阅读说明：本技术 交联的多羧酸化多糖及其使用方法 (Crosslinked polycarboxylated polysaccharides and methods of use thereof ) 是由 S·梅尼尔 S·施奈德 E·拉戈津 于 2019-06-11 设计创作，主要内容包括：本发明涉及包括通过包括与四嗪部分或其衍生物偶联的不饱和部分或其衍生物的连接体交联的第一透明质酸(HA)链和第二HA链的聚合物。在一些实施方式中,本发明的聚合物的特征在于具有0.2至4％的交联度。(The present invention relates to a polymer comprising a first Hyaluronic Acid (HA) chain and a second HA chain cross-linked 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%.)

1. A polymer comprising a first Hyaluronic Acid (HA) chain or derivative thereof and a second HA chain or derivative thereof, wherein the first HA chain and the second HA chain are crosslinked by one or more linkers, wherein the one or more linkers comprise an unsaturated moiety or derivative thereof bound to a tetrazine moiety or derivative thereof, wherein the crosslinking is characterized by a degree of crosslinking of 0.2 to 4%.

2. The polymer of claim 1, wherein the unsaturated moiety comprises: unsaturated cycloalkyl, unsaturated alkaryl, unsaturated alkyl, or combinations thereof.

3. The polymer of any one of claims 1 and 2, wherein the one or more linkers comprise a compound represented by the formula:

formula (A)

Or formula (B):

or a combination thereof; wherein:

represents a single bond or a double bond;

R¹、R²or both are selected from: a bond, alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, C (O) -NH-alkyl-NH and alkane-NZ, wherein Z is a bond, aryl or heteroaryl;

a is selected from: a bond, an alkyl group and an aryl group;

Q¹、Q²or both represent hydrogen or absent;

and wherein R³Selected from: hydrogen, substituted or unsubstituted alkyl, aryl or heteroaryl.

4. The polymer of any one of claims 1 to 3, wherein the unsaturated moiety comprises a styrene moiety or a derivative thereof.

5. The polymer of claim 4, wherein the styrene moiety or derivative thereof is represented by formulas IA-D:

6. the polymer of any one of claims 1 and 2, wherein the one or more linkers comprise a compound represented by the formula:

formula (C)

Or formula (D)

Or a combination thereof; wherein:

represents a single bond or a double bond;

R¹、R²or both are selected from: bond, alkyl, alkenyl, ringAlkyl, aryl, heteroaryl, heteroalicyclic, C (O) -NH-alkyl-NH, and alkyl-NZ, wherein Z is a bond, aryl, or heteroaryl;

Q¹、Q²or both represent hydrogen or absent;

and wherein

R³Selected from: hydrogen, substituted or unsubstituted alkyl, aryl or heteroaryl.

7. The polymer of any one of claims 1, 2, and 6, wherein the one or more linkers comprise:

(a) a first compound derived from a norbornene moiety selected from the group consisting of formulas IIA-G:

and

b) a second compound derived from a tetrazine moiety selected from formulas IIIA-F:

8. the polymer of any one of claims 1, 2, 6, and 7, wherein the first compound and the second compound are covalently bonded.

9. The polymer of any one of claims 1, 2, 6 to 8, wherein the norbornene moiety and the tetrazine moiety are bound to the first and second HA chains by covalent bonds.

10. The polymer of any one of claims 1, 2, 6 to 9, wherein the covalent bond is selected from the group consisting of: amides, amines, esters, ethers, ureas, thioureas, and carbamates.

11. The polymer of any one of claims 1 to 10, wherein the average molecular weight of the first and second HA chains is from 100,000 to 4,000,000 daltons (Da).

12. The polymer according to any one of claims 1 to 11, having a phase angle (δ) of from 0.1 to 10 °.

13. The polymer of any one of claims 1 to 12, having an elastic modulus (G') of from 10 to 1,000 Pa.

14. The polymer of any one of claims 1 to 13, comprising an HA content of 1 to 20 mg/gr.

15. The polymer of any one of claims 1 to 14, wherein the degree of crosslinking is determined by¹H NMR determination.

16. A composition comprising the polymer of any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

17. The composition of claim 16, comprising one or more from the group consisting of:

a. a polymer comprising a linker represented by formula (a);

b. a polymer comprising a linker represented by formula (B);

c. a polymer comprising a linker represented by formula (C);

d. a polymer comprising a linker represented by formula (D).

18. The composition of any one of claims 16 and 17, further comprising non-crosslinked HA in an amount of 0.1-30% (w/w) of the total HA content in the composition.

19. The composition of any one of claims 16 to 18, further comprising a compound selected from the group consisting of: amino acids, minerals, vitamins, antioxidants, nucleic acids, coenzymes, enzymes, growth factors, proteins, antineoplastic agents, steroids, non-steroidal anti-inflammatory drugs, antibiotics, anesthetics, antimicrobial drugs, or any combination thereof.

20. A method of filling or engorging tissue in a subject in need thereof, comprising administering to the tissue a composition according to any one of claims 16 to 19 or a polymer according to any one of claims 1 to 15, thereby filling or engorging tissue in a subject in need thereof.

21. The method of claim 20, wherein the tissue is selected from the group consisting of: skin, gums, cartilage and ophthalmic tissues, muscle and subcutaneous tissues.

22. A method of making the polymer of any one of claims 1 to 15, the method comprising: mixing a first Hyaluronic Acid (HA) chain or derivative thereof with a second HA chain or derivative thereof, wherein the first HA chain or derivative thereof comprises an unsaturated moiety or derivative thereof, and the second HA chain or derivative thereof comprises a tetrazine moiety or derivative thereof; wherein the unsaturated moiety or derivative thereof and the tetrazine moiety or derivative thereof are present in a molar ratio of from 3: 1 to 1: 3; thereby crosslinking the first HA chain or derivative thereof with the second HA chain or derivative thereof.

23. The method of claim 22, wherein the unsaturated moiety comprises a norbornene moiety or a derivative thereof.

24. The method of any one of claims 22 and 23, wherein the first HA chain or derivative thereof and the second HA chain or derivative thereof have an average molecular weight, M, of 100,000 to 4,000,000 daltons (Da)_w。

25. The method of any one of claims 22 to 24, wherein the crosslinking comprises forming a covalent bond between the norbornene moiety or derivative thereof and the tetrazine moiety or derivative thereof.

26. A kit comprising a first HA chain or derivative thereof coupled to an unsaturated moiety or derivative thereof, and a second HA chain or derivative thereof coupled to a tetrazine moiety or derivative thereof.

27. The kit of claim 26, wherein the unsaturated moiety comprises a norbornene moiety or a derivative thereof.

28. The kit of any one of claims 26 and 27, further comprising an injection device.

29. The kit of any one of claims 26 to 28, further comprising a pharmaceutically acceptable carrier.

30. The kit according to any one of claims 26 to 29, further comprising a component selected from the group consisting of: free HA chains, amino acids, minerals, vitamins or anesthetics.

31. The kit of any one of claims 26 to 30, wherein the kit comprises instructions for:

a. mixing said first HA chain or derivative thereof coupled to said norbornene moiety or derivative thereof and said second HA chain or derivative thereof coupled to said tetrazine moiety or derivative thereof in a ratio of 3: 1 to 1: 3; and

b. applying a composition formed by mixing said first HA chain or derivative thereof coupled to said norbornene moiety or derivative thereof and said second HA chain or derivative thereof coupled to said tetrazine moiety or derivative thereof to a filling or filling method.

32. The kit of any one of claims 26 to 31, further comprising instructions for mixing the composition with free HA chains, amino acids, minerals, vitamins, anesthetics, or any combination thereof.

33. The kit of any one of claims 26 to 32, wherein the mixing is performed in the injection device.

Technical Field

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

Background

The cross-linking of hyaluronic acid in dermal fillers imparts desirable mechanical properties, in particular lifting (or filling) properties, to the material.

The need to use materials as close as possible to the endogenous hyaluronic acid dictates that materials with a low degree of modification (e.g.% cross-linking) are to be produced. Both hyaluronic acid and cross-linked hyaluronic acid are known to be highly hygroscopic. Thus, when these materials are injected, they tend to absorb water in the tissue and cause swelling and edema. The water absorption rate is in positive correlation with the content of hyaluronic acid and cross-linked hyaluronic acid. Thus, due to the need to reduce the risk of swelling and edema, it is desirable to produce dermal fillers with low hyaluronic acid and cross-linked hyaluronic acid content.

It is known that the materials currently on the market contain a high degree of crosslinking (4 to 10%) or a total hyaluronic acid content (equal to or greater than 20mg/gR) to provide sufficient lifting properties

Disclosure of Invention

In some embodiments thereof, the present invention relates to cross-linked hyaluronic acid.

In one aspect of the invention, there is provided a polymer comprising a first Hyaluronic Acid (HA) chain and a second HA chain cross-linked via a linker, the linker comprising an unsaturated moiety or derivative thereof bound to a tetrazine moiety or derivative thereof, and wherein the cross-linking is characterized by a degree of cross-linking of 0.2 to 4%.

