Aldehyde-modified hyaluronic acid, method for preparing same and applications thereof

1 . A modified hyaluronic acid derivative, wherein the —CH 2 —OH group of at least one N-acetyl-D-glucosamine unit is modified into an aldehyde group having a structure —CH 2 —O—CH 2 —CHO, wherein the degree of modification is 2.0% to 6.9%, the degree of modification being defined as the number of —CH 2 —O—CH 2 —CHO groups divided by the total number of N-acetyl-D-glucosamine units present in the modified hyaluronic acid derivative, wherein the number of aldehyde groups having the structure —CH 2 —O—CH 2 —CHO of the at least one N-acetyl-D-glucosamine unit divided by the total number of aldehyde groups present in the modified hyaluronic acid derivative is from 0.95 to 1, wherein the modified hyaluronic acid derivative has a weight average molecular weight of 0.4 MDa to 1.3 MDa, and wherein the modified hyaluronic acid derivative comprises at least one disaccharide unit of the following structure

wherein Ac denotes —C(O)CH 3 and R is selected from the group consisting of hydrogen, an alkali metal ion and an alkaline earth metal ion, and further comprises at least one disaccharide unit of the following structure

wherein Ac denotes —C(O)CH 3 , R 1 , R 2 , R 3 and R 4 are independently selected from the group consisting of H and —CH 2 —CHO, and R 5 is selected the group consisting of from hydrogen, an alkali metal ion, an alkaline earth metal ion, and —CH 2 —CHO, wherein, at least one of R 1 , R 2 , R 3 , R 4 and R 5 is —CH 2 —CHO and, if R 1 is —CH 2 —CHO, at least one of R 2 , R 3 , R 4 , and R 5 is —CH 2 —CHO.

2 . A method for preparing the modified hyaluronic acid derivative of claim 1 , comprising:

a) providing a glycerol-modified hyaluronic acid, wherein a —CH 2 —OH group of at least one N-acetyl-D-glucosamine unit is modified into a group of the following formula —CH 2 —O—CH 2 —CHOH—CH 2 OH;

b) dissolving the glycerol-modified hyaluronic acid in an aqueous medium to obtain solubilized glycerol-modified hyaluronic acid; and

c) reacting said solubilized glycerol-modified hyaluronic acid with an oxidizing agent, optionally a periodate, optionally sodium periodate, to convert at least a part of said —CH 2 —O—CH 2 —CHOH—CH 2 OH groups into one or more aldehyde groups having the formula —CH 2 —O—CH 2 —CHO, thereby obtaining an aldehyde-modified hyaluronic acid derivative.

3 . The method of claim 2 , further comprising one or more of:

d) stopping the reaction of c), optionally by adding a vicinal diol (1,2-diol), optionally ethylene glycol;

e) purifying the modified hyaluronic acid derivative, optionally by precipitating the modified hyaluronic acid derivative in an organic solvent, optionally ethanol, isopropanol or a mixture thereof, re-dissolving the precipitate in saline and re-precipitating the modified hyaluronic acid derivative in said organic solvent; or

f) drying the modified hyaluronic acid derivative obtained in e).

4 . The method of claim 2 , wherein the glycerol-modified hyaluronic acid has a weight average molecular weight of 0.1 to 5.0 MDa, optionally 1.0 to 3.0, optionally 1.0 to 2.0 MDa, optionally 1.1 to 1.9, optionally 1.2 to 1.8 MDa, optionally 1.3 to 1.7 MDa, optionally 1.4 to 1.6 MDa and/or a degree of modification of 5 to 25%, optionally 10 to 20%, the degree of modification being defined as the number of —CH 2 —O—CH 2 —CHOH—CH 2 OH groups divided by the total number of N-acetyl-D-glucosamine units present in the glycerol-modified hyaluronic acid and/or wherein

(i) c) is performed at a temperature of 4 to 35° C., optionally 15 to 35° C., optionally 20 to 30° C., optionally 20 to 25° C., optionally about 21 to 23° C., optionally about 22° C.; and/or

