Method of crosslinking glycosaminoglycans

Abstract

A new hydrogel made of crosslinked glycosaminoglycans, particularly crosslinked hyaluronic acid, chondroitin or chondroitin sulfate, having reversible linkages using boronic acid or boroxole derivatives leading to new benefits. Glycosaminoglycans that are crosslinked via an alkoxyboronate ester anion formed between a diol portion of a diol-functional moiety grafted to a first glycosaminoglycan and a boronate hemiester grafted to a second glycosaminoglycan.

Claims

1. Crosslinked glycosaminoglycans, wherein said glycosaminoglycans are crosslinked via an alkoxyboronate ester anion formed between a diol portion of a diol-functional moiety grafted to a first glycosaminoglycan and a boronate hemiester grafted to a second glycosaminoglycan, said glycosaminoglycans having a structure of Formula (I) ##STR00051## wherein the boronate hemiester is selected from: ##STR00052## wherein the boronate hemiester is grafted to said second glycosaminoglycan by the —NH.sub.2 group of the boronate hemiester and forms an amide with a backbone carboxylate group of said second glycosaminoglycan, each GAG is hyaluronic acid and, diol functional moiety is selected from maltose, fructose, lactose and sorbitol.

2. Crosslinked glycosaminoglycans according to claim 1, said crosslinked glycosaminoglycans having a structure of Formula (II) ##STR00053##

3. A method of crosslinking a first glycosaminoglycan grafted with a diol-functional moiety having a diol portion and a second glycosaminoglycan grafted with a boronate hemiester, comprising crosslinking said first glycosaminoglycan with said second glycosaminoglycan by forming an alkoxyboronate ester anion linkage between the boronate hemiester of said second glycosaminoglycan and the diol portion of said diol-functional moiety of said first glycosaminoglycan, whereby the crosslinked glycosaminoglycans according to claim 1 are obtained.

4. Polymer composition comprising crosslinked glycosaminoglycans according to claim 1 and an aqueous buffer.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1A-1D: Rheological analyses of HA-BOR/HA-fructose and HA-PBA/HA-fructose mixtures in 0.01M HEPES buffer containing 0.15M NaCl at pH 7.4, using HA-BOR and HA-PBA with DS of 0.15 (A) and with higher DS of 0.4 and 0.5, respectively (B). Viscosity measurements of the HA-BOR and HA-PBA derivatives alone with DS of 0.15 (C) and with higher DS of 0.4 and 0.5, respectively (D).

(2) FIG. 2: Photo of a gel obtained with HA-BOR/HA-polyol

(3) FIG. 3: Self-healing properties of a dynamic gel of HA-BOR/HA-fructose (CHA=15 g/L) at 25° C.: stress recovery of the hydrogel after four cycles of recovery from stress-induced breakdowns at pH 7.4.

(4) FIGS. 4A-4C: Rheological analyses of HA-BOR/HA-fructose (A), HA-FBOR/HA-fructose (B) and HA-PBA/HA-fructose (C) mixtures in 0.01M HEPES buffer containing 0.15M NaCl at different pH (from 4 to 8).

(5) The following terms and characteristics will be used in the examples and results shown. The definitions are the one hereafter:

(6) Mw—Molecular Weight: The mass average molecular mass

(7) DS—Degree of Substitution The term “degree of substitution” (DS) as used herein in connection with various polymers, e.g. polysaccharides, refers to the average number of substituting group per repeating disaccharide unit.

(8) [PS]— The polysaccharide concentration (g/l).

(9) G′: storage (elastic) modulus (in Pa)

(10) G″: loss (viscous) modulus (in Pa)

(11) G′ 1 Hz: storage modulus (in Pa) measured at a frequency of 1 Hz

(12) G″ 1 Hz: loss modulus (in Pa) measured at a frequency of 1 Hz

(13) Gel-like behavior: G′>G″ within the whole range of frequency covered (0.01-10 Hz)

(14) Viscoelastic behavior: viscous (G′<G″) and elastic (G′>G″) behavior observed within the range of frequency covered (0.01-10 Hz).

(15) The IUPAC names of the benzoboroxol derivatives in example 4-11 are generated using Biovia DRAW 4.2.

EXAMPLES

(16) Without desiring to be limited thereto, the present invention will in the following be illustrated by way of examples.