In some embodiments, the unsaturated moiety comprises: unsaturated cycloalkyl, unsaturated alkaryl, unsaturated alkyl, or combinations thereof.

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

formula (A)

Or formula (B):

or a combination thereof; wherein:

- - -represents a single bond or a double bond;

R¹、R²or both are selected from: a bond, alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, C (O) -NH-alkyl-NH, and alkyl-NZ, wherein Z is a bond, aryl, or heteroaryl;

a is selected from: a bond, an alkyl group and an aryl group;

Q¹、Q²or both represent hydrogen or absent;

and wherein R³Selected from: hydrogen, substituted or unsubstituted alkyl, aryl and heteroaryl.

In some embodiments, the unsaturated moiety comprises a styrene moiety or a derivative thereof.

In some embodiments, the styrene moiety or derivative thereof is represented by formulas IA-D:

in some embodiments, one or more linkers comprise a compound represented by the formula:

formula (C)

Or formula (D):

or a combination thereof; wherein:

- - -represents a single bond or a double bond;

R¹、R²or both are selected from: a bond, alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, C (O) -NH-alkyl-NH, and alkyl-NZ, wherein Z is a bond, aryl, or heteroaryl;

Q¹、Q²or both represent hydrogen or absent;

and wherein R³Selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, aryl and heteroaryl.

In some embodiments, the one or more linkers comprise:

a first compound derived from a norbornene moiety selected from the group consisting of formulas IIA-G:

and

b) a second compound derived from a tetrazine moiety selected from formulas IIIA-F:

in some embodiments, the first compound and the second compound are covalently bound.

In some embodiments, the norbornene moiety and the tetrazine moiety are bound to the first HA chain and the second HA chain by covalent bonds.

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

In some embodiments, the average molecular weight Mw of the first and second HA chains is from 100,000 to 4,000,000 daltons (Da).

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

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

In some embodiments, the polymer comprises an HA content of 1mg/gR to 20 mg/gR.

In some embodiments, the degree of crosslinking is determined by¹And (4) HNMR determination.

According to another aspect, there is provided a composition comprising a polymer of the invention and a pharmaceutically acceptable carrier.

In some embodiments, the composition comprises one or more from the following:

1) a polymer comprising a linker represented by formula (a);

2) a polymer comprising a linker represented by formula (B);

3) a polymer comprising a linker represented by formula (C);

4) a polymer comprising a linker represented by formula (D).

In some embodiments, the composition further comprises non-crosslinked HA in an amount of 0.1-30% (w/w) of the total HA content of the composition.

In some embodiments, the composition further comprises one or more compounds selected from the group consisting of: amino acids, minerals, vitamins, antioxidants, nucleic acids, coenzymes, enzymes, growth factors, proteins, antineoplastic agents, steroids, non-steroidal anti-inflammatory drugs, antibiotics, anesthetics, antimicrobial drugs, or any combination thereof.

According to another aspect, there is provided a method of filling or engorging tissue in a subject in need thereof, comprising administering to the tissue a composition of the invention or a polymer of the invention, thereby causing filling or engorgement of tissue in the subject in need thereof.

In some embodiments, the tissue is selected from: skin, gums, cartilage and ophthalmic tissues, muscle and subcutaneous tissues.

According to another aspect, there is provided in an embodiment thereof a method of making a polymer, the method comprising: mixing a first Hyaluronic Acid (HA) chain or derivative thereof with a second HA chain or derivative thereof, wherein the first HA chain comprises an unsaturated moiety or derivative thereof and the second HA chain comprises a tetrazine moiety or derivative thereof; wherein the unsaturated moiety or derivative thereof and the tetrazine moiety or derivative thereof are present in a molar ratio of from 3: 1 to 1: 3; thereby crosslinking the first HA chain or derivative thereof with the second HA chain or derivative thereof.

In some embodiments, the unsaturated moiety comprises a norbornene moiety or a derivative thereof.

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

In some embodiments, crosslinking comprises forming a covalent bond between a norbornene moiety or derivative thereof and a tetrazine moiety or derivative thereof.

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

In some embodiments, the unsaturated moiety comprises a norbornene moiety or a derivative thereof.

In some embodiments, the kit further comprises a pharmaceutically acceptable carrier.

In some embodiments, the kit further comprises an injection device.

In some embodiments, the kit further comprises a component selected from the group consisting of: free HA chains, amino acids, minerals, vitamins or anesthetics.

In some embodiments, the kit comprises instructions for:

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

b. a composition formed by mixing a first HA chain or derivative thereof coupled to a norbornene moiety or derivative thereof and a second HA chain or derivative thereof coupled to a tetrazine moiety or derivative thereof is applied to a filling or filling method.

In some embodiments, the kit further comprises instructions for mixing the composition with free HA chains, amino acids, minerals, vitamins, anesthetics, or any combination thereof.

In some embodiments, the mixing is performed in an injection device.

Unless defined otherwise, 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 belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be necessarily limiting.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Drawings

FIG. 1: method for preparing hyaluronic acid modified with tetrazine (HA-tetrazine)¹HNMR spectra showing an aromatic peak of tetrazine and an aliphatic peak of N-acetylglucosamine residue.

FIG. 2: process for preparing hyaluronic acid (HA-norbornene) modified with norbornene¹HNMR spectra showing an olefinic peak of norbornene and an aliphatic peak of the N-acetylglucosamine residue.

FIG. 3: method for preparing hyaluronic acid (HA-styrene) modified with styrene¹HNMR spectra showing an olefinic peak of styrene and an aliphatic peak of the N-acetylglucosamine residue.

FIG. 4: a graph depicting the starting point (onset point) in the Linear Viscoelastic Region (LVR) of a polymer.

FIG. 5: graph depicting the size of subcutaneous gel mass during 95 days after in vivo administration of commercial product vs. inventive Polymer

Detailed Description

In some embodiments, the present invention relates to a polymer comprising a first Hyaluronic Acid (HA) chain and a second HA chain cross-linked by a linker comprising an unsaturated moiety or derivative thereof bound 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%.

In some embodiments, the unsaturated moiety comprises: unsaturated cycloalkyl, unsaturated alkaryl, unsaturated alkyl, or combinations thereof. In some embodiments, the unsaturated moiety comprises a cyclic or acyclic alkyne. In some embodiments, the unsaturated moiety comprises a cyclic or acyclic alkene. In some embodiments, the unsaturated moiety comprises an optionally fused cyclic olefin.

In some embodiments, the unsaturated moiety is selected from: substituted or unsubstituted styrenes, cyclohexenes, cyclopentenes, cyclohexadienes, cyclopentadienes, norbornadienes, fused norbornadienes, norbornenes and fused norbornenes. In an exemplary embodiment, the unsaturated moiety is norbornene.

Polymer and method of making same

In some embodiments, the present invention relates to a polymer comprising Hyaluronic Acid (HA) chains or derivatives thereof. As used herein, HA chains or derivatives thereof include D-glucuronic acid and N-acetyl-glucosamine.

As used herein, derivatives of HA chains relate to chemically modified HA. In some embodiments, the chemically modified HA comprises a side chain modification (e.g., acetylation of a hydroxyl group, decarboxylation of a carboxyl group, esterification, or amidation). In some embodiments, the chemically modified HA comprises one or more of the 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 comprises a combination of modified side chains.

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

As used herein, the term "molecular weight" includes any one of the following average weight values: 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 cut-off). Unless otherwise indicated, the term refers to Mw.

In one embodiment, the present invention relates to a polymer comprising a polycarboxylated polysaccharide or derivative thereof, wherein the molecular weight is within the above-described range.

In some embodiments, the polymers of the present invention comprise one or more HA chains. In some embodiments, "one or more" is two. In some embodiments, the two HA chains of the present invention are crosslinked. In one embodiment, the crosslinks are intercorosslinked. As defined herein, the term "mutual" refers to the formation of a bond between two moieties 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 via a linker.

"linker" as defined herein refers to a molecule or macromolecule used to link different moieties or functional groups of one or more polycarboxylated polysaccharides. In one embodiment, the linker may also facilitate other functions, including but not limited to preservation of biological activity, maintenance of interactions, and others.

In some embodiments, the polymer of the present invention comprises a first HA chain linked to a second HA chain by one or more linkers, the linkers comprising a compound represented by the formula:

formula (A)

Or formula (B)

Or a combination thereof; wherein:

represents a single bond or a double bond.

In some embodiments, the polymer of the present invention comprises a first HA chain crosslinked to a second HA chain via one or more linkers, the linkers comprising a compound represented by formula (a) and/or (B).

In some embodiments, R¹、R²Or both are selected from: a bond, alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, C (O) -NH-alkyl-NH, and alkyl-NZ, wherein Z is a bond, aryl, or heteroaryl.

In some embodiments, a is selected from: a bond, an alkyl group, and an aryl group.

In some embodiments, Q¹、Q²Or both represent hydrogen or are absent.

In some embodiments, R³Selected from: hydrogen, substituted or unsubstituted alkyl, aryl and heteroaryl.

In some embodiments, the polymer of the present invention comprises a first HA chain linked to a second HA chain by one or more linkers, the linkers comprising a compound represented by the formula:

formula (C)

Or formula (D)

Or a combination thereof;

whereinRepresents a single bond or a double bond.

