foams, pastes, sustained-release formulations, etc. Examples of suitable drug carriers are described in Remington's Pharmaceutical Sciences, by E.W. Martin, the contents of which are incorporated herein by reference. Such compositions will comprise a therapeutically effective amount of the polymer of the present invention, preferably in a substantially purified form, and an appropriate amount of carrier to provide a form suitable for administration to the subject.

[0182] According to an embodiment of the present invention, the pharmaceutical composition comprises 0.1-95% of one or more polymers of the present invention. According to another embodiment of the present invention, the pharmaceutical composition comprises 1-70% of the polymer. According to another embodiment of the present invention, the composition or formulation to be administered may contain a certain amount of polymer, according to an embodiment of the present invention, to effectively treat the condition or disease of the treated subject.

[0183] Embodiments of this invention relate to the polymers of this invention presented in unit dosage forms and prepared by any method known in the pharmaceutical field. In embodiments of this invention, the unit dosage form is in the form of tablets, capsules, lozenges, ampoules, vials, or pre-filled syringes. Additionally, in vitro assays may optionally be employed to aid in identifying the optimal dosage range. The precise dosage to be used in the formulation will also depend on the route of administration and the nature of the disease or condition, and should be determined based on the practitioner's judgment and the individual patient's circumstances. The effective dose can be extrapolated from dose-response curves derived from in vitro or in vivo animal model bioassays or systems.

[0184] According to one embodiment, the compositions of the present invention are administered as pharmaceutical compositions comprising at least one of the active ingredients (polymers) of the present invention and a pharmaceutically acceptable carrier or diluent. In another embodiment, the compositions of the present invention may be administered alone or together with any conventional transdermal dosage form.

[0185] As used herein, the terms “give,” “administer,” and similar terms refer to any method of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect, in reasonable medical practice.

[0186] Depending on the location of the target tissue, the polymers of the present invention can be administered in any manner suitable for delivering the polymer to the target tissue. Thus, for example, a composition comprising the polymers of the present invention can be introduced (e.g., injected) into the target tissue, which will cause the polymer to distribute within the tissue.

[0187] In some embodiments, the pharmaceutical composition comprising the polymer is administered via ophthalmic, percutaneous, intradermal, subcutaneous, intramuscular, or intraperitoneal routes. The route of administration of the pharmaceutical composition will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injection, such as intradermal, intravenous, intramuscular, intra-injury, subcutaneous, intrathecal, and any other injection method known in the art. Although the bioavailability of polymers administered via other routes may be lower than that administered via parenteral injection, it is possible to administer the compositions of the invention via percutaneous, oral, rectal, vaginal, topical, nasal, inhalation, and ophthalmic therapeutic routes by using appropriate formulations.

[0188] For topical applications, the polymers of the present invention can be combined with pharmaceutically acceptable carriers to deliver an effective dose based on the desired activity. The carrier can be, for example, but not limited to, ointments, creams, gels, pastes, foams, aerosols, suppositories, pads, or gel sticks.

[0189] According to some embodiments, the polymers of the present invention can be delivered in a controlled-release system. In yet another embodiment, the controlled-release system can be placed near the therapeutic target, thus requiring only a portion of the systemic dose.

[0190] In one embodiment, it will be understood that the polymers of the present invention can be administered to an individual together with other active agents to achieve improved therapeutic effects compared to treatment with each agent alone. In another embodiment, measures are taken to address adverse side effects associated with combination therapy (e.g., administration and selection of supplements).

[0191] In one implementation, depending on the severity and responsiveness of the condition to be treated, the medication may be administered once or multiple times, and the treatment process may last for days to weeks or until a cure is achieved or the condition is alleviated.

[0192] In some embodiments, the polymer is administered in a therapeutically safe and effective amount. As used herein, the term "safe and effective amount" refers to an amount of component sufficient to produce the desired therapeutic response without causing excessive adverse side effects (e.g., toxicity, irritation, or anaphylactic response) commensurate with a reasonable benefit / risk ratio when used in the manner described herein. In another embodiment, the therapeutically effective amount of the polymer is the amount of polymer required for a measurable, expected biological or therapeutic effect in vivo. The actual amount administered, as well as the rate and timing of administration, will depend on the nature and severity of the condition being treated. The determination of treatment prescription (e.g., dosage, timing, etc.) is within the responsibility of the general practitioner or specialist and generally takes into account the barrier to be treated or the defect to be corrected, the individual patient's condition, the delivery site, the method of administration, and other factors known to the practitioner. Examples of techniques and protocols can be found in Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005). In some embodiments, the effective amount or dose may be initially estimated from in vitro assays. In one implementation, the dosage can be formulated in an animal model, and this information can be used to more accurately determine the dosage that is useful to humans.

[0193] In one embodiment, the toxicity and therapeutic efficacy of the active ingredient described herein can be determined in vitro in cell cultures or laboratory animals using standard pharmaceutical procedures. In one embodiment, data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a range of doses for human use. In one embodiment, the dose varies depending on the dosage form and route of administration. In one embodiment, individual physicians can select the exact formulation, route of administration, and dose based on the patient's condition. [See, for example, Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1].

[0194] Pharmaceutical compositions containing the polymers described herein as active ingredients can be prepared using conventional pharmaceutical mixing techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990). See also Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa. (2005).

[0195] In one embodiment, the composition, comprising the formulation of the present invention formulated in a compatible drug carrier, is prepared, arranged in a suitable container, and labeled for treating a specified condition.

[0196] In one embodiment, the composition of the invention is presented in a packaging or dispenser device, such as an FDA-approved kit containing one or more unit dosage forms containing the active ingredient. In one embodiment, the packaging includes, for example, metal or plastic foil, such as a blister pack. In one embodiment, the packaging or dispenser device is accompanied by instructions. In one embodiment, the packaging or dispenser is contained in a container-associated label in a form prescribed by a government agency regulating the manufacture, use, or sale of the drug, reflecting that agency's approval of the composition in human or veterinary form. In one embodiment, such a label is a label or approved product insert approved by the U.S. Food and Drug Administration (FDA) for prescription drugs.

[0197] How to use it.

[0198] In some embodiments, the present invention relates to a method of filling or filling tissue into an object in need, the method comprising administering a pharmaceutical composition to the object comprising a therapeutically effective amount of the polymer of the present invention.

