Hyaluronic acid gel with a divalent zinc cation

Abstract

A method of preparing a sterilized injectable hydrogel composition, including the steps: a) covalently crosslinking a glycosaminoglycan using a bi- or polyfunctional crosslinking agent to form a covalently crosslinked glycosaminoglycan, b) swelling the covalently crosslinked glycosaminoglycan in a solution including a divalent cation to form a hydrogel composition, and c) sterilizing the hydrogel composition by autoclaving to form a sterilized injectable hydrogel composition, and to sterilized injectable hydrogel compositions obtainable by such method.

Claims

1. A sterilized injectable hydrogel composition, comprising: (i) a covalently crosslinked glycosaminoglycan, wherein the glycosaminoglycan is hyaluronic acid, and (ii) a divalent cation, wherein the divalent cation is Zn.sup.2+, and wherein the concentration of Zn.sup.2+ in the composition is in the range of 0.5 to 2 mM; wherein the composition releases the divalent cation at a rate below 0.1 mmol/day upon injection into a subject, and wherein the composition has been subjected to sterilization by autoclaving.

2. The composition according to claim 1, wherein the concentration of the divalent cation in the composition is in the range of 0.8 to 2 mM.

3. The composition according to claim 2, wherein the concentration of Zn.sup.2+ in the composition is in the range of 0.8 to 1.5 mM.

4. The composition according to claim 3, wherein the concentration of Zn.sup.2+ in the composition is about 1.0 mM.

5. The composition according to claim 1, further comprising a therapeutically relevant concentration of a local anesthetic selected from the group consisting of lidocaine and ropivacaine.

6. The composition according to claim 1, wherein the composition exhibits increased stability compared to an identical composition without the divalent cation.

7. A method of preparing a sterilized injectable hydrogel composition comprising: (i) a covalently crosslinked glycosaminoglycan, wherein the glycosaminoglycan is hyaluronic acid, and (ii) a divalent cation, wherein the divalent cation is Zn.sup.2+, and the concentration of Zn.sup.2+ in the composition is in the range of 0.5 to 2 mM; wherein the composition releases the divalent cation at a rate below 0.1 mmol/day upon injection into a subject, the method comprising: (a) covalently crosslinking a glycosaminoglycan using a bi- or polyfunctional crosslinking agent to form a covalently crosslinked glycosaminoglycan, wherein the glycosaminoglycan is hyaluronic acid, and (b) swelling the covalently crosslinked glycosaminoglycan in a solution comprising Zn2+, to form a hydrogel composition, wherein the concentration of Zn2+ in the solution is in the range of 0.5 to 2 mM; and (c) sterilizing the hydrogel composition by autoclaving to form the sterilized injectable hydrogel composition.

8. The method according to claim 7, wherein the concentration of Zn.sup.2+ in the solution is in the range of 0.8 to 2 mM.

9. The method according to claim 8, wherein the concentration of Zn.sup.2+ in the solution is in the range of 0.8 to 1.5 mM.

10. The method according to claim 8, wherein the concentration of Zn.sup.2+ in the solution is about 1.0 mM.

11. The method according to claim 7, wherein the solution further comprises a therapeutically relevant concentration of a local anesthetic selected from the group consisting of lidocaine and ropivacaine.

12. The method according to claim 7, wherein the sterilized injectable hydrogel composition exhibits increased stability compared to an identical composition without the divalent cation.

13. A method of cosmetically treating skin, the method comprising administering to the skin the sterilized injectable hydrogel composition according to claim 1.

14. The composition according to claim 1, wherein the glycosaminoglycan is in the range of 5 to 30 mg/mL.

15. The composition according to claim 1, wherein the divalent cation is added as a zinc salt.

16. The composition according to claim 15, wherein the zinc salt is selected from zinc chloride, zinc gluconate, or zinc citrate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention is further illustrated by FIGS. 1-6. FIGS. 1-6 represent exemplary embodiments only.

(2) FIG. 1a is a diagram showing the stabilizing effect of Zn on a hyaluronic acid gel with a degree of modification of 1 mole %.

(3) FIG. 1b is a diagram showing the stabilizing effect of Zn on a hyaluronic acid gel with a degree of modification of 5 mole %.