Example 1: Synthesis of HA-BOR

(17) ##STR00035##

(18) The amine-acid coupling agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) was dissolved in 1 mL of water and was added to a solution of native HA in a mixture of water/DMF (3/2, v/v). A concentration of HA in the reaction medium of 3 g/L was used for HA samples of 75 and 100 kg/mol, whereas 2 g/L was used for HA with 600 kg/mol. Then, 5-amino-2-hydroxymethylphenylboronic acid hydrochloride (1-hydroxy-3H-2,1-benzoxaborol-amine, ABOR) solubilized in 1 mL of water was added to the reaction medium. The pH was adjusted to 6.5 using 0.5 M HCl or NaOH and the reaction was kept under stirring at room temperature for 24 h. The product was purified by diafiltration with ultrapure water and was recovered by freeze-drying. The degree of substitution (DS) of HA-BOR was determined by .sup.1H NMR (DS.sub.NMR), and were also estimated from the reaction kinetics performed using 2,4,6-Trinitrobenzene Sulfonic Acid (DS.sub.TNBS). This method consisted in quantifying the free primary amines in the reaction medium as a function of time. Table 1 summarizes the DMTMM/HA and BOR/HA molar ratios used for the syntheses with different M.sub.w HA, as well as the DS and the yields of HA-BOR conjugates.

(19) HA-BOR: .sup.1H NMR (400 MHz, D.sub.2O) δ.sub.H (ppm) 4.55 (H-1 from N-acetylglucosamine unit), 4.25 (H-1 from glucuronic acid), 3.9-3.1 (H-2, H-3, H-4, H-5, H-6 protons of HA), 2.08 (CH.sub.3—CO from HA), 7.95 (s, 1H, NH—C—CH—C—B from Ph), 7.72 (m, 1H, C—CH—CH—C—C—B from Ph), 7.55 (m, 1H, C—CH—CH—C—C—B from Ph), 5.13 (s, 2H, CH.sub.2—O—B).

Example 2: Synthesis of HA-PBA (Comparative Example)

(20) ##STR00036##

(21) Grafting of phenylboronic acid was done according to Example 1, but using 3-aminophenylboronic acid hemisulfate salt (APBA) instead of 5-amino-2-hydroxymethylphenylboronic acid hydrochloride (ABOR). The degree of substitution (DS) of HA-PBA was determined by .sup.1H NMR (DS.sub.NMR), and were also estimated from the reaction kinetics performed using 2,4,6-Trinitrobenzene Sulfonic Acid (DS.sub.TNBS). This method consisted in quantifying the free primary amines in the reaction medium as a function of time. Table 1 summarizes the DMTMM/HA and PBA/HA molar ratios used for the syntheses with different M.sub.w HA, as well as the DS and the yields of HA-PBA conjugates.

(22) HA-PBA: .sup.1H NMR (400 MHz, D.sub.2O) δ.sub.H (ppm) 4.55 (H-1 from N-acetylglucosamine unit), 4.25 (H-1 from glucuronic acid), 3.9-3.1 (H-2, H-3, H-4, H-5, H-6 protons of HA), 2.08 (CH.sub.3—CO from HA), 7.93 (s, 1H, NH—C—CH—C—B from Ph), 7.7 (m, 2H, C—CH—CH—CH—C—B from Ph), 7.55 (m, 1H, C—CH—CH—CH—C—B from Ph).

(23) TABLE-US-00001 TABLE 1 Syntheses of HA-BOR and HA-PBA. HA-boronic BOR or acid M.sub.w HA DMTMM/HA PBA/HA molar derivative (Kg/mol) molar ratio ratio DS.sub.NMR.sup.a DS.sub.TNBS Yield (%).sup.b HA-BOR  75 1 0.16 0.16 0.16 75 HA-BOR 100 1 0.16 0.12 0.14 85 HA-BOR 600 1 0.14 0.11 0.13 75 HA-PBA  75 1 0.16 0.16 0.16 75 HA-PBA 100 1 0.16 0.16 0.16 77 HA-PBA 600 1 0.14 0.14 0.14 78 .sup.aDS by .sup.1H NMR: 10% of accuracy. .sup.bHA-BOR or HA-PBA yield: calculation considering the DS.sub.NMR.

