Method of Preparing Single-Phase Modified Sodium Hyaluronate Gel

Abstract

A method of preparing a single-phase modified sodium hyaluronate gel, comprising preparing a sodium hyaluronate solution with a mass fraction between 5% to 15% in an alkaline condition at a pH value between 11 to 14, wherein the sodium hyaluronate has a molecular weight between 1.5 million to 4 million Daltons; adding a cross-linking agent to a solution of step (1), wherein the cross-linking agent and the sodium hyaluronate has a molar ratio between 9% to 15%; rapidly mixing for 20 to 40 minutes to form a gel; allowing to stand after subjecting to a water bath at constant temperature; dialyzing with a dialysis membrane to remove un-reacted cross-linking agent and hydroxide ion; homogenizing; and adding a mobile phase and mixing sufficiently to obtain a high viscosity stabilized single-phase modified sodium hyaluronate gel.

Claims

1. A method of preparing a single-phase modified sodium hyaluronate gel, comprising the following steps: (1) preparing a sodium hyaluronate solution with a mass fraction between 5% to 15% in an alkaline condition at a pH value between 11 to 14, wherein the sodium hyaluronate has a molecular weight between 1.5 million to 4 million Daltons; (2) adding a cross-linking agent to a solution of step (1), wherein the cross-linking agent and the sodium hyaluronate has a molar ratio between 9% to 15%; rapidly mixing for 20 to 40 minutes to form a gel; (3) allowing the gel of step (2) to stand after subjecting the gel of step (2) to a water bath at constant temperature; (4) dialyzing the gel of step (3) with a dialysis membrane to remove un-reacted cross-linking agent and hydroxide ion; (5) homogenizing; and (6) adding a mobile phase and mixing sufficiently to obtain a high viscosity stabilized single-phase modified sodium hyaluronate gel.

2. The method according to claim 1, wherein in step (1), the sodium hyaluronate is a sodium hyaluronate produced by bacterial fermentation.

3. The method according to claim 1, wherein in step (1), the alkaline condition is selected from potassium hydroxide or sodium hydroxide, wherein the pH value is between 13 to 14.

4. The method according to claim 1, wherein in step (2), the cross-linking agent is selected from epoxide, halohydrin or divinyl sulfone.

5. The method according to claim 4, wherein the epoxide is a compound selected from the group consisting of 1,4-butanediol diglycidyl ether, 1-(2, 3-epoxypropyl) 2,3-epoxycyclohexane and 1,2-ethanediol diglycidyl ether.

6. The method according to claim 1, wherein in step (3), the constant temperature is between 27 to 60 C.

7. The method according to claim 6, wherein the constant temperature is between 30 to 50 C.

8. The method according to claim 1, wherein in step (4), the dialysis membrane has a dialysis molecular weight of 20,000 Daltons.

9. The method according to claim 1, wherein in step (4), the dialysis membrane has a dialysis molecular weight of 15,000 Daltons.

10. The method according to claim 1, wherein step (5) further comprising adding an anesthetic before the homogenizing.

11. The method according to claim 10, wherein the anesthetic is lidocaine hydrochloride; the anesthetic has a mass content between 0.1% to 0.5%.

12. The method according to claim 11, wherein the mass content is between 0.2% to 0.4%.

13. The method according to claim 1, wherein in step (6), the mobile phase is the same as a raw material of the sodium hyaluronate which has been cross-linked, and the mobile phase is prepared by bacterial fermentation; wherein the mobile phase and the raw material have a same molecular weight; wherein the mobile phase and the gel has a consistent content; wherein the mobile phase has a total mass ratio of 5% to 50%.

14. The method according to claim 13, wherein the mobile phase has a total mass ratio of 10% to 30%.

15. The method according to claim 1, comprising dissolving the sodium hyaluronate with a molecular weight of 2 million Daltons in an alkaline solution at pH 14; adding 12% of the cross-linking agent, wherein the cross-linking agent is 1,4-butanediol diglycidyl ether; rapidly mixing for 30 minutes; cross-linking at 40 C. in the water bath; dialyzing with the dialysis membrane of 1.5 million Daltons; adding 0.2% to 0.4% of lidocaine hydrochloride; adding 20% of the mobile phase; thereby obtaining the single-phase modified sodium hyaluronate gel.

