CROSSLINKED HYALURONIC ACID AND USE THEREOF

Abstract

Provided are a cross-linked hyaluronic acid and use thereof.

Claims

1. A cross-linked hyaluronic acid having a critical strain of 0.1% to 8% and a tack force of 3 N to 15 N.

2. The cross-linked hyaluronic acid of claim 1, wherein an elastic modulus (G′) of the cross-linked hyaluronic acid is 400 Pa to 2,000 Pa.

3. The cross-linked hyaluronic acid of claim 1, wherein a viscous modulus (G″) of the cross-linked hyaluronic acid is 100 Pa to 600 Pa.

4. The cross-linked hyaluronic acid of claim 1, wherein compression of the cross-linked hyaluronic acid is 15 N.Math.s to 100 N.Math.s.

5. The cross-linked hyaluronic acid of claim 1, wherein an injection force of the cross-linked hyaluronic acid is 1 N to 50 N.

6. The cross-linked hyaluronic acid of claim 1, wherein the cross-linked hyaluronic acid is cross-linked by a cross-linking agent having a difunctional epoxy group.

7. The cross-linked hyaluronic acid of claim 6, wherein the cross-linking agent having a difunctional epoxy group is at least one selected from the group consisting of 1,4-butanediol diglycidyl ether, poly(ethylene glycol) diglycidyl ether, poly(propylene glycol) diglycidyl ether, poly(tetramethylene glycol) diglycidyl ether, polyglycerol polyglycidyl ether, glycerol diglycidyl ether, triethylene diglycidyl ether, trimethylolpropane triglycidyl ether, ethylene diglycidyl ether, neopentyl glycol diglycidyl ether, and 1,6-hexanediol diglycidyl ether.

8. A filler composition comprising the cross-linked hyaluronic acid of claim 1.

9. The filler composition of claim 8, wherein a concentration of the cross-linked hyaluronic acid is 10 mg/mL to 30 mg/mL.

10. The filler composition of claim 8, not comprising additional non-crosslinked hyaluronic acid.

11. The filler composition of claim 8, further comprising a local anesthetic.

12. The filler composition of claim 8, wherein the filler composition is filled in a syringe.

13. The filler composition of a claim 8, wherein the composition is for one or more uses selected from the group consisting of facial plastic surgery, wrinkle improvement, facial contouring, breast plastic surgery, breast augmentation, genital enlargement, glans enlargement, urinary incontinence treatment, and arthritis treatment.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0053] FIG. 1 is a diagram showing measured maximum volumes of the injection site compared to the initial volume after injecting cross-linked hyaluronic acid into hairless mice.

[0054] FIG. 2 is a diagram showing volumes of the injected hyaluronic acid gels according to the time elapsed after cross-linked hyaluronic acid samples are injected into hairless mice.

BEST MODE

[0055] Hereinafter, the present disclosure will be described in more detail through embodiments. However, these embodiments are intended to illustrate the present disclosure, and the scope of the present disclosure is not limited to these embodiments.

[0056] Materials and Methods

[0057] The following materials and methods were used in the following embodiments.

[0058] (1) Material: Cross-Linked Hyaluronic Acid Product

[0059] As control groups, commercially available cross-linked hyaluronic acid products as follows were used. As monophasic hyaluronic acids, Allergan's Juvederm Voluma Lidocaine™, Hanmi Pharmaceutical's Gugufill™, and Medytox's Neuram is Volume Lidocaine™ were purchased. In addition, as a biphasic hyaluronic acid, Galderma's Restylane Lyft Lidocaine™ was purchased.

[0060] (2) Method

[0061] (2.1) Critical Strain Measurement Method

[0062] Critical strain was measured by a strain-sweep test. Specifically, a DHR-2 rheometer instrument (TA instruments) was operated. For the strain-sweep test, temperature of the device was set to 25° C., and a geometry of a diameter of 25 mm was equipped to perform calibration. An appropriate amount of a sample was loaded in the center between an upper Peltier plate and a lower geometry. The sample was overloaded with a sufficient amount so as not to be insufficient, and an appropriate amount was loaded so that it does not stain the side and top surfaces of the geometry, even if the residue outside the lower area of the geometry was to be trimmed. After lowering the geometry to a set distance, and checking whether the sample is well filled under the geometry, the remaining sample outside the geometry was trimmed and removed. Then, when a strain of 0.1% to 1,000% was applied to the sample by using the geometry, the value of the elastic modulus measured at each strain was measured. A semi-log graph was drawn with the value obtained by taking log of the strain % as the x-axis and the elastic modulus as the y-axis. When a graph differentiated from this graph value was drawn, the lowest strain % value among the strain % values having the minimum y value was defined as the critical strain.