(4) FIGS. 2a and 2b are diagrams showing that the stabilizing effect of Zn is also present when lidocaine or ropivacaine is added to the composition.

(5) FIG. 3 is a diagram showing that the stabilizing effect of Zn is also present when tris is used as the buffer.

(6) FIG. 4 is a diagram showing that different Zn salts have a similar stabilizing effect.

(7) FIG. 5a is a diagram showing the concentration dependence of the stabilizing effect of Zn.

(8) FIG. 5b is a diagrams showing the effect of Zn on the storage stability of the composition.

(9) FIG. 6 is a diagram showing the fraction of Zn released from the gel containing 1 mM zinc chloride as a function of time.

EXAMPLES

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

Example 1Hyaluronic Acid Gel with MoD 1%

(11) A BDDE (1,4-butandiol diglycidylether) crosslinked hyaluronic acid gel with a degree of modification of 1 mole % and a hyaluronic acid content of 20 mg/mL was prepared by first transferring hyaluronic acid (Mw 1 MDa) to a plastic jar. A solution of 1% NaOH and 0.3% BDDE was added and the mixture was homogenized. The jar was transferred to an incubator to perform the crosslinking step. The resulting gel was divided in two parts. The first part was allowed to swell to a HA concentration of about 20 mg/mL in a buffer solution containing sodium phosphate, HCl and NaCl, at a pH about 7.4 (Reference). The second part was allowed to swell to a HA concentration of about 20 mg/mL in a buffer solution containing sodium phosphate, HCl, NaCl and 1 mM ZnCl.sub.2. The pH of the formulations was adjusted to 7.4 and the formulations were filled in glass syringes and autoclaved in a Getinge 6610 ERC-1 autoclave (F.sub.019).

(12) The rheological properties of the formulations were analyzed using an Ahres G2 reometer (measure system PP 40, gap 1.00 mm). Initially a strain sweep was made to find the linear viscoelastic region (LVR) and then the viscoelastic properties were measured within the LVR.

(13) FIG. 1a shows the difference in G between the HA gel sample swelled in buffer with ZnCl.sub.2 and the Reference sample swelled in buffer only.

(14) A stabilizing effect on the gel can be seen when 1 mM ZnCl.sub.2 is used in the formulation.

Example 2Hyaluronic Acid Gel with MoD 5%

(15) A BDDE (1,4-butandiol diglycidylether) crosslinked hyaluronic acid gel with a of modification of 5 mole % was prepared by mixing 3 g of hyaluronate (Mw 2 MDa) with a mixture of BDDE (1,4-butandiol diglycidylether) in 1% NaOH. The BDDE/NaOH solution was prepared by diluting BDDE in 1% NaOH to get to give a basic BDDE solution diluted to 1/100. 19 g of the prepared BDDE solution was then added to hyaluronate and homogenized. The mixture was placed in a water bath at 50 C. for 2 h.

(16) The resulting crosslinked hyaluronic acid was swelled in phosphate buffer saline (1 mM PBS) and pH was adjusted to 7. The gel was then purified by dialysis (MWCO 15000) for 2 days. The gel was then divided in two parts, to one of the gels 1 M ZnCl.sub.2 was added to get a concentration of 1 mM ZnCl.sub.2 in the final gel, PBS buffer was added to get a final hyaluronic acid concentration of 20 mg/m L. To the second part of the gel PBS buffer was added to get a final hyaluronic acid concentration of 20 mg/mL. The pH of the formulations was adjusted to 7.4 and the formulations were filled in glass syringes and autoclaved in a Getinge 6610 ERC-1 autoclave (F.sub.019).

(17) The rheological properties of the formulations were analyzed using an Ahres G2 reometer (measure system PP 40, gap 1.00 mm). Initially a strain sweep was made to find the linear viscoelastic region (LVR) and then the viscoelastic properties were measured within the LVR.

(18) FIG. 1b shows the difference in G between the HA gel sample swelled in buffer with ZnCl.sub.2 and the Reference sample swelled in buffer only.

(19) A stabilizing effect on the gel can be seen when 1 mM ZnCl.sub.2 is used in the formulation. The stabilizing effect on the gel from the addition of ZnCl.sub.2 is thus present both in the case of a gel with 1% and 5% degree of modification.