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

In some embodiments, the linker comprises a first compound derived from an unsaturated moiety and a second compound derived from a tetrazine moiety, as described below. In some embodiments, the first compound and the second compound are covalently bound. In some embodiments, the first compound and the second compound are covalently bound, thereby forming a compound represented by any one of formulas (a) to (D).

In some embodiments, R¹、R²And R³Comprising substituents selected from the group consisting of: alkyl, cycloalkyl, aryl, heteroalicyclic, heteroaryl, alkoxy, hydroxy, phosphonate, thiol, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amide, amine, alkylamine, carboxy, sulfonyl, sulfoxy (sulfoxy), sulfinyl, and sulfonamide.

In some embodiments, Q¹、Q²Or both represent hydrogen or are absent.

In some embodiments, the polymer of the present invention comprises a first HA chain linked to a second HA chain by a linker comprising a compound selected from the group consisting of:

in some embodiments, R¹Selected from: -C₀-C₆alkyl-NZ-, -C₀-C₆alkyl-O-and C₀-C₃alkyl-C (O) -.

In some embodiments, Z is selected from: a bond, aryl or heteroaryl, wherein said aryl and heteroaryl are optionally substituted with halogen, hydroxy, C₁-C₆Alkyl radical, C₁-C₆Alkoxy group, (C)₁-C₆Alkyl) amino and di (C)₁-C₆Alkyl) amino substituted.

In some embodiments, R²Selected from: c₀-C₆alkyl-NZ, -C₀-C₆Alkyl radicals O-and C₀-C₃alkyl-C (O) -.

In some embodiments, R³Selected from: hydrogen, C₁-C₆Alkyl, aryl or heteroaryl, wherein said aryl and heteroaryl are optionally substituted by halogen, hydroxy, C₁-C₆Alkyl radical, C₁-C₆Alkoxy group, (C)₁-C₆Alkyl) amino and di (C)₁-C₆Alkyl) amino substituted.

In one embodiment of the method of the present invention,refers to a single bond, or in certain embodiments, a double bond, where feasible.

In some embodiments, R¹、R²Or both are selected from: -NZ-, -C_i-C₆alkyl-NZ-, -O-, -C₁-C₆alkyl-O-, -C (O) -or-C₁-C₃alkyl-C (O) -; -methyl-O, -pentyl-O-; -c (o) -; and-methyl-C (O) -.

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.

Another embodiment provides a linker comprising a compound of formula (C) or (D) according to any of the preceding embodiments, wherein R is¹、R²Or both are selected from: -NZ-, -C₁-C₆alkyl-NZ-, -O-, -C₁-C₆alkyl-O-, -C (O) -or-C₁-C₃alkyl-C (O) -; -C₁-C₆alkyl-NZ-; c₁-C₃alkyl-NZ-; -methyl-NH-or-pentyl-NH-; -C₁-C₆alkyl-O-; -C₁-C₃alkyl-O-; -methyl-O or-pentyl-O-; -C₀-C₃alkyl-C (O) -; c (O) -; and-methyl-C (O) -.

In some embodiments, R³Is hydrogen.

In some embodiments, R³Selected from: c₁-C₆Alkyl, aryl or heteroaryl, wherein the aryl or heteroaryl may be optionally substituted; aryl or heteroaryl, wherein aryl and heteroaryl are optionally substituted; a phenyl group; pyridyl, pyrimidinyl, or pyrazinyl.

In some embodiments, R in the linker comprising a compound of formula (C) or (D)³Selected from: c₁-C₆Alkyl radical, C₁-C₃Alkyl and methyl.

In some embodiments, the polymer comprises a plurality of linkers comprising a compound of formula (C) or (D).

In some embodiments, the present invention relates to a polymer comprising a first HA chain and a second HA chain linked to each other by a linker resulting in cyclization, such as but not bound by any particular mechanism, including reverse electron-demand Diels-AldeR cyclization of unsaturated moieties (e.g., norbornene moieties) or derivatives thereof and tetrazine moieties or derivatives thereof.

In some embodiments, the first HA chain of the present invention comprises an unsaturated moiety, as described above. In some embodiments, the first HA chain of the present invention comprises a styrene moiety or derivative thereof.

In some embodiments, the styrene moiety is represented by formulas IA-D:

in some embodiments, the derivative of a styrene moiety comprises a styrene moiety of formula IA-ID bound to the first HA chain via a covalent bond.

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

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

in some embodiments, the first HA chain of the present invention comprises a norbornene moiety or derivative thereof. In some embodiments, the number of norbornene moieties or derivatives 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 derivative thereof. In some embodiments, the number of tetrazine moieties or derivatives 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, 1,000-5,000, 500-1000 or 1-500 norbornene moieties or derivatives 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, 1,000-5,000, 500-1,000, or 1-500 tetrazine moieties or derivatives thereof. Each possibility represents a separate embodiment of the invention.

In some embodiments, the norbornene moiety is in an endo, exo, or mixed conformation thereof. Non-limiting examples of norbornene moieties include, but are not limited to, compounds of formulas IIA-IIG:

non-limiting examples of tetrazine moieties include, but are not limited to, compounds of formulas IIIA-IIIF:

in some embodiments, the norbornene moiety is a compound of formula IIG:

in some embodiments, the derivative of a norbornene moiety comprises a norbornene moiety represented by formulas IIA-IIG covalently bonded to the first HA chain.

In some embodiments, the amine of the norbornene moiety is bound to the first HA chain. In some embodiments, the norbornene moiety is bound to the carboxyl group of the first HA chain. In some embodiments, a derivative of a norbornene moiety refers to a norbornene moiety (e.g., a compound of formula IIG) bound to a first HA chain via an amide linkage, as shown in formula IF:

in some embodiments, the norbornene moiety is bonded to the first HA strand by reacting the first HA strand and the norbornene moiety (e.g., norbornene methylamine, norbornene methanol) with a suitable coupling agent.

Non-limiting examples of coupling agents include, but are not limited to, 1-ethyl-3- (3-dimethylamino-propyl) carbodiimide (EDC), carbonyldiimidazole, N ' -Dicyclohexylcarbodiimide (DCC), N ' -Diisopropylcarbodiimide (DIC), 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methyl-morpholine hydrochloride (DMTMM), and N, N ' -tetramethyl-O- (1H benzotriazol-1-yl) urea Hexafluorophosphate (HBTU). In one embodiment, a derivative of a tetrazine moiety is associated with a tetrazine moiety, as defined above, which is bound to the second HA chain by a covalent bond. In some embodiments, the covalent bond is as defined above.

In some embodiments, the derivative of a tetrazine moiety includes a tetrazine moiety of formulae IIIA-IIIF bound to a second HA chain by a covalent bond. In some embodiments, the amine of the tetrazine moiety is bound to the second HA chain. In some embodiments, the tetrazine moiety is bound to the carboxyl group of the second HA chain. In some embodiments, a derivative of a tetrazine moiety refers to a tetrazine moiety (e.g., a compound of formula IIIB) bound to a second HA chain through an amide bond, as shown in formula IG:

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.

In some embodiments, the first HA chain coupled to the norbornene moiety or derivative thereof and the second HA chain coupled to the tetrazine moiety or derivative thereof are present in the polymers of the present invention in a ratio of 3: 1, 3: 2, 2: 1, 1: 2, 2: 3, or 1: 3, including any values and ranges therebetween. Each possibility represents a separate embodiment of the invention. As defined herein, a ratio is any ratio selected from the group consisting of: moles, weight or concentration.

As defined herein, the term "degree of crosslinking" refers to the molar ratio between the linker and the repeating polycarboxylated polysaccharide that form the polymer of the present invention. In some embodiments, the degree of crosslinking refers to the molar ratio between the linker comprising unreacted norbornene and tetrazine and the repeating polycarboxylated polysaccharide forming the polymer of the present invention. In some embodiments, the degree of crosslinking of the polymer of the 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 polymers of the invention have a degree of crosslinking of 0.01 to 0.1%, 0.01 to 0.5%, 0.05 to 0.1%, 0.1 to 0.3%, 0.1 to 0.5%, 0.1 to 0.75%, 0.1 to 1%, 1 to 1.75%, 1 to 2%, 1 to 2.5%, 2 to 2.5%, 2.25 to 3%, 2.5 to 3.25%, 3 to 3.75%, 3.6 to 4.2%, 4 to 5.25%, 5 to 6.5%, 6 to 7.5%, 7 to 8.5%, 8 to 9.25%, 9 to 10.5%, or 10 to 12.5%. Each possibility represents a separate embodiment of the invention. In one embodiment, the degree of crosslinking of a polymer refers to the calculated average of the degree of crosslinking of a plurality of HA chains within the polymer of the present invention.