Example 3: Synthesis of Pentenoate-Modified HA

(24) ##STR00037##

(25) HA (1 g, 2.5 mmol, M.sub.w=100 kg/mol) was dissolved in ultrapure water (50 mL) under continuous stirring overnight at 4° C. DMF (33 mL) was then added dropwise in order to have a water/DMF ratio of (3/2, v/v). 4-pentenoic anhydride (0.454 g, 2.5 mmol) was added while maintaining the pH between 8 and 9 by adding 1 M NaOH for at least 4 h. The reaction was kept at 4° C. under stirring for one night. The product was purified by diafiltration with ultrapure water and was recovered by freeze-drying. The degree of substitution (DS) of HA-pentenoate was found to be 0.18±0.01 by .sup.1H NMR. A yield of 49% was calculated considering its DS.

(26) .sup.1H NMR (400 MHz, D.sub.2O) δ.sub.H (ppm) 4.71 (H-1 from N-acetylglucosamine unit), 4.53 (H-1 from glucuronic acid), 4.13-3.2 (H-2, H-3, H-4, H-5, H-6 protons of HA), 2.1 (CH.sub.3—CO from HA), 6.0 (m, 1H, CH═CH.sub.2), 5.18 (m, 2H, CH═CH.sub.2), 2.62 (m, 2H, CH.sub.2—C═O), 2.45 (m, 2H, OCF.sub.2—CH.sub.2).

Example 4: Synthesis of HA-Maltose

(27) ##STR00038##

(28) a. Maltose-Disulfide

(29) To an aqueous solution of maltose (0.25 g, 0.694 mmol) in 25 mL of ultrapure water at room temperature, O-(carboxymethyl)hydroxylamine hemihydrochloride (0.0768 g, 0.694 mmol) was added. The pH was adjusted to 4.8 using 0.5 M NaOH. The reaction mixture was stirred for 24 hours at room temperature and then, was neutralized to pH 7 by addition of 0.5 M NaOH. The maltose-COOH derivative was then recovered by freeze-drying without further purification as a white powder (46 mol % of maltose-COOH/maltose). To a solution of maltose-COOH (0.25 g, 0.622 mmol) in dry DMF (50 mL), hydroxybenzotriazole (HOBt) (0.1875 g, 1.39 mmol), diisopropylcarbodiimide (DIC) (0.3483 g, 2.8 mmol) and cystamine dihydrochloride (0.094 g, 0.42 mmol) were successively added. The resulting mixture was stirred overnight at room temperature under nitrogen. After evaporation of most of the solvent, the residual syrup was poured dropwise into acetone (500 mL) under stirring. The white precipitate was collected by filtration, washed three times with acetone and dried to give the desired maltose-disulfide in 60% yield (0.295 g, 0.625 mmol).

(30) .sup.1H NMR (400 MHz, D.sub.2O).sup.6H (ppm) 7.75 (1H, anomeric Hβ from linked glucose unit, N═CH.sub.β—), 7.13 (1H, anomeric Hα from linked glucose unit, N═CH.sub.α—), 5.4 (1H, anomeric H from pendant glucose unit of maltose), 5.19 (1H, anomeric Hα from linked glucose unit), 5.14 (1H, anomeric H from pendant glucose unit of maltose-disulfide), 4.7 (1H, anomeric Hβ from pendant glucose unit), 4.66 (2H, N—O—CH.sub.2), 4.6 (1H, N═CH.sub.α,β—CH(OH) from linked glucose group), 3.4-4.2 (8H, H-3, H-4, H-5, H-6 from linked and pendant glucose groups), 2.95 (4H, NH—CH.sub.2—CH.sub.2).

(31) b. HA-Maltose

(32) The first step consisted in reducing the disulfide bond of maltose-disulfide. Thus, to an aqueous solution of this derivative (0.2 g, 0.211 mmol) in 4 mL of degassed phosphate buffered saline (PBS) pH 7.4 at room temperature, a solution of TCEP (91 mg, 0.317 mmol) in 1 mL of degassed PBS was added and the pH was adjusted to 5-5.5. The mixture was stirred for 15 min under nitrogen at room temperature to give maltose-SH. The pH was adjusted to 7.4 using 0.5 M NaOH and the mixture was added to HA-pentenoate solubilized in PBS in the presence of Irgacure 2959 (0.1%, w/v) as a photoinitiator. The grafting of maltose-SH moieties was performed under UV radiation (X=365 nm, at 20 mW/cm.sup.2 for 15 min). The product was purified by diafiltration with ultrapure water and was recovered by freeze-drying. The degree of substitution (DS) of HA-maltose was found to be 0.1±0.01 by .sup.1H NMR.