Example 3Hyaluronic Acid Gels with Different ZnCl.SUB.2 .Concentrations

(20) A BDDE (1,4-butandiol diglycidylether) crosslinked hyaluronic acid gel with a degree of modification of 1 mole % and a hyaluronic acid content of 20 mg/mL was prepared according to Example 1, with the difference that the gel was divided into 8 parts after the crosslinking step. The gels parts were swelled to about 20 mg/mL with PBS buffer containing 0, 0.01, 0.05, 0.1, 0.5, 1, 2, 4 and 8 mM ZnCl.sub.2, respectively. The pH of the formulations was adjusted to 7.4 and the formulations were filled in glass syringes and autoclaved in a Getinge 6610 ERC-1 autoclave (F.sub.019).

(21) FIG. 5a shows the effect on the rheological properties following autoclaving of the gel at different concentrations of ZnCl.sub.2.

(22) A stability study was also performed on the gels. Autoclaved syringes were put in an incubator for 1 and 2 weeks and the rheological properties were then analyzed.

(23) FIG. 5b shows the rheological properties of the gel at different concentrations of ZnCl.sub.2 as a function of time when the gels is subjected to 60 C. for 1 and 2 weeks.

(24) A stabilizing effect in the gel was seen with 0.05-2 mM ZnCl.sub.2 while degradation of the gel was observed with concentration at 4 mM ZnCl.sub.2 and above. The degradation rate of the gel was higher when a higher concentration of ZnCl.sub.2 was used.

Example 4Hyaluronic Acid Gel with Different Zn Salts

(25) A BDDE (1,4-butandiol diglycidylether) crosslinked hyaluronic acid gel with a degree of modification of 1 mole % and a hyaluronic acid content of 20 mg/mL was prepared according to Example 1, with the difference that the gel was divided into 4 parts after the crosslinking step. As shown in Table 1, one part was swelled in PBS buffer (1 mM phosphate), one part was swelled in PBS buffer with 1 mM ZnCl.sub.2, one part was swelled in PBS buffer with 1 mM Zn-gluconate and one part was swelled with PBS buffer with 1 mM Zn-citrate. All gels were swelled to a HA concentration of about 20 mg/mL. The pH of the formulations was adjusted to 7.4 and the formulations were filled in glass syringes and autoclaved in a Getinge 6610 ERC-1 autoclave (F.sub.019).

(26) The rheological properties of the formulations were analyzed using an Ahres G2 reometer (measure system PP 40, gap 1.00 mm). Initially a strain sweep was made to find the linear viscoelastic region (LVR) and then the viscoelastic properties were measured within the LVR.

(27) TABLE-US-00001 TABLE 1 HA Gel Zn-salt PBS G at 0.1 Hz Formulation (mg/mL) Zn-salt (mM) (mM) (kPa) 4a 20 0 1 0.80 4b 20 ZnCl.sub.2 1 1 0.99 4c 20 Zn-gluconate 1 1 1.04 4d 20 Zn-citrate 1 1 1.12

(28) FIG. 4. shows the difference in G between an the samples swelled in PBS with 1 mM ZnCl.sub.2, 1 mM Zn-gluconate and Zn-citrate and the reference sample swelled in PBS only. A stabilizing effect can be seen when Zn is added either as ZnCl.sub.2, Zn-gluconate or Zn-citrate.

Example 5Hyaluronic Acid Gel with ZnCl.SUB.2 .and Different Buffers

(29) A BDDE (1,4-butandiol diglycidylether) crosslinked hyaluronic acid gel with a degree of modification of 1 mole % and a hyaluronic acid content of 20 mg/mL was prepared and analyzed according to Example 1, with the difference that Tris buffer (50 mM) in saline was used instead of PBS during the swelling of the gel.

(30) As shown in FIG. 3, the stabilizing effect of 1 mM ZnCl.sub.2 after autoclaving is present also when Tris buffer (50 mM) in saline is used in the formulation.

