METHOD OF PREPARING A HYDROGEL PRODUCT

Abstract

A method of preparing a hydrogel product comprising crosslinked glycosaminoglycan molecules, comprising the steps of: (a) providing a mixed solution of glycosaminoglycan molecules, a di- or multinucleophilic functional crosslinker, and a mononucleophilic functional graft chain; (b) activating carboxyl groups on the glycosaminoglycan molecules with a coupling agent to form activated glycosaminoglycan molecules; and (c) simultaneously crosslinking the activated glycosaminoglycan molecules and grafting the graft chain to the activated glycosaminoglycan molecules by reacting the nucleophiles with the activated carboxyl groups.

Claims

1. A method of preparing a hydrogel product comprising crosslinked glycosaminoglycan molecules, comprising the steps of: (a) providing a mixed solution of glycosaminoglycan molecules, a di- or multinucleophilic functional crosslinker, and a mononucleophilic functional graft chain; (b) activating carboxyl groups on the glycosaminoglycan molecules with a coupling agent to form activated glycosaminoglycan molecules; and (c) simultaneously crosslinking the activated glycosaminoglycan molecules and grafting the graft chain to the activated glycosaminoglycan molecules by reacting the nucleophiles with the activated carboxyl groups.

2. The method according to claim 1, wherein the glycosaminoglycan molecules are selected from the group consisting of hyaluronic acid, chondroitin, and chondroitin sulfate, and mixtures thereof.

3. The method according to claim 1, wherein the crosslinker comprises a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides.

4. The method according to claim 1, wherein the nucleophilic groups of the crosslinker are selected from the group consisting of primary amine, hydrazine, hydrazide, carbazate, semi-carbazide, thiosemicarbazide, thiocarbazate, and aminoxy.

5. The method according to claim 4, wherein the nucleophilic groups of the crosslinker are primary amine.

6. The method according to claim 1, wherein the crosslinker is a dinucleophilic functional crosslinker.

7. The method according to claim 6, wherein the crosslinker is selected from the group consisting of diamino hyaluronic acid tetrasaccharide, diamino hyaluronic acid hexasaccharide, diamino trehalose, diamino lactose, diamino maltose, diamino sucrose, chitobiose, and diamino raffinose.

8. The method according to claim 5, wherein the crosslinker is an at least partially deacetylated hyaluronic acid.

9. The method according to claim 1, wherein the crosslinking and grafting of step (c) provides amide bonds between glycosaminoglycan molecules and crosslinkers and between glycosaminoglycan molecules and graft chains.

10. The method according to claim 1, wherein the mononucleophilic functional graft chain is a mononucleophilic functional carbohydrate.

11. The method according to claim 1, wherein the mononucleophilic functional graft chain is an aminodextran and/or an aminocyclodextrin.

12. The method according to claim 11, wherein the molar ratio of the aminodextran and/or aminocyclodextrin to the disaccharides of the glycosaminoglycan is 0.1-50%.

13. The method according to claim 11, wherein the aminodextran and/or aminocyclodextrin contain a linking group having an amino group, and wherein the linking group of the aminodextran and/or aminocyclodextrin forms an amide bond with a carboxyl group of the glycosaminoglycan.

14. The method according to claim 13, wherein the linking group contains a C1-6 alkyl.

15. The method according to claim 11, wherein the aminodextran and/or aminocyclodextrin is an aminodextran.

16. The method according to claim 15, wherein the aminodextran has an average molecular weight of less than 10 kDa.

17. The method according to claim 16, wherein the aminodextran is covalently grafted to the activated glycosaminoglycan by single end-point attachment.

18. The method according to claim 15, wherein the aminodextran is functionalized at the reducing end with a diamine.

19. The method according to claim 11, wherein the aminodextran and/or aminocyclodextrin is an aminocyclodextrin.

20. The method according to claim 19, wherein the aminocyclodextrin is constituted by 5-32 glucopyranoside units.

21. The method according to claim 20, wherein the aminocyclodextrin is constituted by 6 glucopyranoside units (-cyclodextrin).

22. The method according to claim 20, wherein the aminocyclodextrin is constituted by 7 glucopyranoside units (-cyclodextrin).

23. The method according to claim 20, wherein the aminocyclodextrin is constituted by 8 glucopyranoside units (-cyclodextrin).

24. The method according to claim 20, wherein the aminocyclodextrin is 6-aminocyclodextrin.

25. The method according to claim 1, wherein at least 90% of the bonds between glycosaminoglycan molecules and crosslinks and between glycosaminoglycan molecules and graft chains are amide bonds.

26. The method according to claim 1, wherein less than 5% of the bonds between glycosaminoglycan molecules and crosslinks and between glycosaminoglycan molecules and graft chains are ester bonds.