16. A method of filling, replacing or isolating a biological tissue, or increasing a volume of the biological tissue, or supplementing or replacing a biological material in medical cosmetology, comprising administering an effective amount of the single-phase modified sodium hyaluronate gel prepared by the method of claim 1 to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 is a figure showing the results of a bi-phase granular gel prepared according to the existing method observed under the microscope.

[0031] FIG. 2 is a figure showing the results of a single-phase homogenous gel prepared according to the method of the present application observed under the microscope.

[0032] FIG. 3 is a graph comparing the curves of enzymatic degradation between Example 1, Example 3, and the bi-phase gel control.

DETAILED DESCRIPTION

[0033] The present application provides a biocompatible single-phase cross-linked gel, which effectively avoids the disadvantages of the bi-phase gel described in the background. Meanwhile, the biocompatible single-phase cross-linked gel has the advantages of being easy to use and having longer duration in clinical application.

[0034] Starting from researching the degree of modification of hyaluronic acid in the human body itself, the present application has adopted a non-saturated cross-linking method through the control of the preliminary parameters, and further perform homogenization, to obtain a single-phase, polymeric, high-viscosity, injectable and long-lasting biocompatible sodium hyaluronate gel. The biocompatibility and physicochemical properties of the product are greatly improved. Furthermore, in order to enhance the comfort level of experience in clinical use, anesthetic lidocaine hydrochloride is added during the production process. The entire process is easy to implement and the result is stable and reliable.

[0035] The single-phase modified sodium hyaluronate gel prepared according to the above method may fill, separate or replace a biological tissue, or increase the volume of the tissue, or supplement or replace a biological fluid. Preferably, the gel is used for filling, separating or replacing the biological tissue or increasing the volume of the tissue, e.g. as a material for therapeutic application, increasing the volume of the vocal cord, increasing the volume of the esophagus, urethral sphincter or other organs, etc., or for filling wrinkle, covering scar or enriching the lip for cosmetic purposes. Preferably, the gel constitutes a matrix including at least one dispersed active body. The gel is then used as a carrier for the active body gradually released from the injected liquid or biological tissue.

[0036] The single-phase gel is different from the conventional bi-phase gel. Observation of the microscopic structure shows that the conventional bi-phase gels are distinguished by the particle sizes as shown in FIG. 1, while the single-phase homogenized gel is shown in FIG. 2.

[0037] The bi-phase contains solid particles and a liquid phase. This leads to localized area which is not smooth during application. Thus, absorption and degradation of the bi-phase gel are different in the body. Meanwhile, the single-phase gel is a very stable colloidal phase, which is very similar to the degree of modification in hyaluronic acid present in the human body itself. The single-phase gel has a stable structure, a high affinity to the tissue, a more natural shaping effect, a significant lifting effect, is effective in improving the unevenness of the postoperative skin surface, and is soft and elastic. The high viscosity of the gel means that the gel has a strong tendency of restructuring, instead of spreading or separating. The excellent corresponding shear viscosity may effectively resist the shear force generated after the injection, thereby reducing diffusion and movement, resisting deformation caused by external force, and being more stable. Therefore, the high viscosity cohesiveness of the gel contributes to the high compatibility and long-term sustainability in vivo.

[0038] It should be specifically noted that the property of high viscosity of the single-phase gel of the present application does not solely mean that the gel has high absolute value of viscosity in specific test conditions, but it needs to be combined with its elastic properties for a comprehensive evaluation. In the industry, the phase angle is a measure of the rheological properties of the gel. tg is the ratio of viscosity to elasticity. The gel of the present application generally has a larger phase angle than the bi-phase gel, and the viscosity performance is more outstanding.

[0039] The single-phase sodium hyaluronate gel prepared according to the present application has outstanding performance, which further reduces the risk of inflammatory reaction and the appearance of granuloma. The prolonged retaining period in the body provides opportunities for longer-interval medical interventions, thereby improving the quality of life of the patients.

[0040] The modified sodium hyaluronate gel prepared according to the present application is more injectable in vivo than other gels having the same degree of cross-linking, and has longer persistence.

[0041] The following examples are given to illustrate the method and product according to the present application and to assist with the understanding of the present application by way of illustration only, but they are by no means intended to limit the scope of the present application.