[0063] (2.2) Tack Force Measurement Method

[0064] Tack force was measured as follows. A DHR-2 rheometer instrument (TA instruments) was operated. For the tack force test, temperature of the device was set to 25° C., and a geometry of a diameter of 40 mm was equipped to perform calibration. An appropriate amount of the sample was loaded in the center between an upper Peltier plate and a lower geometry. The sample was overloaded with a sufficient amount so as not to be insufficient, and an appropriate amount was loaded so that it does not stain the side and top surfaces of the geometry. After lowering the geometry to a set distance, and checking whether the sample is well filled under the geometry, the remaining sample outside the geometry was trimmed and removed. Thereafter, the force applied to the geometry was measured when the geometry gap was set to a height of 1,000 μm and the geometry was pulled from the lower Peltier plate at a constant speed of 100.0 μm/s in the vertical axis direction for 180 seconds. Due to the tack force of the sample between the geometry and the Peltier plate, the greatest force is measured at the moment the initial sample is separated, and the corresponding force was defined as the tack force.

[0065] (2.3) Viscoelasticity Measurement Method

[0066] A DHR-2 rheometer instrument (TA instruments) was operated. For the viscoelasticity test, temperature of the device was set to 25° C., and a geometry of a diameter of 40 mm was equipped to perform calibration. An appropriate amount of the sample was loaded in the center between an upper Peltier plate and a lower geometry. The sample was overloaded with a sufficient amount so as not to be insufficient, and an appropriate amount was loaded so that it does not stain the side and top surfaces of the geometry. After lowering the geometry to a set distance, and checking whether the sample is filled under the geometry, the remaining sample outside the geometry was trimmed and removed. Then, a shear strain was periodically applied to the sample according to a specific frequency with a constant strain of the geometry, and among the modulus values obtained, the storage modulus and loss modulus values at a frequency of 0.1 Hz were defined as elasticity and viscosity, respectively.

[0067] (2.2) Injection Force Measurement Method

[0068] Injection force was measured by using Mecmesin's Multitest 2.5-i Universal Testing Machine. As a load cell for measuring the force applied to the device, an appropriate load cell with a higher allowable value than the measurement range of injection force was used. A syringe containing the sample was placed under the load cell of the instrument, and a 27G ½″ needle was attached to the syringe. A flat-tipped push bar was placed with the syringe so that a constant force was applied when the load cell applied force. After adjusting the distance so that the load cell part was placed just before it would touch the tip of the push bar, the force applied was measured by pressing the push bar by using the load cell at a speed of 12 mm/min. In case of the initial part and the end part of the measured value, the pressure value generated when the sample flows into the needle was lower than the pressure applied when the syringe was injected. In addition, when the injection of the infusion is almost completed, there may be a case in which a force is continuously applied even when there is no sample, and in this case, the applied force is a force not related to the injection force of the sample. Therefore, the values corresponding to parts where the sample flowed into the needle, and where the injection was almost completed were excluded, and the average value of the injection force measured in the middle part of the syringe filling solution was defined as the injection force, excluding the measured values of the initial injection force and end injection force.

[0069] (2.2) Compression Measurement Method

[0070] A DHR-2 rheometer instrument (TA instruments) was operated. For the compression test, temperature of the device was set to 25° C., and a geometry of a diameter of 25 mm was equipped to perform calibration. 1 mL of the sample was loaded in the center between an upper Peltier plate and a lower geometry. After the geometry was lowered to a set distance, the geometry was rotated at a slow speed so that the sample could come to the center with respect to the geometry. Thereafter, while lowering the geometry from 2,500 μm to 900 μm at a constant speed of 13.33 μm/s, the force applied to the geometry was measured. When a graph was drawn with the force measured from the beginning to the end of the test as the Y axis and the movement time as the X axis, compression was defined as a value corresponding to the value of the area under the graph, which is an integral value of the graph.