27. The method according to claim 1, wherein the coupling agent is DMTMM.

28. The method according to claim 27, wherein the DMTMM/disaccharide repeating unit ratio is in the range of 1-3.

29. The method according to claim 27, wherein the DMTMM/disaccharide repeating unit ratio is in the range of 3-8.

Description

EXAMPLES

Characterization

Reductive Amination on Dextran

[0088] The total DEX-HMDA (including mono- and cross-linked HMDA to dextran and free dextran) was determined by .sup.1H NMR by comparing the integral of the signals from the anomeric proton of dextran (4.94 ppm), hexamethylenediamine signal (1.33 ppm) and KHP (7.62 ppm, internal standard)
LC-QToF-MS analysis was made to evaluate the residual hexamethylenediamine (HMDA) in the powder after reductive amination.
Amide Crosslinked Hyaluronan Hydrogels with Grafted Dextran
Swelling was done in saline
MoD.sub.DEX/diHAdegree of modification of dextran on hyaluronan is measured with .sup.1H NMR. The integral of the anomeric proton of dextran is compared to the integral of the N-acetyl group of hyaluronan. The average number of glucose units in the dextran and thus anomeric protons is 3.7, which the integral is divided with according to the equation below. The hydrogels were degraded with chondroitinase ABC or HCl prior to the analysis.

[00001]${\mathrm{MoD}}_{\mathrm{DEX}/\mathrm{diHA}}\left(\%\right)=\left(\frac{\frac{\mathrm{Integral}.Math..Math.\mathrm{of}.Math..Math.\mathrm{anomeric}.Math..Math.\mathrm{proton}.Math..Math.\mathrm{of}.Math..Math.\mathrm{dextran}}{3.7}}{\frac{\mathrm{Integral}.Math..Math.\mathrm{of}.Math..Math.N.Math.\text{-}.Math.\mathrm{acetyl}.Math..Math.\mathrm{of}.Math..Math.\mathrm{hyaluronan}}{3}}\right)*100$

GelPGel part is a description of the percentage of polysaccharide that is a part of the gel network. A number of 90% means that only 10% of polysaccharide is not a part the network. The amount of free polysaccharide in the gel was quantified with .sup.1H NMR. The hydrogels were degraded with chondroitinase ABC or HCl prior to the analysis.
SwFSwelling factor analysis was done in saline.
SwCCCorrected swelling capacity is the total liquid uptake of one gram polysaccharide, corrected for gel part.
MwThe mass average molecular mass
SwCSwelling capacity in saline, total liquid uptake per gram PS (mL/g).

[0089] Diaminotrehalose (DATH) is synthesized as described in Synthetic Carbohydrate Polymers Containing Trehalose Residues in the Main Chain: Preparation and Characteristic Properties; Keisuke Kurita, * Naoko Masuda, Sadafumi Aibe, Kaori Murakami, Shigeru Ishii, and Shin-Ichiro Nishimurat; Macromolecules 1994, 27, 7544-7549.

Example 1Reductive Amination of Dextran

[0090] ##STR00001##

[0091] Dextran (100.0 g) with a number average molecular weight (M.sub.n) of 849 Da, hexamethylenediamine dihydrochloride (HMDA, 193.3 g) and NaCNBH.sub.3 (64.8 g) were added to a reaction vessel. The reagents were dissolved in water (500 g) and the pH was adjusted to pH 10.0 by adding 1 M NaOH. The reaction mixture was incubated at 60 C.

[0092] After 4 hours, the reaction was neutralized to pH 7 by adding HCl (aq. 1.2 M) and NaCl (approx. 10 g) was added to facilitate precipitation. The crude reaction was precipitated by slowly addition of the mixture to ethanol under agitation by an over-head stirrer until the final ethanol concentration was 90%. The precipitate was washed with ethanol (90%) numerous times to completely remove residual chemicals and subsequently dried in under vacuum.

[0093] The dry powder was dissolved in D.sub.2O and analyzed by .sup.1H NMR. The total DEX-HMDA content was 75 w/w %. The residual HMDA in the powder was 0.03 w/w %.

Example 2One-Pot Crosslinking and Drafting of Hyaluronan with Dextran

[0094] ##STR00002##

[0095] Hyaluronic acid (HA) was weighed in a reaction vessel. A stock solution of the crosslinker (DATH) was prepared by dissolving it in buffer pH 7. DMTMM and DEX-HMDA were weighed in a PTFE-container and the DATH-solution was added to the DMTMM and DEX-HMDA (for details, see Table 1). The pH of the reagent solution was adjusted to 6-7 with HCl (aq. 1.2 M) and then added to the HA. The mixture was thoroughly homogenized and then incubated at 50 C. for 24 h.