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

In some embodiments, the polymers of the present invention have an elastic modulus of 10-500Pa, 20-1,000Pa, 30-600Pa, 40-1,000Pa, 40-5,000Pa, 50-10,000Pa, 500-50,000Pa, 500-10,000Pa, 500-5,000Pa, 500-1,000Pa, 1,000-50,000Pa, 1,000-10,000Pa, 1,000-5,000Pa, 50-20,000Pa, 500-20,000Pa, or 1,000-20,000 Pa. In some embodiments, the polymers of the present invention have an elastic modulus of up to 10,000Pa, up to 20,000Pa, up to 30,000Pa, up to 40,000Pa, or up to 50,000 Pa. Each possibility represents a separate embodiment of the invention.

As defined herein, the term "phase angle" or "δ" refers to the degree of viscoelasticity of a material. As will be apparent to one of ordinary skill in the art, δ can be calculated according to the following equation:

wherein G 'is the viscosity modulus and G' is the elastic modulus. In some implementationsIn this way, G' and G "are obtained by oscillatory rheology and are measured in the viscoelastic domain of low oscillation of stress or amplitude.

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

In some embodiments, the present invention relates to a process for preparing a polymer of the present invention, the process comprising the steps of: (i) attaching an unsaturated moiety (e.g., a norbornene moiety) (or derivative thereof) to a first Hyaluronic Acid (HA) chain (or derivative thereof) and a tetrazine moiety (or derivative thereof) to a second HA chain (or derivative thereof); (ii) mixing a first (HA) chain (or derivative thereof) comprising a norbornene moiety (or derivative thereof) and a second HA chain (or derivative thereof) comprising a tetrazine moiety (or derivative thereof); and (iii) cross-linking the first HA chain with the second HA chain.

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

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

In some examples, the crosslinking reaction occurs without input of external energy at temperatures and conditions within a useful range for forming polymers. In one embodiment, the crosslinking reaction is heated to increase the reaction effectiveness.

In some embodiments, the process for preparing the polymers of the present invention comprises a spontaneous crosslinking reaction. In some embodiments, "spontaneous chemical reaction" means a process without assistance from, for example, light, heat, or free radicals. In some embodiments, the crosslinking reaction may 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's balanced salt solution, DulbeCCO modified Eagle's medium, and the like. In some embodiments, crosslinking may occur in an organic solvent. Non-limiting examples of organic solvents include, but are not limited to, methanol, ethanol, dichloromethane, dimethylformamide, and the like.

In some embodiments, the crosslinking reaction can occur at a wide range of temperatures of at least-80 ℃, at least-50 ℃, at least-20 ℃, at least 0 ℃, at least 4 ℃, at least 22 ℃, at least 37 ℃, or at least 45 ℃ and not more than 60 ℃. In some embodiments, the crosslinking reaction can occur at a wide range of temperatures (-80) - (-50) ° c, (-60) - (-15) ° c, (-20) - (-4) ° c, (-5) -0 ℃, (-2) -4 ℃, 2-8 ℃, 5-20 ℃, 15-30 ℃, 25-40 ℃, or 35-55 ℃.

In some embodiments, steps (ii) and (iii) are performed in situ, thereby forming a crosslinked polymer by mixing the first (HA) chains and the second (HA) chains. In some embodiments, the preparation method is free of post-processing steps, such as sieving or homogenization.

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

In some embodiments, the norbornene or derivative thereof and/or the tetrazine or derivative thereof may be modified. In some embodiments, the post-crosslinking reaction modification comprises the binding of one or more molecules to unbound norbornene or derivatives thereof and/or unbound tetrazine or derivatives thereof on the crosslinked polymer.

Non-limiting examples of the one or more molecules include, but are not limited to: amino acids, anesthetics, minerals, vitamins, and the like. The amount of unreacted norbornene or derivative thereof and/or tetrazine or derivative thereof on the HA chains can be adjusted by varying the ratio of the first HA chain to the second HA chain or vice versa during the crosslinking reaction.

Composition comprising a metal oxide and a metal oxide

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

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

According to another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a polymer of the present invention together with a pharmaceutically acceptable carrier and/or diluent. In some embodiments, the pharmaceutical composition can facilitate administration of the polymer to an organism.

In some embodiments, the compositions of the present invention further comprise an amino acid. In some embodiments, an amino acid includes 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-Cbz protected aminopentanoic acid (Nva), ornithine (O), aminocaprylic acid (AoC), 2, 4-diaminobutyric acid (Abu), homoarginine, norleucine (Nle), N-methylaminobutyric acid (MeB), 2-naphthylalanine (2Np), aminoheptanoic acid (Ahp), phenylglycine, β -phenylproline, tert-leucine, 4-aminocyclohexylalanine (Cha), N-methyl-norleucine, 3, 4-dehydroproline, N-dimethylaminoglycine, N-methylaminoglycine, 4-aminopiperidine-4-carboxylic acid, 6-aminocaproic acid, trans-4- (aminomethyl) -cyclohexanecarboxylic acid, trans-methyl-norleucine, 3, 4-dehydroproline, N-dimethylaminoglycine, 4-aminopiperidine-4-carboxylic, 2-, 3-and 4- (aminomethyl) -benzoic acid, 1-aminocyclopentanecarboxylic acid, 1-aminocyclopropanecarboxylic acid, cyanopropionic acid, 2-benzyl-5-aminopentanoic acid, norvaline (Nva), 4-O-methylthreonine (TMe), 5-O-methyl-homoserine (hSM), tert-butylalanine (tBu), cyclopentylalanine (Cpa), 2-aminoisobutyric acid (Aib), N-methylglycine (MeG), N-methyl-alanine (MeA), N-methyl-phenylalanine (MeF), 2-thienyl-alanine (2Th), 3-thienyl-alanine (3Th), O-methyl-tyrosine (YMe), 3-benzothienyl-alanine (Bzt), and D-alanine (DAl). In some embodiments, the amino acid is an amino acid oligomer or dimer linked by peptide bonds. In some embodiments, the oligomer is a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, or, in some embodiments, a polymer having more than 11 amino acids bound to each other by peptide bonds. In some embodiments, the amino acid is included in the composition in the form of a peptide, polypeptide, or protein. In some embodiments, the peptide, polypeptide or protein included in the composition of the invention is in a form selected from, but not limited to: native, denatured, neutralized, digested, cross-linked, unfolded, reduced, oxidized or inactivated forms.

In some embodiments, the compositions of the present invention further comprise 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.

In some embodiments, the compositions of the present invention further comprise a vitamin. Non-limiting examples of vitamins include, but are not limited to: vitamin A (retinol, retinal and four carotenoids, including beta-carotene), vitamin B₁(thiamine), vitamin B₂(Riboflavin) and vitamin B₃(Niacin, nicotinamide riboside), vitamin B₅(pantothenic acid), vitamin B6 (pyridoxine, pyridoxamine, pyridoxal), vitamin B₇(Biotin) and vitamin B₉(Folic acid)Salt (ester)), vitamin B₁₂(cyanocobalamin, hydroxycobalamin, methylcobalamin, adenosylcobalamin), vitamin C (ascorbic acid), vitamin D (cholecalciferol (D3), ergocalciferol (D2)), vitamin E (tocopherol, tocotrienol) or vitamin K (phylloquinone, menaquinone).

In some embodiments, the compositions of the present invention further comprise an anesthetic. As used herein, the term "anesthetic" refers to any molecule or substance that prevents pain (e.g., during surgery) or completely blocks any sensation. In one embodiment, the anesthetic is a general anesthetic. In one embodiment, the anesthetic is a local anesthetic. In some embodiments, a local anesthetic causes reversible sensory loss confined to a region of the body while maintaining consciousness.

Non-limiting examples of anesthetics include, but are not limited to, articaine, procaine (procaine), amethocaine (amethocaine), lidocaine (lidocaine), bupivacaine (bupivacaine), levobupivacaine (levobupivacaine), ropivacaine (ropivacaine), mepivacaine (mepivacaine), dibucaine (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) anesthetic. In some embodiments, the compositions of the present invention comprise at most 0.01% (w/w), at most 0.05% (w/w), at most 0.75% (w/w), at most 1% (w/w), at most 1.5% (w/w), at most 2% (w/w), at most 3% (w/w), at most 4% (w/w), or at most 5.5% (w/w) anesthetic. Each possibility represents a separate embodiment of the invention.

In some embodiments, the compositions of the present invention further comprise 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 through an unsaturated moiety (e.g., a norbornene moiety) or derivative thereof and a tetrazine moiety or derivative thereof. In some embodiments, the percentage of non-crosslinked HA, as defined herein, is the calculated proportion of non-crosslinked HA in the total HA content in the final product (i.e., composition). In some embodiments, the composition comprises a total HA content of 1 to 6mg/gr, 2 to 7mg/gr, 3 to 8mg/gr, 4 to 9mg/gr, 5 to 10mg/gr, 6 to 11mg/gr, 7 to 12mg/gr, 8 to 13mg/gr, 9 to 14mg/gr, 10 to 15mg/gr, 11 to 16mg/gr, 12 to 17mg/gr, 13 to 18mg/gr, 14 to 19mg/gr, 15 to 20mg/gr, 16 to 21mg/gr, 17 to 22mg/gr, 18 to 25mg/gr, or any range therebetween. In some embodiments, the non-crosslinked HA content in the compositions of the invention is: at most 5%, at most 7%, at most 10%, at most 15%, at most 20%, at most 25%, at most 27%, at most 30%, or 35%. In some embodiments, the non-crosslinked HA is present in the composition at a concentration 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 separate embodiment of the invention.