[0199] In some embodiments, filling or hydration methods, as defined herein, refer to the injection of soft filler materials into skin tissue. In some embodiments, filling is filling wrinkles, such as facial wrinkles. In some embodiments, filling is restoring the smooth appearance or texture of the skin.

[0200] In some embodiments, the method involves injecting the polymer or composition of the present invention into skin tissue. In some embodiments, the method involves injecting the polymer or composition of the present invention into gingival tissue. In some embodiments, the method involves injecting the polymer or composition of the present invention into cartilage tissue. In some embodiments, the method involves injecting the polymer or composition of the present invention into ophthalmic tissue.

[0201] According to some embodiments, injection can be performed using any method and any injection device known in the art. Non-limiting examples of injection devices include, but are not limited to, the use of syringes, microinjectors, needle-free devices, microneedles, needles, cannulas, and catheters. Non-limiting examples of needle gauges include, but are not limited to, 18G, 19G, 20G, 21G, 22G, 23G, 24G, 25G, 26G, 27G, 28G, 29G, 30G, 31G, 32G, 33G, or 34G.

[0202] In some embodiments, the method of the present invention relates to treating arthritis in a subject in need. Non-limiting examples of arthritis include, but are not limited to, acute infectious arthritis, calcium pyrophosphate arthritis, temporomandibular joint (TMJ) arthritis, reactive arthritis, psoriatic arthritis, chronic infectious arthritis, juvenile idiopathic arthritis (JIA), rheumatoid arthritis (RA), or prosthetic joint infectious arthritis.

[0203] In one embodiment, the method of the present invention relates to treating a person suffering from osteoarthritis.

[0204] In some embodiments, the method of the present invention relates to preventing or treating ophthalmic tissue damage to a subject before or after a surgical procedure.

[0205] In some embodiments, the method involves topically applying the polymer or composition of the present invention. In some embodiments, the polymer of the present invention is applied to skin tissue. In some embodiments, the method involves promoting / enhancing wound healing in a subject in need. In some embodiments, the method involves promoting / enhancing wound closure in a subject in need.

[0206] In one embodiment, the polymer of the present invention is provided to the subject in its own form. In one embodiment, one or more polymers of the present invention are provided to the subject in its own form. In one embodiment, the polymer of the present invention is provided to the subject as part of a pharmaceutical composition (in which it is mixed with a pharmaceutically acceptable carrier). In one embodiment, one or more polymers of the present invention are provided to the subject as part of a pharmaceutical composition (in which it is mixed with a pharmaceutically acceptable carrier).

[0207] As used herein, the term "object" refers to an animal, and more specifically to non-human mammals and human organisms. Non-human animal objects may also include prenatal forms of animals, such as embryos or fetuses. Non-limiting examples of non-human animals include, but are not limited to: horses, cattle, camels, goats, sheep, dogs, cats, non-human primates, mice, rats, rabbits, hamsters, guinea pigs, or pigs. In one embodiment, the object is a human. Human objects may also include fetuses. In one embodiment, the object in need is an object suffering from arthritis-related conditions and / or at risk of developing arthritis-related conditions. In some embodiments, the object in need is an object suffering from tissue volume reduction. In some embodiments, tissue volume reduction is referred to as "defilling." In some embodiments, tissue defilling includes fat loss, water loss, moisture loss, extracellular matrix degradation, collagen loss, or others. In some embodiments, defilling causes skin sagging and deterioration. In some embodiments, the object suffers from burns. In some embodiments, the object requiring wound closure methods suffers from fluid leakage, such as bleeding. As used herein, the term “treatment” or “management” for a disease, disorder, or condition encompasses the reduction of at least one symptom, a decrease in its severity, or the inhibition of its progression. Treatment does not imply a complete cure of the disease, disorder, or condition. As an effective treatment, the compositions useful herein need only reduce the severity of the disease, disorder, or condition, decrease the severity of its associated symptoms, or provide an improvement in the quality of life of the patient or subject.

[0208] As used herein, the term “prevention” for a disease, disorder, or condition includes delaying, preventing, suppressing, or inhibiting the onset of a disease, disorder, or condition. As used in light of the subject matter currently described, the term “prevention” refers to a method of prevention in which an object is exposed to the polymer currently described or a composition comprising the polymer prior to the onset or progression of the disease / disorder. In any case, the term “prevention” may be used to encompass prevention. Conversely, the term “treatment” refers to the clinical administration of an active agent to counteract a condition that is already present in the patient’s body and whose clinical symptoms have occurred.

[0209] As used herein, the term “condition” includes anatomical and physiological deviations from the normal state that constitute an impairment of the normal state of a living animal or part thereof, which interrupts or alters the performance of bodily functions.

[0210] Reagent test kit

[0211] According to some embodiments, the present invention provides a kit comprising a first HA chain or a derivative thereof coupled to an unsaturated moiety or a derivative thereof, and a second HA chain or a derivative thereof coupled to a tetrazine moiety or a derivative thereof. The terms “HA chain or derivative thereof,” “unsaturated moiety or derivative thereof,” and “tetrazine moiety or derivative thereof” are as defined above.

[0212] In some embodiments, the present invention provides a kit comprising a first HA chain or a derivative thereof coupled to a norbornene moiety or a derivative thereof and a second HA chain or a derivative thereof coupled to a tetrazine moiety or a derivative thereof, and a device designated for administering a dose for injection into or through the skin or for microperforation of the skin.

[0213] According to some embodiments, the present invention provides a kit comprising a polymer comprising a first hyaluronic acid (HA) chain or a derivative thereof and a second HA chain or a derivative thereof, wherein the first HA chain and the second HA chain are crosslinked via one or more linkers, wherein the one or more linkers comprise a norbornene moiety or a derivative thereof coupled to a tetrazine moiety or a derivative thereof, and wherein the polymer is characterized by being crosslinked via one or more linkers. 1 The degree of crosslinking, as determined by H NMR, ranged from 0.2% to 4%.

[0214] In some embodiments, the injection device is as disclosed above. In one embodiment, the injection device is disposable. In some embodiments, the injection device is adapted for intradermal and / or intradermal and / or subcutaneous injection. In some embodiments, the injection device is adapted for mesotherapy techniques. As used herein, the term "mesotherapy" refers to non-surgical cosmetic pharmaceutical treatment.

[0215] In some embodiments, the kit also includes the amino acids disclosed above.

[0216] In some embodiments, the kit also includes the minerals disclosed above.