[0071] (2.6) Particle Size Measurement Method

[0072] Particle size measurement was performed by using Microtrac's Particle Size Analyzer S3500. Particle size measurement was performed by adding a sample to a solvent, and was performed by using laser diffraction analysis method. The solvent was measured using distilled water. After thoroughly washing the sample inlet with distilled water before performing particle size measurement, 1.33 and 1.37 were input as refractive index of the distilled water and the refractive index of the sample, respectively, and the sample type was set to be transparent and irregularly shaped particles. Before measurement, after filling the sample inlet of the device with distilled water, the sample was put into a tube, etc., and diluted with excess distilled water, and then was sufficiently dispersed by using a Vortex, etc., and then the sample was put into the device for measurement. Among the measurement results, the average particle size (D50) was used as data.

Example: Preparation of Cross-Linked Hyaluronic Acid and Identification of Physical Properties

[0073] 1. Preparation of Cross-Linked Hyaluronic Acid

[0074] A cross-linked hyaluronic acid having a critical strain of 0.1% to 8% and a tack force of 3 N to 15 N (hereinafter referred to as “experimental group”) was prepared as follows.

[0075] Experimental Group 1:

[0076] First, a 1% (w/w) NaOH solution was prepared. 5 g of sodium hyaluronate (IV 1.7 to 1.9) was mixed with the prepared 1% NaOH solution to 14.50% (w/w), and then stirred to sufficiently dissolve. Here, IV represents intrinsic viscosity. 0.528 g of butanediol diglycidyl ether (BDDE) (Sigma-Aldrich) was added to this solution and further stirred to mix well, and then the solution was taken out and a cross-linking reaction was proceeded at 25° C. for 18 hours to prepare a cross-linked hyaluronic acid gel. Next, the obtained cross-linked gel was placed in a dialysis membrane and sealed, and dialysis was performed using 1 mol/kg NaCl aqueous solution and 1×PBS aqueous solution as dialysis solutions. After dialysis is completed, the weight of sodium hyaluronate, in which cross-linking and dialysis has been completed, was calculated by considering the loss rate, a content calibration was proceeded using 1×PBS so that the concentration of the total sodium hyaluronate would be 2% (w/w), at this time, the content calibration was proceeded so that a content of lidocaine hydrochloride hydrate would be 0.3% (w/w). As a result, a PBS solution containing 2% (w/w) of cross-linked hyaluronic acid and 0.3% of lidocaine was prepared. After filling 1 ml of the prepared cross-linked hyaluronic acid-containing solution into a glass syringe, high-temperature steam sterilization was performed.

[0077] Experimental Group 2:

[0078] First, a 1% (w/w) NaOH solution was prepared. 5 g of sodium hyaluronate (IV 1.7 to 1.9) was mixed with the prepared 1% NaOH solution to 14.0% (w/w), and then stirred to dissolve for 6 hours. 0.428 g of BDDE (Sigma-Aldrich) was added to this solution, and the mixture was further stirred for 10 minutes to mix well, and then the solution was taken out and left at 25° C. for 18 hours to proceed a cross-linking reaction to prepare a cross-linked hyaluronic acid gel. Next, after dialysis and content calibration were performed in the same manner as with Experimental Group 1, a glass syringe was filled.

[0079] Experimental Group 3:

[0080] First, a 1% (w/w) NaOH solution was prepared. 5 g of sodium hyaluronate (IV 1.7 to 1.9) was mixed with the prepared 1% NaOH solution to 15.0% (w/w), and then stirred to dissolve for 6 hours. 0.251 g of BDDE (Sigma-Aldrich) was added to this solution, and the mixture was further stirred for 10 minutes to mix well, and then the solution was taken out and left at 40° C. for 18 hours to proceed a cross-linking reaction to prepare a cross-linked hyaluronic acid gel. Next, after dialysis and content calibration were performed in the same manner as with Experimental Group 1, a glass syringe was filled.

[0081] Comparison Groups 1 to 3

[0082] Cross-linked hyaluronic acids of Comparison Groups 1 to 3 were prepared in the same manner as with Experimental Group 1, except that the hyaluronic acid dissolution concentration, amount of added BDDE, cross-linking temperature, and cross-linking time were as described in Table 1 below. Table 1 shows the materials and conditions used for preparing the cross-linked hyaluronic acids of Experimental Groups 1 to 3 and Comparison Groups 1 to 3. In case of the BDDE concentration, it represents % of the number of moles of the added BDDE to the number of moles of the added hyaluronic acid (HA) monomers.