(33) .sup.1H NMR (400 MHz, D.sub.2O) δ.sub.H (ppm) 4.55 (H-1 from N-acetylglucosamine unit), 4.25 (H-1 from glucuronic acid), 3.9-3.1 (H-2, H-3, H-4, H-5, H-6 protons of HA), 1.85 (CH.sub.3—CO from HA), 1.52 (m, 2H, CH.sub.2—CH.sub.2—CH.sub.2—S), 1.62 (m, 2H, CH.sub.2—CH.sub.2—CH.sub.2—S), 2.35 (m, 2H, OC—CH.sub.2) 2.63 (m, 2H, CH.sub.2—CH.sub.2—CH.sub.2—S), 2.82 (m, 2H, S—CH.sub.2—CH.sub.2—NH), 7.63 (m, 1H, H anomer of maltose).

Example 5: Synthesis of HA-Lactobionic

(34) ##STR00039##

(35) a. Lactobionic-Disulfide

(36) To a solution of lactobionic acid (0.5023 g, 1.39 mmol) in dry DMF (50 mL), hydroxybenzotriazole (HOBt) (0.3768 g, 2.79 mmol), diisopropylcarbodiimide (DIC) (0.705 g, 5.56 mmol) and cystamine dihydrochloride (0.141 g, 0.626 mmol) were successively added. The resulting mixture was stirred overnight at room temperature under nitrogen. After evaporation of most of the solvent, the residual syrup was poured dropwise into acetone (500 mL) under stirring. The white precipitate was collected by filtration, washed three times with acetone and dried to give the desired lactobionic-disulfide in 29% yield (0.2362 g, 0.585 mmol).

(37) b. HA-Lactobionic

(38) A first step of reduction of the disulfide bond of the lactobionic-disulfide derivative (0.2 g, 0.211 mmol) dissolved in 1 mL of degassed PBS was performed by adding TCEP (91 mg, 0.317 mmol) in 1 mL of degassed PBS, with pH adjusted to 5-5.5. The mixture was stirred for 15 min under nitrogen at room temperature to give lactobionic-SH. The pH was adjusted to 7.4 using 0.5 M NaOH and the mixture was added to HA-pentenoate solubilized in PBS in the presence of Irgacure 2959 (0.1%, w/v) as a photoinitiator. The grafting of lactobionic-SH moieties was performed under UV radiation (X=365 nm, at mW/cm.sup.2 for 15 min). The product was purified by diafiltration with ultrapure water and was recovered by freeze-drying. The degree of substitution (DS) of HA-lactobionic was found to be 0.2 t 0.01 by .sup.1H NMR.

Example 6: Synthesis of HA-Fructose

(39) ##STR00040##

(40) 1-amino-1-deoxy-D-fructose hydrochloride (0.0121 g, 0.056 mmol) dissolved in 1 mL of ultrapure water was added to a solution of native HA (0.15 g, 0.374 mmol) in a mixture of water/DMF (3/2, v/v) in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (0.1035 g, 0.374 mmol) as an amine-acid coupling agent. The pH was adjusted to 6.5 using 0.5 M HCl or NaOH and the reaction was kept under stirring at room temperature for 24 h. The product was purified by diafiltration with ultrapure water and was recovered by freeze-drying. The degree of substitution (DS) of HA-fructose was determined by .sup.13C NMR (DS.sub.NMR=0.15±0.01), and was also estimated from the reaction kinetics performed using 2,4,6-Trinitrobenzene Sulfonic Acid (DS.sub.TNBS=0.14). A yield of 84% was determined for HA-fructose (considering its DS.sub.NMR).

(41) .sup.1H NMR (400 MHz, D.sub.2O) δ.sub.H (ppm) 4.62 (H-1 from N-acetylglucosamine unit), 4.46 (H-1 from glucuronic acid), 4.05-3.2 (18H, H-2, H-3, H-4, H-5, H-6 protons of HA and of fructose moieties), 2.02 (CH.sub.3—CO from HA).