[0096] The resulting material was pressed through a 1 mm steel mesh two times and then treated with NaOH solution (pH 13 for 60 min). The gel was neutralized with HCl (aq. 1.2 M) to pH 7 and washed with an excess of sodium chloride solution to remove excess of reagents. The washed gel was precipitated with ethanol. The resulting precipitate was washed with ethanol (70 w/w %) to remove salts and subsequent ethanol to remove water. The obtained powder was dried in under vacuum over night.

[0097] The precipitate was swelled in 0.7% NaCl in 8 mM phosphate buffer pH 7.4 and then pressed through a 125 m filter mesh three times. The gel was filled on syringes and sterilized (125 C., 5.5 min, F0 23). Gel properties is presented in Table 2.

TABLE-US-00001 TABLE 1 Reaction Conditions HA Start DEX- <Mw> DATH/HA DMTMM/HA HMDA HA Example (k) (mol %) (mol %) (mol %) (w/w %) 2-1 240 0.6 125 41 15.0 2-2 170 0.9 125 41 7.5 2-3 170 0.6 125 41 7.5

TABLE-US-00002 TABLE 2 Gel Properties MoD (DEX/diHA) [PS] SwF Example (%) (mg/mL) (mL/g) 2-1 20% 62 1.9 2-2 17% 53 4.3 2-3 20% 55 4.8

Example 3aComparative Example, Two Step Process for Manufacturing of Amide Crosslinked Hyaluronan Gels Grafted with Dextran; Step 1

[0098] ##STR00003##

[0099] Hyaluronic acid was weighed in a reaction vessel. A stock solution of the crosslinker (DATH) was prepared by dissolving it in buffer pH 7. DMTMM was weighed in a PTFE-container and the crosslinker-solution was added to the DMTMM to dissolve it. The pH of the DMTMM-crosslinker solution was adjusted to 6-7 with 1.2 M HCl and then added to the HA. The contents was thoroughly homogenized and then incubated at 23 C. for 24 h. The resulting material was pressed through a 1 mm steel mesh two times, swelled in 0.9% NaCl and the pH adjusted to 7.3-7.5 with diluted acid/base. The gel was subjected to heat (70 C., 24 h) in order to hydrolyze any potential ester bonds. The gel was particle size reduced through a 125 m mesh followed by precipitation with ethanol and the precipitate was washed with 100 mM NaCl in ethanol (70% w/w) to remove excess reagents and then with ethanol (70% w/w) to remove salts and finally with ethanol to remove water. The precipitate was then dried in vacuum over night. The gels were swelled in phosphate saline buffer to 20 mg/mL HA, filled on syringes and sterilized at (F.sub.0 20). Reaction conditions and gel properties is provided in Table 3.

TABLE-US-00003 TABLE 3 Reaction conditions DMTMM/ DATH/ Results <Mw> [HA] diHA diHA GelP SwCC Example (k) (w/w %) (mol %) (mol %) (%) (mL/g) 3-1 240 15.0 4.9 0.6 89 64 3-2 170 7.5 7.7 0.9 85 166 3-3 170 7.5 5.1 0.6 61 443 3-4 240 15.0 4.9 0.6 89 63

Example 3bComparative Example, Two Step Process for Manufacturing of Amide Crosslinked Hyaluronan Gels Grafted with Dextran; Step 2

[0100] ##STR00004##

[0101] Dextran functionalized with hexamethylenediamine according to Example 1 was grafted to hyaluronic acid hydrogels from Example 3-1 to 3-4. A general manufacturing procedure is described below.

[0102] Dextran modified at the reducing end with hexamethylenediamine (from Example 1) and DMTMM were weighed in a glass or PTFE bottle. 1 mM phosphate buffer pH 7.4 was added to dissolve the reagents. Precipitated hyaluronan hydrogel (from Example 3a) was added to the reaction solution while stirring so that a concentration of 20 mg/mL hyaluronan was obtained. The sample was incubated at 50 C. or stirred at ambient temperature for approx. 24 hrs.

[0103] The reaction was stopped by increasing the pH to 13.0 with 0.25 M NaOH, and after stirring for 60-90 min the mixture was neutralized with diluted HCl to neutral pH. The gel was purified by continuously washing with 0.9% NaCl and then precipitated by slowly adding ethanol up to 70% ethanol or only precipitated by slowly adding ethanol up to 70% ethanol. The precipitate was washed with 70% ethanol. The precipitated gel powder was subsequently washed with ethanol and dried under vacuum. The gel powder was swelled in phosphate saline buffer to 20 mg/mL HA and filled on syringes. and sterilized (F.sub.0 20). A short summary of the. Summary of reaction conditions and gel properties are presented in Table 4.