[0217] In some embodiments, the kit also includes the vitamins disclosed above.

[0218] In some embodiments, the kit also includes the anesthetic agent disclosed above.

[0219] In some embodiments, the kit also includes a pharmaceutically acceptable carrier as disclosed above.

[0220] In some embodiments, the packaging is scored to allow sampling of a first HA chain or a derivative thereof coupled to the norbornene moiety or a derivative thereof, a second HA chain or a derivative thereof coupled to the tetrazine moiety or a derivative thereof, free HA chains, amino acids, vitamins, minerals, anesthetics, or any combination thereof.

[0221] In one embodiment, the packaging is in the form of ampoules, vials, or capsules. In one embodiment, the capsule is a soft capsule.

[0222] In some embodiments, the components of the kits disclosed above are sterile. As used herein, the term "sterile" refers to a state free from biological contaminants. Any sterilization method is applicable and will be apparent to those skilled in the art.

[0223] In some embodiments, the kit also includes a free HA chain or a derivative thereof. As defined herein, a free HA chain or a derivative thereof is an HA chain not coupled to the tetrazine moiety, norbornene moiety, or a derivative thereof.

[0224] According to some embodiments, the kit is used by mixing a first HA chain or derivative thereof coupled with a norbornene moiety or a derivative thereof and a second HA chain or derivative thereof coupled with a tetrazine moiety or a derivative thereof, and applying the composition formed by mixing the first HA chain or derivative thereof coupled with a norbornene moiety or a derivative thereof and the second HA chain or derivative thereof coupled with a tetrazine moiety or a derivative thereof to a filling or filling method.

[0225] In some embodiments, the kit is utilized by further mixing a composition formed by mixing a first HA chain or a derivative thereof coupled with a norbornene moiety or a derivative thereof and a second HA chain or a derivative thereof coupled with a tetrazine moiety or a derivative thereof with free HA chains, amino acids, vitamins, minerals, anesthetics, or any combination thereof, and applying the composition formed by mixing the first HA chain or a derivative thereof coupled with a norbornene moiety or a derivative thereof and a second HA chain or a derivative thereof coupled with a tetrazine moiety or a derivative thereof, along with free HA chains, amino acids, vitamins, minerals, anesthetics, or any combination thereof, to a filling or filling method.

[0226] In some embodiments, the kit includes instructions for mixing a first HA chain or a derivative thereof coupled to a norbornene moiety or a derivative thereof and a second HA chain or a derivative thereof coupled to a tetrazine moiety or a derivative thereof in a ratio of about 3:1. In some embodiments, the kit includes instructions for mixing a first HA chain or a derivative thereof coupled to a norbornene moiety or a derivative thereof and a second HA chain or a derivative thereof coupled to a tetrazine moiety or a derivative thereof in a ratio of about 3:2. In some embodiments, the kit includes instructions for mixing a first HA chain or a derivative thereof coupled to a norbornene moiety or a derivative thereof and a second HA chain or a derivative thereof coupled to a tetrazine moiety or a derivative thereof in a ratio of about 2:1. In some embodiments, the kit includes instructions for mixing a first HA chain or a derivative thereof coupled to a norbornene moiety or a derivative thereof and a second HA chain or a derivative thereof coupled to a tetrazine moiety or a derivative thereof in a ratio of about 1:1. In some embodiments, the kit includes instructions for mixing a first HA chain or a derivative thereof coupled to a norbornene moiety or a derivative thereof and a second HA chain or a derivative thereof coupled to a tetrazine moiety or a derivative thereof in a ratio of about 1:2. In some embodiments, the kit includes instructions for mixing a first HA chain or a derivative thereof coupled to a norbornene moiety or a derivative thereof and a second HA chain or a derivative thereof coupled to a tetrazine moiety or a derivative thereof in a ratio of about 2:3. In some embodiments, the kit includes instructions for mixing a first HA chain or a derivative thereof coupled to a norbornene moiety or a derivative thereof and a second HA chain or a derivative thereof coupled to a tetrazine moiety or a derivative thereof in a ratio of about 1:3. In some embodiments, the kit includes instructions for mixing a first HA chain or a derivative thereof coupled to a norbornene moiety or a derivative thereof and a second HA chain or a derivative thereof coupled to a tetrazine moiety or a derivative thereof in any of the above-described ratios or any ratio between them.

[0227] In some embodiments, the kit includes instructions for mixing a first HA chain or a derivative thereof coupled with a norbornene moiety or a derivative thereof, a second HA chain or a derivative thereof coupled with a tetrazine moiety or a derivative thereof, as well as free HA chains, amino acids, vitamins, minerals, anesthetics, or any combination thereof.

[0228] In some embodiments, the kit is utilized by mixing a first HA chain or a derivative thereof coupled with a norbornene moiety or a derivative thereof, a second HA chain or a derivative thereof coupled with a tetrazine moiety or a derivative thereof, as well as a free HA chain, amino acids, vitamins, minerals, anesthetics, or any combination thereof, wherein mixing includes introducing the components into an injection device.

[0229] In some embodiments, the kit includes instructions for mixing a first HA chain or a derivative thereof coupled with a norbornene moiety or a derivative thereof, a second HA chain or a derivative thereof coupled with a tetrazine moiety or a derivative thereof, as well as free HA chains, amino acids, vitamins, minerals, anesthetics, or any combination thereof, wherein the mixing is carried out in an injection device.

[0230] In some embodiments of this subject kit, the degree of crosslinking of the composition formed by mixing a first HA chain or a derivative thereof coupled with a norbornene moiety or a derivative thereof, a second HA chain or a derivative thereof coupled with a tetrazine moiety or a derivative thereof, as well as free HA chains, amino acids, vitamins, minerals, anesthetics, or any combination thereof, is 0.01-0.1%, 0.01-0.5%, 0.05-0.1%, 0.1-0.3%, etc. 0.1-0.5%, 0.1-0.75%, 0.1-1%, 1-1.75%, 1-2%, 1-2.5%, 2-2.5%, 2.25-3%, 2.5-3.25%, 3-3.75%, 3.6-4.2%, 4-5.25%, 5-6.5%, 6-7.5%, 7-8.5%, 8-9.25%, 9-10.5%, 10-12.5% ​​or any range therebetween.