In another embodiment, the pharmaceutical compositions of the invention may be formulated in the form of pharmaceutically acceptable salts of the polymers of the invention. In another embodiment, pharmaceutically acceptable salts include those derived from non-toxic inorganic or organic acids such as hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like. In some embodiments, the salts are formed with free carboxyl groups, such as salts derived from non-toxic inorganic or organic bases (e.g., sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like).

As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound of the disclosure is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions as well as dextrose and glycerol aqueous solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. If desired, the compositions may also include minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; and agents for adjusting tonicity, such as sodium chloride or dextrose, are also contemplated. The carriers may collectively comprise from about 0.1% to about 99.9% by weight of the pharmaceutical compositions presented herein.

As used herein, the term "pharmaceutically acceptable" means suitable for administration to a subject, e.g., a human. For example, the term "pharmaceutically acceptable" may mean approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals and more particularly in humans.

In another embodiment, the compositions of the present invention take the form of solutions, suspensions, emulsions, tablets, powders, gels, foams, pastes, sustained release formulations, and the like. Examples of suitable pharmaceutical carriers are described in: remington's Pharmaceutical Sciences ", e.w. martin, the contents of which are incorporated herein by reference. Such compositions will comprise a therapeutically effective amount of a polymer of the invention, preferably in substantially purified form, and an appropriate amount of a carrier to provide a form for proper administration to a subject.

According to an embodiment of the invention, the pharmaceutical composition comprises 0.1-95% of the polymer(s) of the invention. According to another embodiment of the invention, the pharmaceutical composition comprises 1-70% of the polymer. According to another embodiment of the invention, the composition or formulation to be administered may comprise an amount of polymer, according to embodiments of the invention, effective to treat the condition or disease in the subject being treated.

Embodiments of the present invention relate to polymers of the present invention presented in unit dosage form and prepared by any method known in the pharmaceutical art. In an embodiment of the invention, the unit dosage form is in the form of a tablet, capsule, lozenge, ampoule, vial or pre-filled syringe. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration and the nature of the disease or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or in vivo animal model test bioassays or systems.

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

As used herein, the terms "administration," "administering," and similar terms refer to any method of delivering a composition comprising an active agent to a subject in a manner that provides a therapeutic effect in sound medical practice.

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

In some embodiments, the pharmaceutical composition comprising the polymer is administered by ophthalmic, transdermal, intradermal, subcutaneous, intramuscular, or intraperitoneal routes of administration. 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, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal and any other injection means known in the art. Although the bioavailability of polymers administered by other routes may be lower than when administered by parenteral injection, it is contemplated that administration of the compositions of the present invention by transdermal, oral, rectal, vaginal, topical, nasal, inhalation, and ocular treatment is possible by use of appropriate formulations.

For topical application, the polymers of the present invention may be combined with a pharmaceutically acceptable carrier to deliver an effective dose based on the desired activity. The carrier may be in the form of, for example, but not limited to, an ointment, cream, gel, paste, foam, aerosol, suppository, pad, or gel stick.

According to some embodiments, the polymers of the present invention may be delivered in a controlled release system. In yet another embodiment, the controlled release system may be placed in the vicinity of the target of treatment, thus requiring only a fraction of the systemic dose.

In one embodiment, it will be understood that the polymers of the invention may be provided to an individual with other active agents to achieve improved therapeutic effects as compared to treatment with each agent alone. In another embodiment, measures are taken against the adverse side effects associated with combination therapy (e.g., administration of supplements and selection).

In one embodiment, the administration may be a single or multiple administrations, depending on the severity and responsiveness of the condition to be treated, and the course of treatment lasts from days to weeks or until a cure is induced or remission is achieved.

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 the component sufficient to produce the desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner presently described. In another embodiment, a therapeutically effective amount of the polymer is the amount of polymer required for the desired biological or therapeutic effect measurable in vivo. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated. The therapeutic prescription (e.g., determination of dosage, timing, etc.) is within the responsibility of the general or specialist practitioner and generally takes into account the disorder to be treated or the defect to be corrected, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. 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 preparation of an effective amount or dose can be initially estimated from in vitro assays. In one embodiment, the dose can be formulated in animal models, and this information can be used to more accurately determine a dose useful for humans.

In one embodiment, toxicity and therapeutic efficacy of the active ingredients described herein can be determined in vitro in cell cultures or experimental animals by standard pharmaceutical procedures. In one embodiment, the data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a range of doses for use in humans. In one embodiment, the dosage varies depending on the dosage form used and the route of administration used. In one embodiment, the exact formulation, route of administration, and dosage can be selected by the individual physician in accordance with the patient's circumstances. [ see, for example, Fingl, et al, (1975) "The Pharmacological Basis of Therapeutics", Ch.1p.1 ].

Pharmaceutical compositions containing the presently described polymers as active ingredients may be prepared according to conventional pharmaceutical compounding 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).

In one embodiment, a composition, including a formulation of the present invention formulated in a compatible pharmaceutical carrier, is prepared, disposed in a suitable container, and labeled for treatment of a specified condition.

In one embodiment, the compositions of the present invention are presented in a pack or dispenser device, such as an FDA approved kit, which contains one or more unit dosage forms containing the active ingredient. In one embodiment, the packaging comprises, for example, a metal or plastic foil, such as a blister pack (blister pack). In one embodiment, the package or dispenser device is accompanied by instructions. In one embodiment, the package or dispenser is contained by a container-associated placard in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which placard reflects approval by the agency of the form of the composition or human or veterinary administration. In one embodiment, such a notice is a label or approved product insert approved by the U.S. food and Drug Administration for prescription drugs.

The application method is disclosed.

In some embodiments, the present invention relates to a method of filling or engorging tissue in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a polymer of the present invention.

In some embodiments, the filling or filling method as defined herein refers to the injection of a soft filling material in skin tissue. In some embodiments, the filling is filling wrinkles, such as facial wrinkles. In some embodiments, filling restores the smooth appearance or texture of the skin.

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 a polymer or composition of the invention into gingival tissue. In some embodiments, the method involves injecting a polymer or composition of the invention into cartilage tissue. In some embodiments, the method involves injecting a polymer or composition of the present invention into an ophthalmic tissue.

According to some embodiments, the injection may be performed according to any method and using any injection device known in the art. Non-limiting examples of injection devices include, but are not limited to, syringes, micro-syringes, needle-free devices, microneedles, needles, cannulas, and use of 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.

In some embodiments, the methods of the invention relate to treating arthritis in a subject in need thereof. 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 arthritic arthritis.

In one embodiment, the methods of the invention relate to treating a subject suffering from osteoarthritis.

In some embodiments, the methods of the invention relate to preventing or treating ophthalmic tissue damage in a subject before or after a surgical procedure.

In some embodiments, the method involves topically applying a polymer or composition of the present invention. In some embodiments, the polymers of the present invention are applied to skin tissue. In some embodiments, the method relates to promoting/enhancing wound healing in a subject in need thereof. In some embodiments, the method relates to promoting/enhancing wound closure in a subject in need thereof.

In one embodiment, the polymer of the present invention is itself provided to the subject. In one embodiment, one or more of the polymers of the present invention are provided to the subject by themselves. In one embodiment, the polymer of the invention is provided to a subject as part of a pharmaceutical composition (wherein it is mixed with a pharmaceutically acceptable carrier). In one embodiment, one or more polymers of the invention are provided to a subject as part of a pharmaceutical composition (wherein they are mixed with a pharmaceutically acceptable carrier).

The term "subject" as used herein refers to animals, more specifically to non-human mammals and human organisms. Non-human animal subjects 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: horse, cow, camel, goat, sheep, dog, cat, non-human primate, mouse, rat, rabbit, hamster, guinea pig, or pig. In one embodiment, the subject is a human. The human subject may also include a fetus. In one embodiment, the subject in need thereof is a subject having and/or at risk of having an arthritis-associated condition. In some embodiments, the subject in need thereof is a subject suffering from a reduction in tissue volume. In some embodiments, the reduction in tissue volume is referred to as "decongestion. In some embodiments, tissue decongestion comprises fat loss, water loss, extracellular matrix degradation, collagen loss, or others. In some embodiments, the decongestion causes sagging and deterioration of the skin. In some embodiments, the subject suffers from a burn. In some embodiments, a subject in need of a wound closure method suffers from leakage of bodily fluids, such as bleeding. As used herein, the term "treatment" or "treatment" of a disease, disorder or condition encompasses the alleviation of at least one symptom thereof, the reduction of the severity thereof, or the inhibition of the progression thereof. Treatment does not mean a complete cure for the disease, disorder or condition. As an effective treatment, a composition useful herein need only reduce the severity of a disease, disorder or condition, reduce the severity of its associated symptoms, or provide an improvement in the quality of life of a patient or subject.