TABLE-US-00004 TABLE 4 Reaction conditions Results Gel used MoD from Eq DEX- Eq DMTMM/ (DEX/diHA) GelP [PS] SwCC Example example amine/diHA diHA (%) (%) (mg/mL) (mL/g) 3-5 3-1 0.41 5 30 97 60 29 3-6 3-1 0.41 1.25 26 96 59 43 3-7 3-2 0.41 5 26 95 44 56 3-8 3-2 0.41 1.25 27 94 27 88 3-9 3-3 0.41 5 28 96 42 94 3-10 3-3 0.41 1.25 20 97 33 191 3-11 3-4 0.30 5 18 97 79 N.A 3-12 3-4 0.30 1.25 17 96 68 N.A Eq = mol(X)/mol(Y), PS = polysaccharide (DEX + HA)

Example 4aComparative Example; Simulation Showing the Apparent Mw for HA in Gels for One-Pot Process for Manufacturing of Amide Crosslinked Hyaluronan Gels Grafted with Dextran

[0104] Hyaluronic acid (HA, Mw 260 k) was weighed in a reaction vessel. DMTMM was dissolving it in 1 mM phosphate buffer, the pH measured and, if needed adjusted, to pH 7. The DMTMM-solution was added to the pre-weighted (for details, see Table 5, step 1) HA. The mixture was thoroughly homogenized and then incubated at 50 C. for 24 h. The resulting mixture was pressed through a 1 mm steel mesh two times and then treated with NaOH solution (pH 13 for 60 min). The solution was neutralized with HCl (aq. 1.2 M) to pH 7. The polysaccharide was precipitated with ethanol. The resulting precipitate was washed with ethanol (70 w/w %) to remove salts and subsequent ethanol to remove water. The obtained powder was dried in under vacuum over night. The obtained powder was dissolved in 1 mM phosphate buffer with 0.9% NaCl and the Mw was determined by SEC-UV.

Example 4bComparative Example; Simulation Showing the Apparent Mw for HA in Gels for the Two Step Process for Manufacturing of Amide Crosslinked Hyaluronan Gels Grafted with Dextran

[0105] Hyaluronic acid (HA, Mw 260 k) was weighed in a reaction vessel. DMTMM was dissolving it in 1 mM phosphate buffer, the pH measured and, if needed adjusted, to pH 7. The DMTMM-solution was added to the pre-weighted (for details, see Table 5, step 1) HA. The mixture was thoroughly homogenized and then incubated at 23 C. for 24 h. The resulting mixture was pressed through a 1 mm steel mesh two times and then treated with NaOH solution (pH 13 for 60 min). The solution was neutralized with HCl (aq. 1.2 M) to pH 7. The polysaccharide was precipitated with ethanol. The resulting precipitate was washed with ethanol (70 w/w %) to remove salts and subsequent ethanol to remove water. The obtained powder was dried in under vacuum over night.

[0106] The obtained power was weighed in a reaction vessel. DMTMM was dissolving it in 1 mM phosphate buffer, the pH measured and, if needed adjusted, to pH 7. The DMTMM-solution was added to the pre-weighted (for details, see Table 5, step 2) HA. The mixture was thoroughly homogenized and then incubated at 50 C. for 24 h. The resulting mixture was pressed through a 1 mm steel mesh two times and then treated with NaOH solution (pH 13 for 60 min). The solution was neutralized with HCl (aq. 1.2 M) to pH 7. The polysaccharide was precipitated with ethanol. The resulting precipitate was washed with ethanol (70 w/w %) to remove salts and subsequent ethanol to remove water. The obtained powder was dried in under vacuum over night. The obtained powder was dissolved in 1 mM phosphate buffer with 0.9% NaCl and the Mw was determined by SEC-UV.

[0107] As seen in Table 5, the Mw after the process described in both 4a and 4b shows that the reduction in Mw correlate independently of the one-pot or two step process is applied.

TABLE-US-00005 TABLE 5 Reaction Conditions HA DMTMM/ DMTMM/ HA Start HA HA End <Mw> (mol %) (mol %) HA <Mw> Example (k) step 1 step 2 (w/w %) (k) 4a 260 125 N.A 15.0 110 4b 260 4.9 125 15.0 110

[0108] Conclusion from Example 2 & 3

As can be seen in Table 6, by applying the efficient one-pot process amide crosslinked hyaluronan hydrogels grafted with dextran with properties comparable with the two step procedure can be obtained. This is also seen in Table 5.

TABLE-US-00006 TABLE 6 Reaction Conditions HA MoD Start DATH/ DMTMM/ DEX- HA (DEX/ <Mw> HA HA HMDA (w/w diHA) Example (k) (mol %) (mol %) (mol %) %) (%) 2-1 240 0.6 125 41 15.0 20 3-1 & 3-6 240 0.6 4.9/125 41 15.0 26 2-2 170 0.9 125 41 7.5 17 3-2 & 3-8 170 0.9 7.7/125 41 7.5 27 2-3 170 0.6 125 41 7.5 20 3-3 & 3-10 170 0.6 5.1/125 41 7.5 20