(ii) c) is performed for a duration of 5 to 120 minutes, optionally 5 to 65 minutes, optionally 10 to 60 minutes, optionally 10 to 50 minutes, optionally 10 to 40 minutes, optionally 10 to 30 minutes, optionally 10 to 20 minutes; and/or

(iii) the oxidizing agent is present in an amount of 0.01 to 0.5 molar equivalents, optionally 0.01 to 0.3 molar equivalents, optionally 0.04 to 0.3 molar equivalents, optionally 0.04 to 0.1, based on the molar amount of disaccharide repeating units of the glycerol-modified hyaluronic acid; and/or

(iv) said glycerol-modified hyaluronic acid is present in an amount of 2 to 50 g/L, optionally 2 to 40 g/L, optionally 3 to 38 g/L, optionally 4 to 36 g/L.

5 . A modified hyaluronic acid derivative obtained by a method comprising:

a) providing a glycerol-modified hyaluronic acid, wherein a —CH 2 —OH group of at least one N-acetyl-D-glucosamine unit is modified into a group of the following formula —CH 2 —O—CH 2 —CHOH—CH 2 OH;

b) dissolving the glycerol-modified hyaluronic acid in an aqueous medium to obtain solubilized glycerol-modified hyaluronic acid; and

c) reacting said solubilized glycerol-modified hyaluronic acid with an oxidizing agent, optionally a periodate, optionally sodium periodate, to convert at least a part of said —CH 2 —O—CH 2 —CHOH—CH 2 OH groups into aldehyde groups having the formula —CH 2 —O—CH 2 —CHO, thereby obtaining a modified hyaluronic acid derivative, wherein c) is performed for a duration of 5 to 65 minutes, and wherein the degree of modification is 2.0% to 6.9%, the degree of modification being defined as the number of —CH 2 —O—CH 2 —CHO groups divided by the total number of N-acetyl-D-glucosamine units present in the modified hyaluronic acid derivative; wherein the number of aldehyde groups having the structure —CH 2 —O—CH 2 —CHO of the at least one N-acetyl-D-glucosamine unit divided by the total number of aldehyde groups present in the modified hyaluronic acid derivative is from 0.95 to 1, wherein the modified hyaluronic acid derivative has a weight average molecular weight of 0.4 MDa to 1.3 MDa, and wherein the modified hyaluronic acid derivative comprises at least one disaccharide unit of the following structure

wherein Ac denotes —C(O)CH 3 and R is selected from the group consisting of hydrogen, an alkali metal ion and an alkaline earth metal ion, and further comprises at least one disaccharide unit of the following structure

wherein Ac denotes —C(O)CH 3 , R 1 , R 2 , R 3 and R 4 are independently selected from the group consisting of H and —CH 2 —CHO, and R 5 is selected from the group consisting of hydrogen, an alkali metal ion, an alkaline earth metal ion, and —CH 2 —CHO, wherein, at least one of R 1 , R 2 , R 3 , R 4 and R 5 is —CH 2 —CHO and, if R 1 is —CH 2 —CHO, at least one of R 2 , R 3 , R 4 and R 5 is —CH 2 —CHO.

6 . A product comprising the modified hyaluronic acid derivative of claim 1 for in situ formation of a crosslinked hydrogel in aesthetic applications, optionally for treating wrinkles and lines of the skin, including glabellar lines, nasolabial folds, chin folds, marionette lines, jawlines, buccal commissures, perioral wrinkles and crow's feet, cutaneous depressions, scars, temples, subdermal support of the brows, malar and buccal fat pads, tear troughs, nose, lips, cheeks, chin, perioral region, infraorbital region, and/or facial asymmetries.

7 . A product comprising the modified hyaluronic acid derivative of claim 1 for formation of a pre-formed crosslinked hydrogel.