As used herein, the term "prevention" of a disease, disorder or condition includes delaying, preventing, inhibiting or inhibiting the onset of the disease, disorder or condition. As used in accordance with the presently described subject matter, the term "prophylaxis" relates to a method of prophylaxis that: wherein the subject is exposed to the presently described polymer or composition comprising the polymer prior to initiation or onset of the disease/disorder process. 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 combat a condition already existing in a patient whose clinical symptoms have developed.

As used herein, the term "condition" includes anatomical and physiological deviations from a normal state that constitute a lesion of the normal state of a living animal or one of its parts that interrupts or alters the performance of a bodily function.

Reagent kit

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

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

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 cross-linked by 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 the presence of a ligand attached to the polymer, and wherein the polymer is attached to the polymer by a linker comprising a norbornene moiety or a derivative thereof¹The degree of crosslinking, determined by H NMR, is between 0.2 and 4%.

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 subcutaneous injection. In some embodiments, the injection device is adapted for the technique of cosmopathy (mesotherapy). As used herein, the term "cosmetic therapy" refers to non-surgical cosmetic drug treatment.

In some embodiments, the kit further comprises an amino acid as disclosed above.

In some embodiments, the kit further comprises a mineral as disclosed above.

In some embodiments, the kit further comprises a vitamin as disclosed above.

In some embodiments, the kit further comprises an anesthetic as disclosed above.

In some embodiments, the kit further comprises a pharmaceutically acceptable carrier as disclosed above.

In some embodiments, the package is scored (sorted) to allow sampling of a first HA chain or derivative thereof coupled to a norbornene moiety or derivative thereof, a second HA chain or derivative thereof coupled to a tetrazine moiety or derivative thereof, a free HA chain, an amino acid, a vitamin, a mineral, an anesthetic, or any combination thereof.

In one embodiment, the package is in the form of an ampoule, vial or capsule. In one embodiment, the capsule is a soft capsule.

In some embodiments, the components of the kits disclosed above are sterile. As used herein, the term "sterile" refers to a state of being free of biological contaminants. Any sterilization method is suitable and will be apparent to one of ordinary skill in the art.

In some embodiments, the kit further comprises a free HA chain or derivative thereof. As defined herein, a free HA chain or derivative thereof is an HA chain that is not coupled to a tetrazine moiety, a norbornene moiety, or a derivative thereof.

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

In some embodiments, the kit is utilized by: 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 is further mixed with a free HA chain, an amino acid, a vitamin, a mineral, an anesthetic, or any combination thereof, and the composition formed by mixing the first HA chain or a derivative thereof coupled to the norbornene moiety or a derivative thereof and the second HA chain or a derivative thereof coupled to the tetrazine moiety or a derivative thereof and the free HA chain, the amino acid, the vitamin, the mineral, the anesthetic, or any combination thereof is applied to the filling or filling method.

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

In some embodiments, the kit comprises instructions for mixing a first HA chain or derivative thereof coupled to a norbornene moiety or derivative thereof with a second HA chain or derivative thereof coupled to a tetrazine moiety or derivative thereof and a free HA chain, amino acids, vitamins, minerals, anesthetics, or any combination thereof.

In some embodiments, the kit is utilized by: mixing a first HA chain or derivative thereof coupled to a norbornene moiety or derivative thereof and a second HA chain or derivative thereof coupled to a tetrazine moiety or derivative thereof with the free HA chain, amino acids, vitamins, minerals, anesthetics, or any combination thereof, wherein mixing comprises introducing the components into an injection device.

In some embodiments, the kit comprises instructions for mixing a first HA chain or derivative thereof coupled to a norbornene moiety or derivative thereof and a second HA chain or derivative thereof coupled to a tetrazine moiety or derivative thereof with the free HA chain, amino acids, vitamins, minerals, anesthetic, or any combination thereof, wherein the mixing is performed in an injection device.

In some embodiments of the subject kits, the degree of crosslinking of a composition formed by mixing a first HA chain or derivative thereof coupled to a norbornene moiety or derivative thereof with a second HA chain or derivative thereof coupled to a tetrazine moiety or derivative thereof and free HA chains, amino acids, vitamins, minerals, anesthetics, or any combination thereof is 0.01 to 0.1%, 0.01 to 0.5%, 0.05 to 0.1%, 0.1 to 0.3%, 0.1 to 0.5%, 0.1 to 0.75%, 0.1 to 1%, 1 to 1.75%, 1 to 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.

In some embodiments of the subject kits, the phase angle (δ) of a composition formed by mixing a first HA chain or derivative thereof coupled to an unsaturated moiety (e.g., a norbornene moiety) or derivative thereof with a second HA chain or derivative thereof coupled to a tetrazine moiety or derivative thereof and a free HA chain, amino acid, vitamin, mineral, anesthetic 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.

In some embodiments of the subject kits, the elastic modulus of the composition formed by mixing a first HA chain or derivative thereof coupled to a norbornene moiety or derivative thereof with a second HA chain or derivative thereof coupled to a tetrazine moiety or derivative thereof and the free HA chain, amino acid, vitamin, mineral, anesthetic or any combination thereof is 10-500Pa, 20-1,000Pa, 30-600Pa, 40-1,000Pa, 40-5,000Pa, 50-10,000Pa, 500-50,000Pa, 500-10,000Pa, 500-5,000Pa, 500-1,000Pa, 1,000-50,000Pa, 1,000-10,000Pa, 1,000-5,000Pa, 50-20,000Pa, 500-20,000Pa, or 1,000-20,000Pa, or any range therebetween.

In some embodiments, the components of the kit are packaged in containers.

In some embodiments, the container is made of a material selected from the group consisting of: thin-walled films or plastics (transparent or opaque), cardboard-based, foils, rigid plastics, metals (e.g., aluminum), glass, and the like.

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

In some embodiments, some or all of the components of the kit may be packaged in suitable packaging to maintain sterility.

In some embodiments, the package has a cap that allows for a hermetic seal during storage and that can be pierced by a needle or cannula at the time of use.

In some embodiments, the components of the kit are stored in separate containers within the main kit containment element, such as a cassette or similar structure, which may or may not be an air-tight container, e.g., to further maintain sterility of some or all of the components of the kit.

In some embodiments, the dosage of the first HA chain or derivative thereof coupled to the norbornene moiety or derivative thereof, the second HA chain or derivative thereof coupled to the tetrazine moiety or derivative thereof, the free HA chain, the amino acid, the vitamin, the mineral, or the anesthetic provided in the kit can be sufficient for a single administration or multiple administrations.

In some embodiments, a kit can have multiple doses of a first HA chain or derivative thereof coupled to a norbornene moiety or derivative thereof, a second HA chain or derivative thereof coupled to a tetrazine moiety or derivative thereof, a free HA chain, an amino acid, a vitamin, a mineral, or an anesthetic, packaged in a single container, such as a single tube, vial, Eppendorf, or the like.

In some embodiments, the kits may have multiple doses of a first HA chain or derivative thereof coupled to a norbornene moiety or derivative thereof, a second HA chain or derivative thereof coupled to a tetrazine moiety or derivative thereof, a free HA chain, an amino acid, a vitamin, a mineral, or an anesthetic packaged separately, such that certain kits may have more than one container of the first HA chain or derivative thereof coupled to a norbornene moiety or derivative thereof, the second HA chain or derivative thereof coupled to a tetrazine moiety or derivative thereof, a free HA chain, an amino acid, a vitamin, a mineral, or an anesthetic.

In some embodiments, multiple doses of a first HA chain or derivative thereof coupled to a norbornene moiety or derivative thereof, a second HA chain or derivative thereof coupled to a tetrazine moiety or derivative thereof, a free HA chain, an amino acid, a vitamin, a mineral, or an anesthetic may be contained in a single container.

In some embodiments, the kit includes instructions for preparing the compositions used therein and how to practice the methods of the invention.

In some embodiments, the instructions may be recorded on a suitable recording medium or substrate. For example, the instructions may be printed on a substrate such as paper or plastic.

In some embodiments, the instructions may be present in the kit as a package insert, in a label of a container of the kit or a component thereof (i.e., associated with a package or sub-package), and the like. In other embodiments, the instructions reside as electronically stored data files on a suitable computer readable storage medium, such as a CD-ROM, diskette, and the like. In other embodiments, no actual instructions are present in the kit, but rather means for obtaining the instructions from a remote source (e.g., over the internet). An example of such an embodiment is a kit: which contains a web site where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this manner of obtaining the instructions is recorded on a suitable substrate.

Unless otherwise indicated, any concentration range, percentage range, or ratio range recited herein should be understood to include any integer concentration, percentage, or ratio within the range and fractions thereof, such as tenths and hundredths of integers.

Unless otherwise indicated, any numerical range recited herein in relation to any physical characteristic, such as polymer subunit, dimension, or thickness, should be understood to include any integer within the range recited.

As used herein, the term "subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, such as a human, in need of treatment.

In the discussion, unless otherwise specified, adjectives (e.g., "substantially" and "about") modifying the condition or relationship of one or more features of an embodiment of the invention are understood to mean that the condition or feature is defined to be within a tolerance range that is acceptable for the intended application of operation of the embodiment. Unless otherwise indicated, the word "or" in the specification and claims is considered to be an inclusive "or" rather than an exclusive "or" and denotes at least one or any combination of its associated items.