Example 7: Synthesis of HA-Sorbitol

(42) ##STR00041##

(43) 1-amino-1-deoxy-D-sorbitol hydrochloride (D-glucamine) (0.0088 g, 0.05 mmol) dissolved in 1 mL of ultrapure water was added to a solution of native HA (0.1305 g, 0.325 mmol) in ultrapure water in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (0.09 g, 0.325 mmol) as an amine-acid coupling agent. The pH was adjusted to 6.5 using 0.5 M HCl or NaOH and the reaction was kept under stirring at room temperature for 164 h. The product was purified by diafiltration with ultrapure water and was recovered by freeze-drying. The degree of substitution (DS) of HA-sorbitol was determined by .sup.13C NMR (DS.sub.NMR=0.15±0.1), and was also estimated from the reaction kinetics performed using 2,4,6-Trinitrobenzene Sulfonic Acid (DS.sub.TNBS=0.1). A yield of 76% was determined for HA-sorbitol (considering its DS.sub.NMR).

(44) .sup.1H NMR (400 MHz, D.sub.2O) δ.sub.H (ppm) 4.68 (H-1 from N-acetylglucosamine unit), 4.51 (H-1 from glucuronic acid), 4.1-3.3 (19H, H-2, H-3, H-4, H-5, H-6 protons of HA and of sorbitol moieties), 2.07 (CH.sub.3—CO from HA).

Example 8: Preparation of HA-BOR/HA-Polyol Gel

(45) Solutions of HA-BOR and of the HA-polyol derivatives (HA-maltose or HA-lactobionic or HA-fructose or HA-sorbitol) were prepared at 15 g/L in 0.01 M HEPES buffer containing 0.15 M NaCl pH 7.4, and were kept under stirring overnight at 4° C. Combinations of HA-BOR/HA-polyol derivative, were prepared by mixing a solution containing HA-BOR with a solution containing a HA-polyol derivative at physiological pH, at a total polymer concentration of 15 g/L and with BOR/polyol molar ratio of 1/1.

(46) Results: When gels were formed quasi-instantaneously upon mixing HA-BOR solution with a solution of a HA-polyol derivative. Characteristics of the resulting HA-BOR/HA-polyol mixtures are summarized in Table 2.

Example 9: Preparation of HA-PBA/HA-Polyol Gel (Comparative Example)

(47) HA-PBA/HA-polyol gels were prepared according to example 8, but using HA-PBA instead of HA-BOR.

(48) Results: When gels were formed quasi-instantaneously upon mixing HA-BOR solution with a solution of a HA-polyol derivative. Characteristics of the resulting HA-PBA/HA-polyol mixtures are summarized in Table 2.

(49) TABLE-US-00002 TABLE 2 Characteristics of HA-BOR/HA-polyol and HA-PBA/HA-polyol mixtures in 0.01M HEPES buffer containing 0.15M NaCl pH 7.4 ([PS] = 15 g/L, BOR or PBA/fructose molar ratio = 1/1). HA- DS HA- boronic boronic DS HA- M.sub.w HA G′ 1 Hz G″ 1 Hz Rheological acid acid HA-polyol polyol (kg/mol) (Pa) (Pa) behavior HA-BOR 0.15 HA-maltose 0.12  75  34  20 Viscoelastic HA-BOR 0.15 HA-fructose 0.15  75 515  17 Gel HA-BOR 0.15 HA-sorbitol 0.15  75 250 125 Viscoelastic HA-BOR 0.1 HA-fructose 0.15 100 280  30 Gel HA-BOR 0.15 HA-fructose 0.15 100 275 ± 35 20.5 ± 2.5 Gel HA-BOR 0.12 HA- 0.2 100  87  33 Viscoelastic lactobionic HA-BOR 0.1 HA-fructose 0.08 600 250  80 Gel HA-PBA 0.15 HA-maltose 0.12  75 490  7 Gel HA-PBA 0.15 HA-fructose 0.15  75 447  6.6 Gel HA-PBA 0.15 HA-sorbitol 0.15  75 150  45 Viscoelastic HA-PBA 0.15 HA-fructose 0.15 100 227 ± 12   8 ± 1.5 Gel HA-PBA 0.15 HA- 0.2 100 439  21 Gel lactobionic HA-PBA 0.15 HA-fructose 0.08 600  91  27 Gel

Example 10: The Effect of a Higher Degree of Substitution (DS) on the Behavior of HA-BOR/HA-Fructose and HA-PBA/HA-Fructose Gels

(50) HA-BOR/HA-fructose and HA-PBA/fructose gels were prepared according to example 8, but using HA-BOR and HA-PBA derivatives with higher DS of 0.4 and 0.5, respectively.

