Table 1. Cohesiveness results of hyaluronic acid-containing fillers known in the art. (Among the products produced with the same technology, products with high cohesiveness score values are referred to as soft gels, while products with low cohesiveness scores are generally referred to as hard gels).

The primary object of the inventors of the present invention is to provide a filler production method in which all gel types can be obtained at the highest cohesiveness score values regardless of the product to be produced being soft or hard gel.

Another object of the invention is to provide dermal filler with a reduced risk of nodules due to material accumulation.

Another object of the invention is to produce dermal filler with high intradermal integration properties.

Another object of the invention is to produce dermal filler with high lifting capacity.

BRIEF DESCRIPTION OF THE DRAWING Figure 1 shows the FT-IR spectrum view of samples 2, 5 and 8 of dermal filler specimens, native HA and hydrogels.

DETAILED DESCRIPTION OF THE INVENTION

In this detailed description, the subject of the invention relates to a method for a dermal filler in which the highest cohesiveness score values can be obtained; and is described by way of non-limiting examples only for a better understanding of the subject matter.

Dermal fillers used in the medical aesthetics field in the art are in the form of soft or hard gels depending on the upper, medium or deep dermis to which they will be applied. The most important factor that determines the soft or hard gel form mentioned is the degree of cross-linking of the gel and the viscosity values accordingly. It is also critical to obtain the highest cohesiveness score values in accordance with the viscosity values of dermal fillers in order to obtain high-efficiency products and to perform high- efficiency dermal filler operations depending on the area they will be applied.

In the invention, "cohesiveness" refers to tightness of the internal molecular structure of the dermal filler and its ability to stay together due to the strong attraction between said molecules. Cohesiveness ensures the structural integrity of the gel, helps provide natural tissue support, and reduces post-injection surface irregularities. In this invention, in the cohesiveness score values as is known in the art, the full cohesiveness is 5-point score value, while the poor cohesiveness value is 1 point score value.

In the invention, "rheology" refers to a branch of science that examines the fluidity and deformation properties of materials.

In the main embodiment of the invention, the dermal filler is in the form of a hydrogel. It is the hyaluronic acid component that determines the physical and chemical properties of said hydrogel. For this reason, the cross-linking technology and rheological values of the hyaluronic acid in the hydrogel are of critical importance. In the main embodiment of the invention, the dermal filler contains hyaluronic acid and at least one cross-linker. The amounts of hyaluronic acid and at least one cross-linker in said dermal filler are determined according to the rheological properties of said dermal filler. The rheological properties of said dermal filler include G', G", q* and tan 5 values.

In the invention, a method is provided for producing hyaluronic acid-containing hydrogel with high cohesiveness score values, even if it has variable G', G", q* and tan 5 values depending on the upper, middle or deep dermis. The process steps of the mentioned production method are characterized in the following lines.

- Preparation of the solution containing sodium hyaluronate

In this invention, sodium hyaluronate (abbreviated as NaHA) is preferably dissolved in a solution. Here, at least one basic component is present in the solution. The pH value of the solution to be obtained in this process step should be between 10-13. At least one of the components such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, triethyl amine, pyridine is used as said basic component. In a preferred embodiment sodium hydroxide (abbreviated as NaOH) is present as a basic component.

In a preferred embodiment sodium hydroxide with a value of 0.25 M is used as a basic component.

In a preferred embodiment, NaHA is completely immersed in a NaOH solution at low temperatures. Thus, by performing these processes at low temperatures, it is ensured that the molecular chain is protected from deterioration and the cross-linking reaction efficiency is increased. In a preferred embodiment, the preparation temperature of the solution is at a temperature value between 2 to 8 °C.

In a possible embodiment of the invention, the amount of said sodium hyaluronate compound is at a value in the range of 5% to 15% by weight.

In a preferred embodiment, the prepared solution is held for at least 1 hour. In this way, sodium hyaluronate becomes fully hydrated in the basic solution. Obtaining the homogeneous first mixture

In a possible embodiment of the invention, mixing processes are performed in order to completely homogenize the mixture obtained after being held for at least 1 hour. Here, the mixing process is performed by means of any mixer.

The preferred mixing process is performed under vacuum. Said vacuum pressure value is at least 1.0 bar. By performing the mixing process under vacuum, air removal processes can be performed during mixing. Thus, bubble formation is prevented.