It should be understood that the terms "a" and "an," as used above and elsewhere herein, refer to "one or more" of the listed components. It will be clear to one of ordinary skill in the art that, unless otherwise specifically stated, the use of the singular includes the plural. Thus, the terms "a", "an" and "at least one" are used interchangeably in this application.

For a better understanding of the present teachings without limiting the scope of the teachings in any way, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

In the description and claims of this application, each of the verbs "comprise", "comprise" and "have", and their conjugates, are used to indicate that the subject or subjects of the verb are not necessarily a complete listing of the subject or subject's components, elements or parts of the verb.

Other terms used herein are intended to be defined by their art-recognized meanings.

Other objects, advantages and novel features of the present invention will become apparent to one of ordinary skill in the art upon examination of the following examples, which are not intended to be limiting. In addition, each of the various embodiments and aspects of the present invention as described above and as claimed below is supported experimentally in the following examples.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments, unless the embodiment is incapable of operation without those elements.

In one embodiment, the term "alkyl" includes aliphatic hydrocarbons, including straight and branched chain groups. Preferably, the alkyl group has 21 to 100 carbon atoms, more preferably 21 to 50 carbon atoms. Whenever numerical ranges are described herein; for example, "21-100," which means that the group (in this case, alkyl group) can include 21 carbon atoms, 22 carbon atoms, 23 carbon atoms, and the like, up to and including 100 carbon atoms.

In one embodiment, the term "long alkyl" includes alkyl groups having at least 20 carbon atoms in their backbone (the longest path of consecutive covalently attached atoms). Thus, short alkyl groups have 20 or fewer backbone carbons. In one embodiment, an alkyl group may be substituted or unsubstituted. In one embodiment, the term "alkyl" as used herein also encompasses saturated or unsaturated hydrocarbons, and thus the term further encompasses alkenyl and alkynyl groups.

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 or unsubstituted with one or more substituents, 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 or unsubstituted with one or more substituents.

In one embodiment, the term "unsaturated" describes a compound containing one or more unsaturated bonds. In some embodiments, unsaturated bonds refer to double and/or triple bonds.

In one embodiment, the term "cycloalkyl" describes an all-carbon monocyclic or fused ring (i.e., rings that share an adjacent pair of carbon atoms) group in which one or more rings do not have a fully conjugated pi-electron system. Cycloalkyl groups may be substituted or unsubstituted.

In one embodiment, the term "aryl" describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a fully conjugated pi-electron system. In one embodiment, the aryl group may be substituted or unsubstituted.

In one embodiment, the term "alkoxy" describes-O-alkyl and-O-cycloalkyl. In one embodiment, the term "aryloxy" describes an-O-aryl group. In one embodiment, the terms alkyl, cycloalkyl and aryl in the formulae herein may be substituted with one or more substituents, wherein each substituent may independently be, for example, halo (halide), alkyl, alkoxy, cycloalkyl, alkoxy, nitro, amine, hydroxy, mercapto, thioalkoxy, thiohydroxy, carboxy, amide, aryl and aryloxy, depending on the group being substituted and its position in the molecule.

In one embodiment, "halo/halide," "halogen," or "halo" describes fluorine, chlorine, bromine, or iodine. In one embodiment, "haloalkyl" describes an alkyl group, as defined herein, further substituted with one or more halo groups. In one embodiment, "haloalkoxy" describes an alkoxy group, as defined herein, further substituted with one or more halo groups. In one embodiment, the term "hydroxy" describes an-OH group. In one embodiment, the term "thiol" or "mercapto" describes an — 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 an-NR 'R "group having R' and R". In one embodiment, the term "heteroaryl" describes a monocyclic or fused ring (i.e., rings that share an adjacent pair of atoms) having one or more atoms in the ring, such as, for example, nitrogen, oxygen, and sulfur, and additionally having a fully conjugated pi-electron system. Non-limiting examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, and,Azoles, thiazoles, pyrazoles, pyridines, pyrimidines, quinolines, isoquinolines, and purines.

In one embodiment, the term "heteroalicyclic" or "heterocyclyl" describes a monocyclic or fused ring group having one or more atoms in the ring such as nitrogen, oxygen, and sulfur. The ring may also have one or more double bonds. In one embodiment, the ring does not have a completely conjugated pi-electron system. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholinyl and the like.

In one embodiment, the term "carboxy" OR "carboxylic acid group" describes a-C (═ O) -OR 'group, where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon), OR heteroalicyclic (bonded through a ring carbon).

In one embodiment, the term "carbonyl" describes a-C (═ O) -R 'group, where R' is as defined above. In one embodiment, the above terms also include thio derivatives thereof (thiocarboxyl and thiocarbonyl).

In one embodiment, the term "thiocarbonyl" describes a — C (═ S) -R 'group, where R' is as defined above. In one embodiment, the term "thiocarboxyl" describes a — C (═ S) -OR 'group, where R' is as defined herein. In one embodiment, the term "sulfinyl" describes a-S (═ O) -R 'group, where R' is as defined herein. In one embodiment, the term "sulfonyl" or "sulfonic acid group" describes a — S (═ O)2 — R 'group, where R' is as defined herein. In one embodiment, the term "carbamoyl" or "carbamato" describes a-OC (═ O) -NR ' R "group, where R ' is as defined herein and R" is as generally defined for R '.

In one embodiment, the term "nitro" refers to the group — NO 2. In one embodiment, the term "cyano" or "nitrile" group refers to a-C ≡ 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. Refers to the group-N3. In one embodiment, the term "sulfonamide" refers to a-S (═ O)2-NR 'R "group, where R' and R" are as defined herein.

In one embodiment, the term "phosphono" OR "phosphonyl" describes a-O-P (═ O) (OR ') 2 group, wherein R' is as defined above. In one embodiment, the term "phosphinic group" describes a-PR 'R "group, wherein R' and R" are as defined above.

In one embodiment, the term "alkaryl" describes an alkyl group, as defined herein, substituted with an aryl or heteroaryl group. In one embodiment, the alkaryl group is benzyl.

In one embodiment, the term "heteroaryl" describes a monocyclic or fused ring (i.e., rings that share an adjacent pair of atoms) having one or more atoms in the ring such as, for example, nitrogen, oxygen, and sulfur, and additionally having a fully conjugated pi-electron system. Non-limiting examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, and,Azoles, thiazoles, pyrazoles, pyridines, pyrimidines, quinolines, isoquinolines, and purines. Heteroaryl groups may be substituted or unsubstituted with one or more substituents, as described above. Representative examples are thiadiazoles, pyridines, pyrroles,Oxazoles, indoles, purines, and the like.

In one embodiment, the terms "halo" and "halo" are used interchangeably herein to describe a halogen atom, i.e., fluorine, chlorine, bromine, or iodine, also referred to herein as fluoro, chloro, bromo, and iodo. In one embodiment, the term "haloalkyl" describes an alkyl group as defined above further substituted with one or more halo groups.

Examples

Materials and methods.

The method A comprises the following steps: preparation of HA-tetrazine or HA-norbornene.

Hyaluronic acid sodium salt was dissolved in 2- (N-morpholino) ethanesulfonic acid (MES) buffer (100mM, pH 5.5) at a concentration of 5 to 10 mg/g. After a homogeneous solution is obtained, 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methyl-morpholine hydrochloride (DMTMM) is added, followed by the primary amine to be coupled. The amount of reagent is adjusted according to the desired degree of modification. The reaction was kept under agitation at 10 to 50 ℃ for 24 hours, and then dialyzed against a saturated NaCI solution (MWCO ═ 12kDa) for 24 hours, and then several times against purified water. The solution was transferred to a flask, cooled to-80 ℃ and freeze-dried to provide the modified HA product in solid form.

The method B comprises the following steps: preparation of HA-tetrazine or HA-norbornene or HA-styrene.

Hyaluronic acid sodium salt was dissolved in Double Distilled Water (DDW) at a concentration of 5 to 10 mg/g. After a homogeneous solution is obtained, 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methyl-morpholine hydrochloride (DMTMM) is added, followed by the primary amine to be coupled. The amount of reagent is adjusted according to the desired degree of modification. The reaction was kept under agitation at 20 to 90 ℃ for 4 hours, and then dialyzed against a saturated solution of NaCl (MWCO ═ 12kDa) for 24 hours, and then several times against purified water. The solution was transferred to a flask, cooled to-80 ℃ and freeze-dried to provide the modified HA product in solid form.

The modification degree of HA-tetrazine, HA-norbornene, HA-styrene and the crosslinking degree of the gel were determined.

To determine the degree of modification, a dry sample of modified HA (e.g.HA-tetrazine or HA-norbornene or HA-styrene) is 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 is liquid. By passing¹The samples were analyzed by H NMR. The degree of modification was determined by calculating an integral ratio, as described below.

The degree of modification of HA-tetrazine (formula IIIB) was calculated as the ratio of the area under the peaks corresponding to the aromatic protons of tetrazine (7.5 to 7.7ppm and 8.1 to 8.9ppm, 8H) to the area under the peaks corresponding to the N-acetylated protons of the glucosamine residue of hyaluronic acid (2.0ppm, 3H). Of HA-tetrazine¹The H NMR spectrum is provided in fig. 1.