(51) Results: Gels were formed quasi-instantaneously upon mixing HA-BOR solution with a solution of a HA-fructose. Characteristics of the resulting HA-BOR or HA-PBA/HA-fructose mixtures are summarized in Table 3. Higher dynamic moduli (G′ and G″) were obtained for the HA-BOR/HA-fructose hydrogel when using HA-BOR with a higher DS, compared to the mixture using HA-PBA with a DS of 0.5 (FIG. 1). Viscosity measurements showed that cross-links formed between BOR moieties and HA hydroxyl groups lead to increase the viscosity of HA network (FIG. 1). Therefore, the double crosslinking of BOR/fructose moieties and BOR/HA contribute to improve gel properties, in opposition to the HA-PBA/HA-fructose mixture.

(52) TABLE-US-00003 TABLE 3 Characteristics of HA-BOR/HA-polyol and HA-PBA/HA-polyol mixtures in 0.01M HEPES buffer containing 0.15M NaCl pH 7.4 ([PS] = 15 g/L, BOR or PBA/fructose molar ratio = 1/1). HA- DS HA- boronic boronic DS HA- Mw HA G′ 1 Hz G″ 1 Hz Rheological acid acid HA-polyol polyol (kg/mol) (Pa) (Pa) behavior HA-BOR 0.4 HA-fructose 0.15 100 350 17 Gel HA-PBA 0.5 HA-fructose 0.15 100 185  5 Gel

Example 11: HA-Benzoboroxole (HA-BOR)/HA-Polyol

(53) Gels obtained from mixtures of benzoboroxole modified HA (HA-BOR)/HA-polyol were prepared by simply mixing solutions of the two HA partners solubilized in 0.01 M HEPES buffer with 0.15 M NaCl at physiological pH. When these solutions were mixed at a total polymer concentration of 15 g/L, and with benzoboroxole/polyol molar ratio of 1/1, transparent gels were formed quasi-instantaneously (FIG. 2).

(54) Results: Characteristics of the resulting HA-BOR/HA-polyol mixtures are summarized in Table 4. Self-healing properties of a dynamic gel of HA-BOR/HA-fructose (CHA=15 g/L) at 25° C. were investigated by, while measuring G′ and G″, applying successive stress values from 1800 to 2100 Pa for 2 min. These were intercalated with short time periods in which low stress values (corresponding to 5% strain) were applied for 3 min. This experiment demonstrated the stress recovery of the HA-BOR/HA-fructose gel after 4 cycles of stress-induced breakdowns. Large stress (from 1800 to 2100 Pa) inverted the values of G′ (filled circles) and G″ (empty circles), indicating breakage of crosslinks and conversion to solution state. G′ was recovered under a small strain (5%) within few seconds. These gels provide self-healing properties (FIG. 3).

(55) TABLE-US-00004 TABLE 4 Characteristics of HA-BOR/HA-Polyol hydrogel DS HA- DS HA- Rheo- BOR HA-polyol polyol Mw HA G′ 1 Hz G″ 1 Hz logical derivative derivative derivative (kg/mol) (Pa) (Pa) behavior 0.16 HA- 0.12  75  34  20 Visco- maltose elastic 0.16 HA- 0.15  75 500  17 Gel fructose 0.16 HA- 0.15  75 250 125 Visco- sorbitol elastic 0.12 HA- 0.15 100 490  7 Gel fructose 0.11 HA- 0.08 600 250  80 Gel fructose

Example 12: Synthesis of HA-1-hydroxy-7-methoxy-3H-2,1-benzoxaborol-6-amine

(56) ##STR00042##

(57) Example 12 is performed according to Example 1, but using 1-hydroxy-7-methoxy-3H-2,1-benzoxaborol-6-amine hydrochloride as the ABOR derivative instead of 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride.

Example 13: HA-1-hydroxy-7-methoxy-3H-2,1-benzoxaborol-6-amine/HA-polyol Gel Preparation

(58) Gels are prepared according to example 8, but using HA-1-hydroxy-7-methoxy-3H-2,1-benzoxaborol-6-amine instead of HA-BOR.