[0231] In some embodiments of this subject kit, the phase angle (δ) of the composition formed by mixing a first HA chain or a derivative thereof coupled with an unsaturated portion (e.g., norbornene portion) or a derivative thereof, and a second HA chain or a derivative thereof coupled with a tetrazine portion or a derivative thereof, as well as free HA chains, amino acids, vitamins, minerals, anesthetics, or any combination thereof, is 0.1-0.5°, 0.1-0.9°, 0.5-1°, 0.7-1.5°, 1-2.5°, 2-4.5°, 3-4.75°, 4.7-5.5°, 5-7.5°, 6-8°, 7-8.5°, 8.25-9.5°, 9-10.5°, 9.5-12°, or any range therebetween.

[0232] In some embodiments of this subject kit, the elastic modulus of the composition formed by mixing a first HA chain or a derivative thereof coupled with a norbornene moiety or a derivative thereof, a second HA chain or a derivative thereof coupled with a tetrazine moiety or a derivative thereof, as well as free HA chains, amino acids, vitamins, minerals, anesthetics, or any combination thereof, is 10-500 Pa, 20-1,000 Pa, 30-600 Pa, 40-1,000 Pa, or 40-500 Pa. 0 Pa, 50-10,000 Pa, 500-50,000 Pa, 500-10,000 Pa, 500-5,000 Pa, 500-1,000 Pa, 1,000-50,000 Pa, 1,000-10,000 Pa, 1,000-5,000 Pa, 50-20,000 Pa, 500-20,000 Pa, or 1,000-20,000 Pa or any range thereof.

[0233] In some implementations, the reagent kit components are packaged in containers.

[0234] In some embodiments, the container is made of a material selected from: thin-walled film or plastic (transparent or opaque), cardboard-based, foil, rigid plastic, metal (e.g., aluminum), glass, etc.

[0235] In some implementations, the contents of the kit are packaged as described below to allow the components to be stored until they are needed.

[0236] In some implementations, some or all of the components of the kit may be packaged in suitable packaging to maintain sterility.

[0237] In some implementations, the packaging has a cap that allows for an airtight seal during storage and can be punctured by a needle or cannula when in use.

[0238] In some implementations, the kit components are stored in separate containers, such as boxes or similar structures, within the main kit containing element. These containers may or may not be airtight, for example, to further maintain the sterility of some or all of the kit components.

[0239] In some embodiments, the dosage of the first HA chain or derivative thereof coupled to the norbornene moiety or its derivative, the second HA chain or derivative thereof coupled to the tetrazine moiety or its derivative, the free HA chain, amino acids, vitamins, minerals or anesthetics provided in the kit may be sufficient for single or multiple administrations.

[0240] In some embodiments, the kit may have multiple doses of a first HA chain or derivative thereof coupled with a norbornene moiety or a derivative thereof, a second HA chain or derivative thereof coupled with a tetrazine moiety or a derivative thereof, a free HA chain, amino acids, vitamins, minerals or anesthetics, packaged in a single container, such as a single tube, long bottle, vial, Eppendorf, etc.

[0241] In some embodiments, the kit may have individually packaged multiple doses of a first HA chain or derivative thereof coupled with a norbornene moiety or a derivative thereof, a second HA chain or derivative thereof coupled with a tetrazine moiety or a derivative thereof, a free HA chain, amino acids, vitamins, minerals, or anesthetics, such that certain kits may have more than one container of a first HA chain or derivative thereof coupled with a norbornene moiety or a derivative thereof, a second HA chain or derivative thereof coupled with a tetrazine moiety or a derivative thereof, a free HA chain, amino acids, vitamins, minerals, or anesthetics.

[0242] In some embodiments, multiple doses of a first HA chain or derivative thereof coupled with a norbornene moiety or a derivative thereof, a second HA chain or derivative thereof coupled with a tetrazine moiety or a derivative thereof, a free HA chain, amino acids, vitamins, minerals, or anesthetics may be packaged in a single container.

[0243] In some embodiments, the kit includes a description of the preparation of the compositions used therein and how to carry out the methods of the invention.

[0244] In some implementations, the instruction manual may be recorded on a suitable recording medium or substrate. For example, the instruction manual may be printed on a substrate such as paper or plastic.

[0245] In some embodiments, the instructions may be included in the kit as a packaging insert, on a label of the kit container or its components (i.e., associated with the packaging or sub-packaging), etc. In other embodiments, the instructions exist as an electronic storage data file on a suitable computer-readable storage medium, such as a CD-ROM, disk, etc. In still other embodiments, there is no actual instruction manual in the kit, but rather a method for obtaining the manual from a remote source (e.g., via the Internet). An example of this implementation is a kit that includes a URL where the instructions can be viewed and / or downloaded. Like the instructions themselves, this method of obtaining the instructions is documented on a suitable substrate.

[0246] Unless otherwise stated, any concentration range, percentage range or proportion range mentioned herein shall be understood to include any integer concentration, percentage or proportion and its fractions within that range, such as one-tenth and one-hundredth of an integer.

[0247] Unless otherwise stated, any numerical ranges relating to any physical characteristic such as polymer subunits, size, or thickness described herein should be understood to include any integers within the range.

[0248] As used herein, the terms “object” or “individual” or “animal” or “patient” or “mammal” refer to any object requiring treatment, specifically a mammalian object, such as a human.

[0249] In this discussion, unless otherwise stated, adjectives modifying one or more features of embodiments of the invention (such as “substantially” and “about”) are understood to mean that the situation or feature is limited to within acceptable tolerances for the intended application. Unless otherwise stated, the word “or” in the specification and claims is considered inclusive rather than exclusive and indicates at least one or any combination of its associated items.

[0250] It should be understood that the terms “a” and “an” as used above and elsewhere in this document refer to “one or more” of the listed components. Those skilled in the art will appreciate that, unless otherwise specified, the use of the singular includes the plural. Therefore, the terms “a,” “an,” and “at least one” are used interchangeably in this application.

[0251] To better understand this teaching and without limiting its scope in any way, all numerical values, percentages, or proportions used in the specification and claims, unless otherwise stated, shall be understood to be modified by the term "about" in all cases. Therefore, unless stated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties to be obtained. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying conventional rounding techniques.

[0252] In the specification and claims of this application, each of the verbs “comprising,” “including,” and “having,” and variations thereof, is used to indicate that one or more objects of the verb are not necessarily a complete enumeration of the subject or components, elements, or parts of the subject.