TABLE-US-00001 TABLE 1 HA (%, BDDE Temperature Hour Name w/w) (mol %) (° C.) (h) Experimental 14.5 21 25 18 Group 1 Experimental 14.0 17 25 18 Group 2 Experimental 15.0 10 40 18 Group 3 Comparison 12.0 22 25 18 Group 1 Comparison 11.0 21 25 18 Group 2 Comparison 11.0 16 25 18 Group 3

[0083] Sensory evaluation was performed by using a sense of touch on the samples prepared in the experimental groups and the comparison groups. That is, the phases of the prepared material were identified with a sense of touch, and it was confirmed that, in case of a monophase, the particles were not palpable, and in case of a biphase, the particles were touched like grains of sand. In the sensory evaluation, the cross-linked hyaluronic acid gels of Experimental Groups 1 to 3 had a gel shape that was neither monophasic nor biphasic. On the other hand, the cross-linked hyaluronic acid gels of Comparison Groups 1 to 3 were confirmed through the sensory evaluation as having a monophase.

[0084] Comparison Groups 4 to 7:

[0085] As Comparison Groups 4 to 6, Allergan's Juvederm Voluma Lidocaine™ (Comparison Group 4), Hanmi Pharmaceutical's Gugufill™ (Comparison Group 5), and Medytox's Neuramis Volume Lidocaine™ (Comparison Group 6), which are monophasic cross-linked hyaluronic acids, were respectively used. In addition, as Comparison Group 7, Galderma's Restylane Lyft Lidocaine™ (Comparison Group 7), which is a biphasic cross-linked hyaluronic acid, was used.

[0086] 2. Analysis of Physical Properties of Prepared Cross-Linked Hyaluronic Acid

[0087] The physical properties of the cross-linked hyaluronic acid gel prepared in Section 1 were analyzed. The measured physical properties of the prepared cross-linked hyaluronic acids of Experimental Groups 1 to 3, and Comparison Groups 1 to 7 are as shown in Table 2.

TABLE-US-00002 TABLE 2 Degree Particle of cross- Tack Injection Critical G′ G″ size linking Compression force force strain Name (Pa) (Pa) (μm) (%) (Ns) (N) (N) (%) Experimental 1629 450.3 358.3 3.20 65.20 7.46 16.01 3.36 Group 1 Experimental 1244.0 336.1 303.4 2.62 71.74 10.65 30.28 5.81 Group 2 Experimental 609.4 164.3 241.6 2.73 40.68 6.73 11.95 6.98 Group 3 Comparison 871.8 210.0 269.3 3.38 55.38 11.72 31.90 60.69 Group 1 Comparison 614.5 135.1 304.9 3.47 54.72 11.02 24.19 59.34 Group 2 Comparison 391.6 76.7 366.2 2.74 46.00 8.45 18.57 103.02 Group 3 Comparison 227.4 30.3 660.7 2.08 23.79 4.50  8 (27G) 54.62 Group 4 Comparison 389.0 57.0 N/A 5.00 48.71 7.07 32.59 85.27 Group 5 Comparison 305 50 410 2.75 76.30 6.94 17   112.01 Group 6 Comparison 742.7 203.3 809.0 0.16 22.39 3.22 19 (29G) 8.19 Group 7

[0088] In Table 2, the injection force indicates the injection force when a 13 mm needle of 27 G is used, unless otherwise specified.

[0089] 3. Measurement of Lifting and Volumizing Effects when the Prepared Cross-Linked Hyaluronic Acid is Applied to Animals

[0090] Cross-linked hyaluronic acids of Experimental Groups 2 and 3 and Comparison Groups 1, 2, 3, 4, 5, 6 and 7 and saline as a negative control group were injected into animals, and the height, maximum length, and volume of the injection site were measured. For animals, a total of 84 hairless 6-week-old female (18.0 g to 24.0 g, average 22.3 g) mice (SKH1-hr) were used. Eight mice were used per sample, and four were used for saline, which is a control group.

[0091] Specifically, 0.1 ml of each of the cross-linked hyaluronic acid samples was administered subcutaneously to one dorsal region site per hairless mouse using a glass syringe. After injection, the height, maximμm length and volume of the injection site were measured by using a Primose device (Primos 5.8E (Canfield Scientific Inc, NJ, USA)) according to designated days. In addition, the injection site was visually observed and photographs were taken. As a result of visual observation, Comparison Groups 1, 2, 3, 4, 5, and 6 showed high swelling.