Example 14: Synthesis of HA-7-fluoro-1-hydroxy-3H-2,1-benzoxaborol-6-amine

(59) ##STR00043##

(60) Example 14 is performed according to Example 1, but using 7-fluoro-1-hydroxy-3H-2,1-benzoxaborol-6-amine hydrochloride as the ABOR derivative instead of 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride.

Example 15: HA-7-fluoro-1-hydroxy-3H-2,1-benzoxaborol-6-amine/HA-Polyol Gel Preparation

(61) Gels are prepared according to example 8, but using HA-7-fluoro-1-hydroxy-3H-2,1-benzoxaborol-6-amine instead of HA-BOR.

Example 16: Synthesis of HA-(1-hydroxy-3H-2,1-benzoxaborol-6-yl)methanamine

(62) ##STR00044##

(63) Example 16 is performed according to Example 1, but using (1-hydroxy-3H-2,1-benzoxaborol-6-yl)methanamine hydrochloride as the ABOR derivative instead of 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride.

Example 17: HA-(1-hydroxy-3H-2,1-benzoxaborol-6-yl)methanamine/HA-Polyol Gel Preparation

(64) Gels are prepared according to example 8, but using HA-(1-hydroxy-3H-2,1-benzoxaborol-6-yl)methanamine instead of HA-BOR.

Example 18: Synthesis of HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine

(65) ##STR00045##

(66) Example 18 is performed according to Example 1, but using 1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine hydrochloride as the ABOR derivative instead of 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride.

Example 19: HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine/HA-Polyol Gel Preparation

(67) Gels are prepared according to example 8, but using HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine instead of HA-BOR.

(68) Example 20: Synthesis of HA-1-hydroxy-3,4-dihydro-2,1-benzoxaborinin-7-amine

(69) ##STR00046##

(70) Example 20 is performed according to Example 1, but using 1-hydroxy-3,4-dihydro-2,1-benzoxaborinin-7-amine hydrochloride as the ABOR derivative instead of 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride.

Example 21: HA-1-hydroxy-3,4-dihydro-2,1-benzoxaborinin-7-amine/HA-Polyol Gel Preparation

(71) Gels are prepared according to example 8, but using HA-1-hydroxy-3,4-dihydro-2,1-benzoxaborinin-7-amine instead of HA-BOR.

Example 22: Synthesis of HA-(1-hydroxy-3H-2,1-benzoxaborol-6-yl)methanamine

(72) ##STR00047##

(73) Example 22 was performed according to Example 1, but using (1-hydroxy-3H-2,1-benzoxaborol-6-yl)methanamine hydrochloride (AMBOR) as the ABOR derivative instead of 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride. The molecular weight of the Hyaluronic acid was 100 kg/mol.

Example 23: HA-(1-hydroxy-3H-2,1-benzoxaborol-6-yl)methanamine/HA-Polyol Gel Preparation

(74) Gels were prepared according to example 8, but using HA-(1-hydroxy-3H-2,1-benzoxaborol-6-yl)methanamine (HA-AMBOR) instead of HA-BOR.

(75) Example 24: Synthesis of HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine

(76) ##STR00048##

(77) Example 24 was performed according to Example 1, but using 1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine hydrochloride (DMABOR) as the ABOR derivative instead of 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride. The molecular weight of the Hyaluronic acid was 100 kg/mol.

Example 25: HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine/HA-Polyol Gel Preparation

(78) Gels were prepared according to example 8, but using HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine (HA-100DMABOR) instead of HA-BOR.

Example 26: Synthesis of HA-7-fluoro-1-hydroxy-3H-2,1-benzoxaborol-6-amine

(79) ##STR00049##

(80) Example 26 was performed according to Example 1, but using 7-fluoro-1-hydroxy-3H-2,1-benzoxaborol-6-amine hydrochloride (FBOR) as the ABOR derivative instead of 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride. The molecular weight of the Hyaluronic acid was 100 kg/mol.

Example 27: HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine/HA-Polyol Gel Preparation

(81) Gels were prepared according to example 8, but using 7-fluoro-1-hydroxy-3H-2,1-benzoxaborol-6-amine (HA-FBOR) instead of HA-BOR.

Example 28: Rheological Behavior of Gels Using HA-BOR Derivatives, Compared to HA-PBA

(82) ##STR00050##

(83) Results: Characteristics of the resulting HA-BOR/HA-polyol mixtures are summarized in Table 5.