[0253] Other terms used herein are intended to be defined by their commonly known meanings in the art.

[0254] Other objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following embodiments (which are not intended to be limiting). Furthermore, each of the various embodiments and aspects of the invention as described above and as set forth in the appended claims is experimentally supported in the following embodiments.

[0255] It should be understood that certain features of the invention described in separate implementation settings for clarity may also be provided in combination in a single implementation. Conversely, various features of the invention described in a single implementation setting for brevity may also be provided separately or in any suitable sub-combination or, where appropriate, in any other said implementation of the invention. Certain features described in various implementation settings should not be considered essential features of those implementations unless the implementation would not function without those elements.

[0256] In one embodiment, the term "alkyl" includes aliphatic hydrocarbons, including straight-chain and branched groups. Preferably, the alkyl group has 21 to 100 carbon atoms, more preferably 21 to 50 carbon atoms. Whenever a numerical range is described herein; for example, "21-100" indicates that the group (in this case, the alkyl group) may include 21 carbon atoms, 22 carbon atoms, 23 carbon atoms, etc., up to and including 100 carbon atoms.

[0257] In one embodiment, the term "long alkyl" includes alkyl groups having at least 20 carbon atoms in their main chain (the longest path of consecutively covalently attached atoms). Thus, short alkyl groups have 20 or fewer main chain carbons. In one embodiment, the alkyl group may be substituted or unsubstituted. In one embodiment, the term "alkyl" as used herein also covers saturated or unsaturated hydrocarbons, and therefore the term further covers alkenyl and ynyl groups.

[0258] In one embodiment, the term "alkenyl" describes an unsaturated alkyl group as defined herein, having at least two carbon atoms and at least one carbon-carbon double bond. The alkenyl group may be substituted with one or more substituents or remain unsubstituted, as described above. In one embodiment, the term "alkynyl" as defined herein is an unsaturated alkyl group having at least two carbon atoms and at least one carbon-carbon triple bond. The alkynyl group may be substituted with one or more substituents or remain unsubstituted.

[0259] In one embodiment, the term "unsaturated" describes a compound containing one or more unsaturated bonds. In some embodiments, an unsaturated bond refers to a double bond and / or a triple bond.

[0260] In one embodiment, the term "cycloalkyl" describes an all-carbon monocyclic or fused-ring (i.e., a ring sharing an adjacent pair of carbon atoms) group, wherein one or more rings do not have a fully conjugated π-electron system. Cycloalkyl groups can be substituted or unsubstituted.

[0261] In one embodiment, the term "aryl" describes an all-carbon monocyclic or fused-ring polycyclic group having a fully conjugated π-electron system. In one embodiment, the aryl group may be substituted or unsubstituted.

[0262] In one embodiment, the term "alkoxy" describes -O-alkyl and -O-cycloalkyl. In one embodiment, the term "aryloxy" describes -O-aryl. In one embodiment, the terms alkyl, cycloalkyl, and aryl in the general formula herein may be substituted with one or more substituents, wherein each substituent may independently be, for example, halide, alkyl, alkoxy, cycloalkyl, alkoxy, nitro, amine, hydroxy, mercapto, thioalkoxy, thiohydroxy, carboxyl, amide, aryl, and aryloxy, depending on the substituted group and its position in the molecule.

[0263] In one embodiment, “halogen / halogen ion,” “halogen,” or “halogenated” describes fluorine, chlorine, bromine, or iodine. In one embodiment, “haloalkyl” describes an alkyl group as defined herein that is further substituted with one or more halogen groups. In one embodiment, “haloalkoxy” describes an alkoxy group as defined herein that is further substituted with one or more halogen groups. In one embodiment, the term “hydroxyl” (“hydroxyl” or “hydroxy”) describes an -OH group. In one embodiment, the term “thiohydroxy” or “thiol” describes a -SH group. In one embodiment, the term “thioalkoxy” describes -S-alkyl and -S-cycloalkyl. In one embodiment, the term “thioaryloxy” describes -S-aryl and -S-heteroaryl. In one embodiment, the term “amine” describes a -NR'R” group having R' and R”. In one embodiment, the term “heteroaryl” describes a monocyclic or fused ring (i.e., a ring sharing an adjacent pair of atoms) having one or more atoms such as nitrogen, oxygen, and sulfur in the ring and additionally having a fully conjugated π-electron system. Non-limiting examples of heteroaryl compounds include pyrrole, furan, thiophene, imidazole, Azole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, and purine.

[0264] In one embodiment, the term "heterocyclic group" or "heterocyclic group" describes a monocyclic or fused ring group having one or more atoms such as nitrogen, oxygen, and sulfur in the ring. The ring may also have one or more double bonds. In one embodiment, the ring does not have a fully conjugated π-electron system. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholino, etc.

[0265] In one embodiment, the term "carboxyl" or "carboxylic acid group" describes a -C(=O)-OR′ group, wherein R′ is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (by cyclic carbon bonding) or heterocyclic (by cyclic carbon bonding).

[0266] In one embodiment, the term "carbonyl" describes a -C(=O)-R' group, where R' is as defined above. In one embodiment, the term also includes its thio derivatives (thiocarboxyl and thiocarbonyl).

[0267] In one embodiment, the term "thiocarbonyl" describes a -C(=S)-R' group, wherein R' is as defined above. In one embodiment, the term "thiocarboxyl" describes a -C(=S)-OR' group, wherein R' is as defined herein. In one embodiment, the term "sulfinyl" describes a -S(=O)-R' group, wherein R' is as defined herein. In one embodiment, the terms "sulfonyl" or "sulfonic acid" describe a -S(=O)2-R' group, wherein R' is as defined herein. In one embodiment, the terms "carbamoyl" or "carbamate" describe a -OC(=O)-NR'R" group, wherein R' is as defined herein and R" is as generally defined as R'.

[0268] In one embodiment, the term "nitro" refers to a -NO2 group. In one embodiment, the terms "cyano" or "nitrile" refer to a -C≡ group, which in turn refers to a -N3 group. In one embodiment, the term "sulfonamide" refers to a -S(=O)2-NR'R" group, where R' and R" are as defined herein. (Note: The last part, "sulfanamide," appears to be an error and is left untranslated.)

[0269] In one embodiment, the term "phosphonoyl" or "phosphonic acid" describes the -OP(=O)(OR′)2 group, wherein R′ is as defined above. In one embodiment, the term "phosphonoyl" describes the -PR'R" group, wherein R' and R" are as defined above.