Example 6HA Gel with Zn and Local Anesthetics

(31) A BDDE (1,4-butandiol diglycidylether) crosslinked hyaluronic acid gel with a degree of modification of 1 mole % and a hyaluronic acid content of 20 mg/mL was prepared according to Example 1, with the difference that the gel was divided into 8 parts after the crosslinking step. The gel parts were swelled in 10 mM PBS buffer with or without 1 mM ZnCl.sub.2, according to Table 2.

(32) Stock solutions of lidocaine hydrochloride monohydrate and ropivacaine hydrochloride monohydrate, respectively, were prepared by dissolving lidocaine and ropivacaine in water. The stock solutions were added to the hyaluronic acid gel with 1 mM ZnCl.sub.2 and 10 mM phosphate buffer saline to a final concentration of 1 mg/mL for ropivacaine and 3 mg/mL for lidocaine. The gels were homogenized by stirring and pH of the formulations was adjusted to 6.5 or 7.3 according to Table 2 and the formulations were filled in glass syringes and autoclaved in a Getinge 6610 ERC-1 autoclave (F.sub.019).

(33) The rheological properties of the formulations were analyzed using an Ahres G2 reometer (measure system PP 40, gap 1.00 mm). Initially a strain sweep was made to find the linear viscoelastic region (LVR) and then the viscoelastic properties were measured within the LVR.

(34) TABLE-US-00002 TABLE 2 Formulations and rheological data for gels prepared according to Example 6 G at Formula- HA Gel ZnCl.sub.2 Lidocaine Ropivacaine 0.1 Hz tion (mg/mL) (mM) (mg/mL) (mg/mL) pH (kPa) 6a 20 0 0 0 7.3 0.71 6b 20 0 3 0 7.3 0.74 6c 20 1 0 0 7.3 0.83 6d 20 1 3 0 7.3 0.85 6e 20 0 0 0 6.6 0.80 6f 20 0 0 1 6.6 0.75 6g 20 1 0 0 6.6 0.84 6h 20 1 0 1 6.5 0.86

(35) As shown in Table 2 and FIGS. 2a and 2b the stabilizing effect of the 1 mM ZnCl.sub.2 on the gel is seen also when a caine is present in the formulation.

Example 7Release of Zn from a Gel Containing ZnCl.SUB.2

(36) The zinc release from the gels was measured by the USP-paddle apparatus using special gel containers in which the gel was placed. A gel containing 1 mM ZnCl.sub.2 was manufactured according to Example 1. The gel was filled into 7 gel containers with a fixed volume of 1 ml. The geometry of the filled gel was a cylinder 1 cm in diameter and 3 mm deep. The gels were covered by a mesh-size plastic net (PA80 m=200 mesh, AB Derma) and a coarse stainless steel net, care was taken to assure that no air was trapped between the gel and the plastic net.

(37) Each gel container was immersed in 600 ml thermostated release medium, stirred at 30 rpm and maintained at 37 C. using a Distek Evolution 6100 (North Brunswick, N.J.). At predetermined time points, gel containers were removed and the Zn content in the container was determined using ICP.

(38) The results are shown in FIG. 6. The release of Zn from the gel is very slow. In this experimental setup, a small molecule freely diffusing in a similar gel without any interactions between the gel and the molecule is normally fully released after 6 hours.

(39) After 8 days, less than 50% of the Zn in the gel has been released. This gives a release of 0.06 mol/day. These results indicate that the gel could be safely used in vivo to effect a slow release of Zn to a subject.

Example 8Release of Zn from Gels Containing Different Zinc Salts

(40) Gels prepared according to example 4 containing zinc chloride (1 mM), zinc gluconate (1 mM) and zinc citrate (1 mM) respectively were analyzed as described in Example 7. Table 3 shows the relative release rate for the different zinc salts.

(41) TABLE-US-00003 TABLE 3 Time for 25% release zinc for the different salts % dissociation of salt (free Salt time for 25% released Zn.sup.2+, reference M) zinc chloride ~75 hours 100 zinc gluconate ~24 hours 30 zinc citrate ~3 hours 0

(42) A larger fraction of dissociated zinc (free Zn.sup.2+) results in a slower release, probably as a result of the interaction between Zn and the hyaluronan in the gel. The zinc that is complexed with its counterion will not interact with the gel, and the larger the fraction of zinc that is complexed with its counterion, the more rapid is the release.