(84) TABLE-US-00005 TABLE 5 HA100-boronic acid/HA100-fructose mixtures analyzed by rheology ([PS] = 15 g/L; BOR or DMABOR or AMBOR or FBOR or PBA/ fructose molar ratio = 1; 0.01M HEPES/0.15M NaCl buffer pH 7.4). HA-boronic DS of HA- DS of HA- Rheological G′ 1 Hz G″ 1 acid boronic acid fructose behavior (Pa) Hz (Pa) HA100- 0.15 0.15 Gel 275 ± 35 20.5 ± BOR 2.5 HA100- 0.12 0.15 Gel 525 ± 11  14 ± 2.8 FBOR HA100- 0.14 0.15 Gel 120 ± 32   7 ± 1.3 DMABOR HA100- 0.12 0.15 Gel/ 116 ± 9   12 ± 1.3 AMBOR Viscoelastic HA100- 0.16 0.15 Gel 227 ± 12   8 ± 1.5 PBA

Example 29: The Effect of pH on the Behavior of HA-BOR/HA-Fructose or HA-FBOR/HA-Fructose and HA-PBA/HA-Fructose Gels

(85) Rheological analyses were performed to compare the effect of pH on the behavior of HA-BOR/HA-fructose or HA-FBOR/HA-fructose vs HA-PBA/HA-fructose gels (FIG. 4).

(86) Results: Table 6 summarizes the characteristics of the gels at different pH. These results showed that a higher stability at a pH range from 4 to 8 was observed for the HA-BOR/HA-fructose and HA-FBOR/HA-fructose mixtures, compared to HA-PBA/HA-fructose (FIG. 4).

(87) TABLE-US-00006 TABLE 6 Analyses of HA-BOR or HA-FBOR or HA-PBA/HA-fructose mixtures in 0.01M HEPES buffer containing 0.15M NaCl at different pH (M.sub.w HA = 75 or 100 kg/mol, [PS] = 15 g/L, DS of HA-BOR or HA-PBA or HA-fructose = 0.15, DS of HA-FBOR = 0.12, BOR or FBOR or PBA/fructose molar ratio = 1). Cross-over frequency: <0.01 = near 0.01 Hz or <<0.01 = far below 0.01 Hz. HA-boronic acid Cross-over derivative pH G′ 1 Hz (Pa) G″ 1 Hz (Pa) frequency (Hz) HA-BOR 4 6 2 0.13 (M.sub.w = 75 kg/mol) 5 23.5 9 0.25 6 125 20 0.063 6.5 210 17.5 0.02 7.4 515 17 <0.01 8 572 13 <<0.01 HA-FBOR 4 1.4 4 G′ < G″ (M.sub.w = 100 kg/mol) 5 19 22 1.3 6 126 50 0.2 6.5 332 50 0.025 7.4 533 12 <<0.01 8 545 11 <<0.01 HA-PBA 4 ≤0.1 ≤0.1 G′ < G″ (M.sub.w = 75 kg/mol) 5 ≤0.1 ≤0.1 G′ < G″ 6 2.4 1.2 0.13 6.5 93.5 5 0.016 7.4 447 7 <<0.01 8 564 9 <<0.01

Example 30: Self Healing Properties of Obtained Gels

(88) The variation of G′ and G″ as a function of time immediately after injection through a 27 gauge needle of HA-BOR/HA-fructose or HA-DMABOR/HA-fructose or HA-FBOR/HA-fructose gels was investigated. Gels were prepared in 0.01 M HEPES/0.15M NaCl buffer pH 7.4, at a [PS]=15 g/L and BOR or DMABOR or FBOR/fructose molar ratios of 1/1).

(89) Results:

(90) The hydrogels exhibited self-healing properties. Consequently, they can be injected as preformed solids, because the solid gel can manage external damages and repair itself under a proper shear stress. Due to fast gelation kinetics after extrusion/injection, they recover their solid form immediately. As an example, FIG. 5 shows the variation of G′ and G″ as a function of time immediately after injection of HA-BOR/HA-fructose or HA-DMABOR/HA-fructose or HA-FBOR/HA-fructose gels through a 27 gauge needle. From this Figure, it can be seen that the three samples recovered into a solid gel quasi-instantaneously.