[0270] In one embodiment, the term "alkylaryl" describes an alkyl group as defined herein that has been substituted with an aryl or heteroaryl group. In one embodiment, the alkylaryl group is a benzyl group.

[0271] In one embodiment, the term "heteroaryl" describes a monocyclic or fused ring (i.e., a ring sharing a pair of adjacent atoms) having one or more atoms such as, for example, nitrogen, oxygen, and sulfur, and additionally having a fully conjugated π-electron system. Non-limiting examples of heteroaryls include pyrrole, furan, thiophene, imidazole, etc. Azoles, thiazoles, pyrazoles, pyridines, pyrimidines, quinolines, isoquinolines, and purines. Heteroaryl groups may be substituted with one or more substituents or remain unsubstituted, as described above. Representative examples are thiadiazoles, pyridines, pyrroles, etc. Azole, indole, purine, etc.

[0272] In one embodiment, the terms “halogenated” and “halogenated” are used interchangeably herein to describe a halogen atom, namely fluorine, chlorine, bromine, or iodine, also referred to herein as fluorinyl, chloroyl, bromine, and iodine. In one embodiment, the term “halogenated alkyl” describes an alkyl group as defined above that is further substituted with one or more halogenated groups.

[0273] Example

[0274] Materials and methods.

[0275] Method A: Preparation of HA-tetraazine or HA-norbornene.

[0276] Sodium hyaluronate was dissolved at a concentration of 5 to 10 mg / g in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (100 mM, pH 5.5). After obtaining a homogeneous solution, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholino hydrochloride (DMTMM) was added, followed by the primary amine to be coupled. The reagent volume was adjusted according to the desired degree of modification. The reaction was stirred at 10 to 50 °C for 24 hours, then dialyzed against a saturated NaCl solution (MWCO = 12 kDa) for 24 hours, followed by several dialyzes against purified water. The solution was transferred to a flask, cooled to -80 °C, and lyophilized to provide the modified HA product in solid form.

[0277] Method B: Preparation of HA-tetraazine, HA-norbornene, or HA-styrene.

[0278] Sodium hyaluronate was dissolved in double-distilled water (DDW) at a concentration of 5 to 10 mg / g. After obtaining a homogeneous solution, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) was added, followed by the desired primary amine. The reagent volume was adjusted according to the desired degree of modification. The reaction was stirred at 20 to 90 °C for 4 hours, then dialyzed against a saturated NaCl solution (MWCO = 12 kDa) for 24 hours, followed by several dialyses against purified water. The solution was transferred to a flask, cooled to -80 °C, and lyophilized to provide the modified HA product in solid form.

[0279] The degree of modification of HA-tetraazine, HA-norbornene, and HA-styrene, and the degree of crosslinking of the gel were determined.

[0280] To determine the degree of modification, dried samples modified with HA (e.g., HA-tetraazine, HA-norbornene, or HA-styrene) were dissolved in D2O and treated with hyaluronidase (from bovine testis, type IV-S, from Sigma Aldrich) at a final concentration of 300 units / mL until the solution was liquid. 1The sample was analyzed by 1H NMR. The degree of modification was determined by calculating the integration ratio, as described below.

[0281] The degree of modification of HA-tetraazine (Formula IIIB) was calculated as the ratio of the peak area under the aromatic protons of the tetraazine (7.5 to 7.7 ppm and 8.1 to 8.9 ppm, 8H) to the peak area under the N-acetylated protons of the glucosamine residues of hyaluronic acid (2.0 ppm, 3H). The modification degree of HA-tetraazine... 1 The H NMR spectrum is provided in Figure 1.

[0282] The degree of modification of HA-norbornene (Formula IIA): the ratio of the peak area under the alkyl proton (5.8 to 6.3 ppm, 2H) to the peak area under the N-acyl proton corresponding to the glucosamine residue of hyaluronic acid (2.0 ppm, 3H). The modification degree of HA-norbornene... 1 The H NMR spectrum is provided in Figure 2.

[0283] The degree of modification of HA-styrene (formula IA): the ratio of the peak area corresponding to the alkylene proton (5.36 ppm, 5.90 ppm, and 6.8 ppm, 3H) to the signal area corresponding to the N-acetyl proton of the glucosamine residue of hyaluronic acid (2.0 ppm, 3H). HA-styrene's 1 The H NMR spectrum is provided in Figure 3.

[0284] The degree of crosslinking of the gel is calculated according to the following equation:

[0285]

[0286] [D c =(D m (Tet) / M(Tet)+D m (Nor) / M(Nor)) / (M(Tet)+M(Nor))].

[0287] in:

[0288] -D c It refers to the degree of cross-linking of the gel;

[0289] -D m (Tet) represents the degree of modification of HA-tetraazine, determined by... 1 Determined by H NMR;

[0290] -D m (Nor) represents the degree of modification of HA-norbornene, determined by... 1 Determined by H NMR;

[0291] -M(Tet) is the weight of the HA-tetraazine involved in cross-linking;

[0292] -M(Nor) is the weight of the HA-norbornene involved in the cross-linking.

[0293] Crosslinking of HA-tetraazine and HA-norbornene

[0294] Two samples, HA-tetraazine and HA-norbornene, were dissolved in phosphate buffer solutions with pH values ​​ranging from 6.8 to 7.8 at the concentrations specified below (Table 1). Equal volumes of the two solutions were mixed and stirred at 37°C for 18 hours, followed by further stirring at room temperature until the rheological gel parameters stabilized. For samples containing non-crosslinked hyaluronic acid, the same concentration of non-crosslinked hyaluronic acid was added after the reaction, and the mixture was homogenized with stirring. The degree of crosslinking of the resulting gel was the average of the modification degrees of HA-tetraazine and HA-norbornene used.

[0295] Measurement of rheological parameters.

[0296] Phase angle δ (in °) and elastic modulus G' (in Pa) were measured at 25 °C at a frequency of 1 Hz, with stress scans of 1 to 1,000 Pa or strain scans of 0.35 to 3500%, using a rheometer (The Rmo Haake MARS 6000 or RS1 or TA DHR1) with a serrated or sandblasted plate geometry of 20 mm diameter.