Degree of modification of HA-norbornene (formula IIA): the ratio of the area under the peak corresponding to the alkylene proton (5.8 to 6.3ppm, 2H) to the area under the peak corresponding to the N-acyl proton of the glucosamine residue of hyaluronic acid (2.0ppm, 3H). Process for preparing HA-norbornene¹The H NMR spectrum is provided in fig. 2.

Degree of modification of HA-styrene (formula IA): the ratio of the area under the peaks corresponding to the alkylene protons (at 5.36ppm, 5.90ppm and 6.8ppm, 3H) to the area under the signal corresponding to the N-acetyl proton of the glucosamine residue of hyaluronic acid (2.0ppm, 3H). Of HA-styrene¹The H NMR spectrum is provided in fig. 3.

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

[D_c＝(D_m(Tet)/M(Tet)+D_m(Nor)/M(Nor))/(M(Tet)+M(Nor))]。

wherein:

-D_cis the degree of crosslinking of the gel;

-D_m(Tet) is the degree of modification of HA-tetrazine by¹H NMR determination;

-D_m(Nor) is the degree of modification of HA-norbornene by¹H NMR determination;

-m (tet) is the weight of HA-tetrazine participating in the cross-linking;

-M (Nor) is the weight of HA-norbornene involved in the crosslinking.

Cross-linking of HA-tetrazine and HA-norbornene

Two samples of HA-tetrazine and HA-norbornene were dissolved in phosphate buffered solutions with pH values in the range of 6.8 to 7.8, respectively, at the concentrations specified below (Table 1). The same volume of the two solutions was mixed and kept under agitation at 37 ℃ for 18 hours, then further agitated at room temperature until the rheological gel parameters stabilized. For the sample including the non-crosslinked hyaluronic acid, the same concentration of the non-crosslinked hyaluronic acid was added after the reaction, and homogenized under agitation. The degree of crosslinking of the resulting gel is the average of the degrees of modification of the HA-tetrazine and HA-norbornene used.

Measurement of rheological parameters.

The measurement of the phase angle δ (in ° s) and the elastic modulus G' (in Pa) is carried out at 25 ℃ at a frequency of 1Hz, with a stress sweep of 1 to 1,000Pa or a strain sweep of 0.35 to 3500% using a rheometer (TheRmo haake ars 6000 or RS1 or TA DHR1) with a serrated or sandblasted plate-plate geometry of 20mm diameter.

TABLE 1 summary of rheological measurements

The linkers used in items 7-24 are presented in the appended claims 5 and 7.

Example 1

Rheological parameters of HA Cross-Linked polymers

First, commercially available dermal fillers were examined (table 1, entries 1-6, 25, 26). The data show that δ less than 10 ° is only obtained in commercial products with high HA content (20 mg/gr-entries 1-3) and/or high degree of crosslinking (6-9% -entries 2-5). It was also observed that products with high HA content and high degree of crosslinking resulted in δ greater than 10 ° (entry 6). Products at lower concentrations (12mg/gr, entries 25-26) and high or low degrees of crosslinking (6% -entry 25 or 1% -entry 26) resulted in δ being greater than 10 °.

Samples containing 10mg/gr hyaluronic acid were formulated at various degrees of crosslinking (Table 1, entries 7-10, 20). The degree of crosslinking is in the range of 3.5% to 0.9% giving low delta. However, a very low degree of crosslinking, such as 0.35%, fails to lead to gel formation (entry 10).

The samples were then formulated with 10mg/gr hyaluronic acid and different amounts of non-crosslinked HA were added to the final product (Table 1, entries 11-14). As observed by the increase in δ values, the addition of non-crosslinked HA reduces the lifting capacity of the crosslinked material. Nevertheless, gels containing HA contents up to 20% still show delta values below 10 °.

Samples with a fixed degree of crosslinking were further formulated at 1.3% or 0.9% and various HA levels (Table 1, entries 15-17, 20-22). A reduction from 15mg/gr to 10mg/gr and 5mg/gr (entries 15-17) or from 10mg/gr to 7.5mg/gr and 5mg/gr (entries 20-22) reduced the gel elastic modulus (G') of the crosslinked material. However, even with HA levels as low as 5mg/gr, the delta values remain below 10 deg..

To test the generality of the cross-linking molecules, tetrazine derivatives and norbornene derivatives were mixed with a fixed HA content (10 mg/gr; Table 1, entries 18-19). As shown, a δ value of 10 ° or less was obtained.

To test the substitution of norbornene with another unsaturated moiety, HA was modified by styrene (formula IA). The tetrazine derivative was mixed with the styrene derivative at a degree of crosslinking of 1.1% and an HA content of 10mg/gr or 7.5mg/gr to give a delta value below 10 ° (Table 1, entries 23-24).

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, as it retains its viscoelastic properties with increasing applied force. LVR is measured as the starting point (intersection of 2 tangents to the G' curve) in the standard strain sweep cycle of the rheometer (TA DHR1), as shown in fig. 4.

Commercial dermal fillers provide a range of onset values from very low as 9.4Pa (Table 2, entry 7) to very high as 421Pa (Table 2, entry 2). The commercial LVR data clearly show a correlation with HA content (table 2, entries 3-6 at a degree of crosslinking of 6%, entries 1 and 7 at a degree of crosslinking of 1%). The LVR data of commercial products also showed a clear correlation with the degree of crosslinking (Table 2, entries 1-3, at 20 mg/gr). The gels according to the invention show a similar correlation between the LVR and HA content and the degree of crosslinking (Table 2, entries 8-13). However, the gels according to the invention show a higher LVR compared to the commercial products and therefore have better material properties, while comprising a lower HA content and a lower degree of crosslinking (table 2, entries 8-12vs entries 1, 4-7, and entry 13vs entry 7).

TABLE 2 LVR value comparison

Example 2

Extrudability of HA Cross-Linked polymers

Two samples of HA-tetrazine and HA-norbornene were dissolved in the same concentration in a phosphate buffered solution at pH 7.0, respectively. Subsequently, the same volume of each sample was mixed together and immediately introduced into a 1mL long cycloolefin polymer (COP) syringe. All syringes were incubated at 37 ℃ for 18 hours and then further at room temperature (incubations) until the rheological gel parameters stabilized.

Extrusion force was measured using a Mecmesin pull bench instrument. By fitting with 27G 1/2' or 29¹/₂Data were collected by extruding the gel through a 1mL syringe with a "or 30G 1/2" needle at a standard rate of 12.5 mm/min. For the gels prepared as described above, no sieving or crushing was performed.

Representative extrusion force data are listed in table 3. Restylane and Voluma are provided with 29G1/2 "and 27G 1/2" needles, respectively (table 3, entries 1-2), and have relatively high G values (table 1, entries 1, 3). Volbella is provided with a 30G1/2 "pin (Table 3, entry 3) and a medium range G' (Table 1, entry 5).

Entries 4-9 in table 3 represent gels of the invention, prepared in 1mL syringes without sieving or homogenization. As shown in table 3, the extrusion force values were within acceptable ranges. The gel of entry 4 was extruded through a 27G1/2 "needle, while the gels of entries 5-9 were extruded through a 30G 1/2" needle.

Table 3 extrusion force values.

Example 3

Gel swelling in Water

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

Swelling ratio (weight of swollen gel)/(initial weight of gel).

TABLE 4 gel swell ratio in Water

The results show that the gels prepared according to the invention (table 4, entries 4-6) have a lower gel swell compared to commercial dermal fillers with higher HA content and/or higher degree of crosslinking (table 4, entries 1-3). The reduced gel swelling in water is likely to reduce the risk of swelling and edema in vivo.

Example 4.

In vivo implant data

Gel samples (corresponding to entries 1, 2, 4-6 of table 4) were injected subcutaneously against five Sprague-Dawley rats using 27G1/2 ″. On day 1, each animal received 5 injections of 100 microliters of gel sample. Each gel sample was injected 5 times in various animals. Animals were followed 95 days after injection. No erythema or edema was found at any injection site in any animal. No changes in the overall health status of any animals were found during the study. Histopathological evaluation of the injection sites 95 days after injection did not reveal any pathological changes at the injection sites.

The size of the subcutaneous gel blocks was measured with an electronic ruler during the study from day 1 to day 95 post injection. Subcutaneous gel size was calculated by palpating the length x width of the ball detected under the skin and measured with an electronic ruler. The mean data (fig. 5) of 5 injection sites per product shows that the gel block size of test article 2 (gel described in entry 5 of table 4) and test article 3 (gel described in entry 4 of table 4) is significantly larger than the two controls, namely the commercial dermal fillers with higher HA content and/or higher degree of cross-linking (control 1 is described in entry 2 of table 4, control 2 is described in entry 1 of table 2). Test 1 with a very low HA content of 5mg/gr (gel described in entry 6 of table 4) shows that the size of the subcutaneous gel pellet is the same as control 2 with a significantly higher HA content of 20mg/gr (gel described in entry 1 of table 4).

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.