8 . A method for treatment of stress urinary incontinence, vaginal dryness, vesico-ureteral reflux, vocal fold insufficiency, and/or vocal fold medialization comprising injecting the modified hyaluronic acid derivative of claim 1 into a subject in need thereof and in situ formation of a crosslinked hydrogel.

9 . The product of claim 6 , wherein the modified hyaluronic acid derivative is used together with a second polysaccharide derivative, which comprises one or more nucleophilic functional groups capable of forming a covalent bond with one or more aldehyde groups of the modified hyaluronic acid derivative, wherein the second polysaccharide is optionally a hyaluronic acid derivative and said nucleophilic functional group is optionally a hydrazide functional group, and wherein the second polysaccharide is optionally a hyaluronic acid derivative comprising at least one disaccharide unit having the following structure:

wherein Ac denotes —C(O)CH 3 .

10 . The product of claim 9 , wherein

(i) an aldehyde group of the modified HA derivative and a nucleophilic functional group of the second polysaccharide derivative spontaneously form a covalent bond upon co-injecting the modified hyaluronic acid derivative and the second polysaccharide derivative to a target site in the body of a subject, thereby forming a crosslinked hydrogel at the target site, or

(ii) an aldehyde group of the modified HA derivative and a nucleophilic functional group of the second polysaccharide derivative spontaneously form a covalent bond upon contacting the modified hyaluronic acid derivative and the second polysaccharide derivative, wherein optionally the modified HA derivative and the second polysaccharide derivative are present in a buffer medium, optionally a physiological buffer medium.

11 . A crosslinked hydrogel obtained by contacting the modified hyaluronic acid derivative of claim 1 and a second polysaccharide derivative, wherein the crosslinked hydrogel optionally comprises the following structural unit:

wherein “Ac” denotes —C(O)CH 3 and R is selected from hydrogen, an alkali metal ion, optionally Na, and an alkaline earth metal ion.

12 . The crosslinked hydrogel according to claim 11 , wherein said contacting is performed in situ.

13 . The crosslinked hydrogel according to claim 11 , wherein said contacting is performed in vitro, wherein said contacting is optionally performed in a buffer, optionally a physiological buffer.

14 . A method, optionally an aesthetic method, of preparing a crosslinked hydrogel, the method comprising:

a) providing a first precursor solution comprising the modified hyaluronic acid derivative of claim 1 and, separately thereof, a second precursor solution comprising a second polysaccharide derivative;

b) mixing the first precursor solution and the second precursor solution into an in situ crosslinkable mixture; and

c) injecting the in situ crosslinkable mixture to a target site in the body of a patient to form a crosslinked gel at the target site.

15 . The method of claim 14 , wherein in a) the first precursor solution and the second precursor solution are present in different barrels of a multi-barrel syringe and mixing of b) occurs during extrusion from said multi-barrel syringe.

16 . A method of preparing a crosslinked hydrogel, the method comprising:

a) providing a buffer, optionally a physiological buffer;

b) adding the modified hyaluronic acid derivative of claim 1 and adding a second polysaccharide derivative to the buffer, and optionally adding uncrosslinked polysaccharide, optionally hyaluronic acid, to the buffer;

c) crosslinking the modified hyaluronic acid derivative and the second polysaccharide derivative in the buffer to thereby prepare a crosslinked hydrogel;

d) optionally adding uncrosslinked polysaccharide to the crosslinked hydrogel obtained in c);

e) optionally sieving and degassing the hydrogel obtained in c) or d);

f) optionally filling the hydrogel obtained in c), d), or e) in a container, optionally a syringe, optionally a ready-to-use syringe; and

g) optionally sterilizing the container comprising the gel obtained in f).

17 . A kit for in situ formation of a crosslinked hydrogel, comprising (i) a first container containing a first precursor solution comprising the modified hyaluronic acid derivative of claim 1 and (ii) a second container containing a second precursor solution comprising a second polysaccharide derivative, and optionally, (iii) instructions for use.