[0297] Table 1. Summary of Rheological Testing

[0298]

[0299]

[0300] Example 1

[0301] Rheological parameters of HA crosslinked polymers

[0302] First, commercially available dermal fillers were examined (Table 1, entries 1-6, 25, 26). Data showed that δ values ​​less than 10° were only obtained in products with high HA content (20 mg / gr – entries 1-3) and / or high crosslinking degree (6-9% – entries 2-5). Products with high HA content and high crosslinking degree were also observed to result in δ values ​​greater than 10° (entry 6). Products with lower concentrations (12 mg / gr, entries 25-26) and high or low crosslinking degree (6% – entry 25 or 1% – entry 26) resulted in δ values ​​greater than 10°.

[0303] Samples containing 10 mg / gr of hyaluronic acid were prepared with various degrees of crosslinking (Table 1, entries 7-10, 20). Low δ values ​​were obtained with crosslinking degrees ranging from 3.5% to 0.9%. However, very low crosslinking degrees, such as 0.35%, failed to result in gel formation (entry 10).

[0304] Samples were then prepared using 10 mg / gr of hyaluronic acid, and different amounts of non-crosslinked HA were added to the final products (Table 1, entries 11-14). As observed by the increase in δ value, the addition of non-crosslinked HA reduced the lifting capacity of the crosslinked material. Nevertheless, gels containing up to 20% HA content still showed δ values ​​below 10°.

[0305] Samples with a fixed degree of crosslinking were further formulated with 1.3% or 0.9% HA and various HA contents (Table 1, entries 15-17, 20-22). Reductions from 15 mg / gr to 10 mg / gr and 5 mg / gr (entries 15-17) or from 10 mg / gr to 7.5 mg / gr and 5 mg / gr (entries 20-22) reduced the gel modulus (G') of the crosslinked materials. However, even with HA contents as low as 5 mg / gr, the δ value remained below 10°.

[0306] To test the generality of the crosslinking molecules, tetrazine and norbornene derivatives were mixed with a fixed HA concentration (10 mg / gr; Table 1, entries 18-19). As shown, δ values ​​below 10° were obtained.

[0307] To test the substitution of norbornene with another unsaturated moiety, HA was modified with styrene (Formula IA). Mixing the tetrazine derivative with the styrene derivative at a crosslinking degree of 1.1% and an HA content of 10 mg / gr or 7.5 mg / gr provided δ values ​​below 10° (Table 1, entries 23-24).

[0308] The linear viscoelastic region (LVR) represents the linear region of the elastic modulus (G') under dynamic strain / stress scanning. A longer LVR region is associated with the superior properties of the gel because it retains its viscoelasticity with increasing applied force. LVR is measured as the starting point of the standard strain scan cycle of the rheometer (TA DHR1) (the intersection of the two tangents of the G' curve), as shown in Figure 4.

[0309] Commercial dermal fillers offer a range of starting values, from very low as low as 9.4 Pa (Table 2, entry 7) to very high as high as 421 Pa (Table 2, entry 2). LVR data for these products clearly show a correlation with HA content (Table 2, entries 3-6 at 6% crosslinking, entries 1 and 7 at 1%). LVR data for commercial products also show a significant correlation with crosslinking degree (Table 2, entries 1-3 at 20 mg / gr). The gel according to the invention shows a similar correlation between LVR and HA content with crosslinking degree (Table 2, entries 8-13). However, the gel according to the invention exhibits a higher LVR compared to commercially available products, thus demonstrating superior material properties while including lower HA content and lower crosslinking degree (Table 2, entries 8-12 vs entries 1, 4-7, and entry 13 vs entry 7).

[0310] Table 2. Comparison of LVR values

[0311]

[0312]

[0313] Example 2

[0314] Extrudability of HA crosslinked polymers

[0315] Two samples, HA-tetraazine and HA-norbornene, were dissolved separately at the same concentration in phosphate buffer solution at pH 7.0. Equal volumes of each sample were then mixed together and immediately introduced into 1 mL long cyclic olefin polymer (COP) syringes. All syringes were incubated at 37°C for 18 hours, followed by further incubation at room temperature until the rheological gel parameters stabilized.

[0316] Extrusion force was measured using a Mecmesin traction benchtop instrument. This was achieved by measuring the extrusion force from an instrument equipped with a 27G... 1 / 2” or 29 1 / 2” or 30G 1 Data were collected by extruding the gel at a standard rate of 12.5 mm / min using a 1 mL syringe with a 2” needle. The gel prepared as described above was not sieved or crushed.

[0317] Representative extrusion force data are listed in Table 3. Restylane and Voluma are provided at 29G. 1 / 2” and 27G 1 / 2” needle (Table 3, entries 1-2), and has a relatively high G' value (Table 1, entries 1, 3). Volbella is supplied with 30G 1 / 2” needle (Table 3, entry 3) and medium range G' (Table 1, entry 5).

[0318] Items 4-9 in Table 3 represent the gels of the present invention, prepared in a 1 mL syringe without sieving or homogenization. As shown in Table 3, the extrusion force values ​​are within acceptable ranges. The gel in item 4 was obtained by passing through a 27G syringe. 1 / 2” needle extrusion, while the gel of entries 5-9 is extruded through 30G 1 / 2” needle squeezed out.

[0319] Table 3. Extrusion force values.

[0320]

[0321] Example 3

[0322] Gel swelling in water

[0323] Gel samples were prepared by mixing 200 mg of each product / product with DDW (1.0 ml) and incubating at 37°C for 6 hours. After centrifuging the mixture (twice at 10,000 rpm for 10 minutes), the aqueous supernatant was carefully removed, and the remaining swollen gel was weighed. The swelling ratio was calculated using the following equation:

[0324] Swelling ratio = (weight of swollen gel) / (initial weight of gel).

[0325] Table 4. Gel swelling ratio in water

[0326]

[0327]

[0328] The results showed that the gels prepared according to the present invention (Table 4, Entries 4-6) had lower gel swelling ratios compared to commercial dermal fillers with higher HA content and / or higher crosslinking (Table 4, Entries 1-3). Reduced gel swelling in water likely lowers the risk of swelling and edema in vivo.

[0329] Example 4

[0330] Implanted data

[0331] Gel samples were subcutaneously injected into five Sprague-Dawley rats using 27G 1 / 2” gel (corresponding to entries 1, 2, 4-6 in Table 4). On day 1, each animal received five injections of 100 μL of gel sample. Each gel sample was injected five times in each animal group. Animals were followed up for 95 days post-injection. No erythema or edema was observed at any injection site in any animal. No changes in the overall health status of any animal were observed during the study period. Histopathological evaluation of the injection sites 95 days post-injection showed no pathological changes at the injection sites.