[0092] FIG. 1 is a diagram showing measured maximum volumes of the injection site compared to the initial volume after injecting cross-linked hyaluronic acid into hairless mice. As shown in FIG. 1, it was confirmed that the volume increase of Experimental Groups 2, 3 and Comparison Group 7 was smaller than that of Comparison Groups 1, 2, 3, 4, and 5. This indicates that the cross-linked hyaluronic acid gels of Experimental Groups 2 and 3 caused little swelling at the injection site.

[0093] FIG. 2 is an observation of the volume change up to 180 days after intradermally injecting cross-linked hyaluronic acid gels. As shown in FIG. 2, in case of monophases, the volume of cross-linked hyaluronic acids of Comparison Groups 2 and 5 increased by 168% and 143%, respectively, compared to the initial volume at 4 weeks after injection. As shown in FIG. 2, at 180 days after injection, the cross-linked hyaluronic acids of Experimental Groups 2 and 3 were larger or equivalent in volume to other comparison groups and the control group. This indicates that the cross-linked hyaluronic acids of Experimental Groups 2 and 3 showed relatively small loss during 180 days. That is, the cross-linked hyaluronic acids of Experimental Groups 2 and 3 had superior in vivo durability compared to other comparison groups and the control group.

[0094] In conclusion, it was confirmed that Experimental Groups 2 and 3 exhibited smaller swelling after injection compared to Comparison Groups 1, 2, 3, 4, 5 and 6, and thus, the volume change after injection was small, and it was confirmed that the volumes of Experimental Groups 2 and 3 up to 180 days after injection were equivalent to that of Comparison Groups 1, 2, 3, 4, 5 and 6, and was superior to that of Comparison Group 7, and thus, in vivo durability was confirmed to be excellent.

[0095] 4. Irritation Measurement when Prepared Cross-Linked Hyaluronic Acid Gel is Applied to Animals: Irritation Test

[0096] Cross-linked hyaluronic acids of Experimental Groups 2 and 3 and Comparison Groups 1, 2, 3, 4, 5, 6 and 7 and saline as a negative control group were injected into animals, and the degree of irritation induced at the injection site was identified by measuring erythema and edema. The experiment was performed according to the guideline described in ISO 109993-10 “Tests for irritation and skin sensitization” by entrusting to Knotus Co., Ltd. (Korea). As animals, a total of 12 male New Zealand white rabbits were selected according to ISO 10993-10. Three rabbits were used per sample. Specifically, 200 μl of each of the cross-linked hyaluronic acid samples per rabbit was subcutaneously administered to 5 dorsal region sites and the injection sites were observed for 45 days. After injection, erythema and edema were evaluated and scored according to the guideline. The differences between the score for each sample and the evaluation score for the control group using saline were cumulatively added and averages for each site were obtained. As a result, no erythema was observed in any animal to which the samples were administered. Table 3 is a table showing the edema scores observed on days 3 and 45 after administering cross-linked hyaluronic acid samples to rabbits. There was no statistically significant difference in the edema scores among the administered groups of animals to which samples were administered.

TABLE-US-00003 TABLE 3 Sample Edema score at day 3 Experimental Group 2 0.6 Experimental Group 3 0.9 Comparison Group 1 1.3 Comparison Group 2 1.3 Comparison Group 3 1.8 Comparison Group 4 1.1 Comparison Group 5 0.8 Comparison Group 6 0.9 Comparison Group 7 1.1 Saline 0.0

[0097] According to the ISO 109993-10 guideline, as a result of measuring formation of erythema and edema up to 3 days, the samples of Experimental Groups 2 and 3 and Comparison Groups 5 and 6 met the criteria of the guideline. Therefore, from the edema formation results on the 3rd day of injection, the samples of Experimental Groups 2 and 3 were confirmed to show equal or superior effects compared to the samples of other comparison groups.

[0098] In the above Example, the cross-linked hyaluronic acid gels of Experimental Groups 2 and 3 were confirmed to have superior properties compared to the cross-linked hyaluronic acid gels of other comparison groups in terms of volume increase resistance and low-irritation when injected into animals.