Example 1

[0042] 2.02 g of sodium hyaluronate with a molecular weight of 1.60 million Daltons was weighed in a beaker and 15.05 g of 1% sodium hydroxide solution was added to fully dissolve the sodium hyaluronate. The cross-linking step was carried out in an alkaline medium and very strong ether bonds were easily formed. 60 l of divinyl sulfone was added, thoroughly mixed, and allowed to react for 4 hours at 50 C. and stand overnight to obtain the cross-linked gel.

Example 2

[0043] The gel of Example 1 was placed in a dialysis membrane bag with a dialysis molecular weight of 15,000 Daltons for dialysis, in order to remove un-reacted cross-linking agents and excess hydroxide ions. 0.3% lidocaine hydrochloride, which was filtered by 0.2 m microfiltration membrane, was then added to adjust the pH to neutral, and then homogenized. 10.06 g of mobile phase was added and thoroughly mixed to obtain a homogenous single-phase gel.

[0044] The gel was packaged in pre-filled syringe and subjected to moist heat sterilization at 121 C. for 30 minutes.

[0045] The product was observed under a Winner 99D Particle Image Analyzer, and the results are shown in FIG. 2.

Example 3

[0046] 5.03 g of sodium hyaluronate with a molecular weight of 2.3 million Daltons was weighed in a beaker and 55.2 g of a 1% sodium hydroxide solution was added to fully dissolve the sodium hyaluronate. 321 l of 1,4-butanediol diglycidyl ether was added, thoroughly mixed, and then allowed to react for 4 hours at 40 C. and stand overnight to obtain the cross-linked gel. The gel was then placed in a dialysis membrane bag for dialysis, in order to remove un-reacted cross-linking agents and excess hydroxide ions. 0.3% of lidocaine hydrochloride, which was filtered by 0.2 m microfiltration membrane, was added to adjust the pH to neutral, and then homogenized. 52.1 g of mobile phase was added and thoroughly mixed to obtain a homogenous single-phase gel.

Example 4

[0047] 10.02 g of sodium hyaluronate with a molecular weight of 1.9 million Daltons was weighed in a beaker and 160.1 g of a 1% sodium hydroxide solution was added to fully dissolve the sodium hyaluronate. 550 l of 1,4-butanediol diglycidyl ether was added, thoroughly mixed, and then allowed to react for 4 hours at 40 C. and stand overnight to obtain the cross-linked gel. The gel was then placed in phosphate buffered saline for dialysis, in order to remove un-reacted cross-linking agents and excess hydroxide ions. 0.3% of lidocaine hydrochloride, which was filtered by 0.2 m microfiltration membrane, was added to adjust the pH to neutral, and then homogenized. 105.0 g of mobile phase was added and thoroughly mixed to obtain a homogenous single-phase gel.

[0048] The gels of the above examples were tested for elasticity and viscosity using a rotational rheometer. The test method was dynamic frequency scanning at a test temperature of 25 C. and a frequency change range between 0.05 to 10 Hz. The values of elastic modulus (G) and viscous modulus (G) at 1 Hz were compared.

[0049] For each set of samples in the examples, 5 parallel samples were taken and installed in the injection needles. The syringe was pushed to remove the small amount of air from the front end until a gel droplet appeared at the needle tip. The syringe was placed on a tension machine and the plunger was pushed at a speed of 20 mm/min. The pressure was recorded and the average value was calculated.

[0050] A comparison between the test results of the single-phase gel according to the present application and a bi-phase gel commercially available on the current market is shown in the following table:

TABLE-US-00001 Single-phase Single-phase Gel Gel Single-phase Gel in Example 1 in Example 3 in Example 4 Bi-phase Gel Elasticity 193 186 190 693 (Pa, 1 Hz) Viscosity 50 48 55 130 (Pa, 1 Hz) Pushing 10 9 10 19 Force (N)

[0051] The in vitro resistance to enzymatic degradation of the gel may indirectly reflect its lasting time in the human body. In the present application, the gels of Examples 1 and 3 and the commercially available bi-phase gel control were subjected to an enzymatic degradation test at a hyaluronidase concentration of 7 U/ml. The results are shown in the following table. The curves of enzymatic degradation are shown in FIG. 3.

TABLE-US-00002 Enzymatic Degradation Rate (%) Hour (h) 1 3 5 7 9 Example 1 29 49 72 80 89 Example 3 38 56 70 86 92 Control 64 88 93 100 100