[0332] During the study period from day 1 to day 95 post-injection, the size of the subcutaneous gel spheres was measured using electronic calipers. Subcutaneous gel size was calculated as the length × width of the spheres detected by palpation under the skin and measured with electronic calipers. Average data from five injection sites for each product (Figure 5) showed that the gel sphere sizes of Test Substance 2 (the gel described in entry 5 of Table 4) and Test Substance 3 (the gel described in entry 4 of Table 4) were significantly larger than the two controls, namely commercial dermal fillers with higher HA content and / or higher crosslinking (Control 1 is described in entry 2 of Table 4, and Control 2 is described in entry 1 of Table 2). Test Substance 1 (the gel described in entry 6 of Table 4), with a very low HA content of 5 mg / gr, showed the same subcutaneous gel sphere size as Control 2 (the gel described in entry 1 of Table 4), with a significantly higher HA content of 20 mg / gr.

[0333] Although certain features of the invention have been exemplified and described herein, many modifications, substitutions, alterations, and equivalents will now occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and alterations falling within the true spirit of the invention.

Claims

1. A polymer comprising a first hyaluronic acid (HA) chain or a derivative thereof and a second HA chain or a derivative thereof, wherein: The first HA chain and the second HA chain are crosslinked via one or more linkers; the one or more linkers include a norbornene moiety or a derivative thereof bound to a tetrazine moiety or a derivative thereof, and the one or more linkers are represented by the following formula: Formula (C) Or (D) Or a combination thereof; where: --- indicates a single or double bond; R 1 It is an aryl or heteroaryl group; wherein the aryl or heteroaryl group is optionally substituted with a C1-C6 alkyl, a C1-C6 alkoxy, a (C1-C6 alkyl)amino, or a di(C1-C6 alkyl)amino; R 2 It is C(O)-NH-alkyl-NH; Q 1 Q 2 "Or both" indicates that hydrogen is present or absent; R 3 Selected from: hydrogen, substituted or unsubstituted alkyl, aryl or heteroaryl; and wherein said polymer is characterized by a degree of crosslinking of 0.5 to 4%.

2. The polymer according to claim 1, wherein R 3 Selected from substituted or unsubstituted aryl and heteroaryl groups.

3. The polymer according to claim 1 or 2, wherein the one or more linkers comprise: (a) A first compound derived from the norbornene moiety, said norbornene moiety being represented by the following formula IIA: IIA; and b) a second compound derived from a tetrazine moiety selected from formulas IIIA-D: IIIA、 IIIB、 IIIC and IIID; wherein each of the norbornene moiety and the tetrazine moiety is covalently bonded to the first HA chain or the second HA chain; and wherein the covalent bond is an amide.

4. The polymer according to claim 1, wherein the average molecular weight of the first HA chain and the second HA chain is 400,000 to 5,000,000 Daltons (Da).

5. The polymer of claim 3, wherein the second compound is derived from the tetrazine portion of formula IIIB.

6. The polymer according to claim 1, wherein the degree of crosslinking is the average of the modification degrees of the first HA chain and the second HA chain; and wherein the modification degree is determined by... 1 H NMR confirmed.

7. A composition comprising the polymer according to claim 1 and a pharmaceutically acceptable carrier.

8. The composition according to claim 7, further comprising 0.1-30% w / w of non-crosslinked HA in the total HA content of the composition; and optionally further comprising compounds selected from: amino acids, minerals, vitamins, antioxidants, nucleic acids, coenzymes, enzymes, growth factors, proteins, antitumor drugs, steroids, nonsteroidal anti-inflammatory drugs, antibiotics, anesthetics, antimicrobial drugs, or any combination thereof.

9. The composition according to claim 7 or 8, wherein the composition is in gel form.

10. The composition according to claim 9, characterized in that... At least one of the following: phase angle (δ) is 0.1 to 10°; and elastic modulus (G') is 10 to 1,000 Pa.

11. The composition according to claim 7, wherein the total HA content in the composition is between 1 mg / gr and 20 mg / gr.

12. A kit comprising a first HA chain coupled to a norbornene moiety or a derivative thereof, and a second HA chain coupled to a tetrazine moiety or a derivative thereof; and wherein the kit includes instructions for mixing the first HA chain and the second HA chain in a ratio of 3:1 to 1:3 to obtain a polymer comprising the first HA chain crosslinked to the second HA chain via one or more linkers; wherein: The one or more connectors are represented by the following formula: Formula (C) Or (D) Or a combination thereof; where: --- indicates a single or double bond; R 1 It is an aryl or heteroaryl group; wherein the aryl or heteroaryl group is optionally substituted with a C1-C6 alkyl, a C1-C6 alkoxy, a (C1-C6 alkyl)amino, or a di(C1-C6 alkyl)amino; R 2 It is C(O)-NH-alkyl-NH; Q 1 Q 2 Either indicates hydrogen or its absence; and R represents either hydrogen or its absence. 3 Selected from: hydrogen, substituted or unsubstituted alkyl, aryl or heteroaryl; and wherein said polymer is characterized by a degree of crosslinking of 0.5 to 4%.

13. The kit according to claim 12, wherein the norbornene moiety is represented by the following formula IIA: IIA; and wherein the tetrazine portion is selected from formula IIIA-D: IIIA、 IIIB、 IIIC and IIID; wherein each of the norbornene moiety and the tetrazine moiety is covalently bonded to the first HA chain or the second HA chain; and wherein the covalent bond is an amide.

14. Use of the kit according to claim 12 or 13 for preparing a product for filling or filling tissue in an object in need.

15. The use according to claim 14, wherein the tissue is selected from: skin, gingiva, cartilage and ophthalmic tissues, muscle and subcutaneous tissue.

16. The use according to claim 14, wherein the product is prepared by: mixing the first HA chain and the second HA chain; thereby crosslinking the first HA chain to the second HA chain to obtain the polymer according to any one of claims 1 to 6; wherein the molar ratio between the norbornene moiety or its derivative and the tetrazine moiety or its derivative is about 1:

1.

17. The use according to claim 16, further comprising mixing the polymer with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition; optionally, wherein the pharmaceutical composition is the composition according to any one of claims 7 to 11.

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