In the main embodiment of the invention, as a result of subjecting the mixture to the mixing process by means of a mixer under vacuum after being held for at least 1 hour, the sodium hyaluronate chains become more linear than a complex intertwined structure, and therefore its complete dissolution is achieved. In this way, the first homogeneous mixture is obtained.

- Obtaining the homogeneous second mixture

In this invention, the cross-linker is added to the first mixture obtained. Here, at least one of a group of materials such as 1 ,4-butanediol diglycidyl ether, divinyl sulfone, glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) is used as the cross-linker. In this process step, 1 ,4-butanediol diglycidyl ether (abbreviated as BDDE) is preferably present as a cross-linker.

In this invention, the ratio of the cross-linker to hyaluronic acid by weight is important for obtaining hyaluronic acid-containing dermal fillers at the targeted cohesiveness score values.

In a possible embodiment of the invention, said cross-linker usage values are optimized according to the sodium hyaluronate ratio and G' and q* values. Accordingly, wherein the ratio of cross-linker to hyaluronic acid in said second mixture is as follows; - in order to obtain hydrogels with a G' value at a value between 20 to 150 Pa and a q* value between 3 to 45, m_Cross-iinker/mhyaiuronic acid is at a value between 0.02 to 0.13,

- in order to obtain hydrogels with a G' value at a value between 150 to 400 Pa and a q* value between 25 to 80, m_Cross-iinker/mhyaiuronic acid is at a value between 0.04 to 0.20,

- in order to obtain hydrogels with a G' value at a value between 400 to 1200 Pa and a q* value between 50 to 250, m_Cross-iinker/mhyaiuronic acid is at a value between 0.08 to 0.35.

In a preferred embodiment, the temperature at which the cross-linking reaction takes place is a temperature value between 35 °C to 50 °C. As is known in the art, temperature is one of the parameters that catalyze the cross-linking reaction. However, the temperature values provided in the present invention are critical values at which the optimum conditions are determined for catalyzing the cross-linking reactions and also the non-degradation of sodium hyaluronate.

In a preferred embodiment, the mixing process of the prepared solution is performed. In this process step, the mixing process takes place for at least 1 minute. In this way, the added cross-linker and the NaHA mixture are homogeneously dispersed and the cross-linking reaction is initiated.

In a preferred embodiment, the mixing process of the prepared solution is performed under vacuum. Said vacuum pressure values are at a value of at least 1 .0 bar. Thus, it is ensured that the cross-linking substances are properly mixed in the hyaluronic acid solution together with the bubbles formed during mixing and the risk of preventing their dispersion is removed.

In a preferred embodiment, a cross-linking reaction is initiated in the prepared solution. In this process step, the reaction process takes place for 150 to 200 minutes. In this way, with the completion of said reaction, a homogeneous second mixture is obtained.

Removal of bubbles formed in the second mixture In a preferred embodiment, the mixing process for the mixture prepared where crosslinking processes have taken place is performed under vacuum. Said vacuum pressure values are at a value of at least 1.0 bar. Thus, it is ensured that the cross-linking substances are properly mixed in the hyaluronic acid solution together with the air bubbles formed during mixing and the risk of preventing their dispersion is eliminated.

- Cutting and neutralizing the obtained parts,

In this process step, the cutting process of the component obtained as a result of cross-linking with the second mixture is performed. The cutting process is performed such that the components are at least at a value of 2.5±0.3 cm.

In a preferred embodiment, the resulting parts are added to a buffer solution. In a preferred embodiment, phosphate buffer solution is present as a buffer solution.

In a possible embodiment of the invention, the neutralization process of the obtained parts is performed. An acid solution with a molarity value in the range of 0.1 -1.0 is added in order to neutralize the solution to be obtained in this process step. For said neutralization processes, at least one of the acid solution hydrochloric acid, acetic acid, phosphoric acid, carbonic acid, formic acid group must be used. In a preferred embodiment, hydrochloric acid (abbreviated as HCI) is present as the required component for neutralization processes.

In a possible embodiment of the invention, the molarity value of said HCI compound is 0.1 mol/L.

In a preferred embodiment, the holding time of the gel particles in the buffer solution continues until the swelling amount of the hydrogels reaches between 2 to 10 times of value. In this way, it is ensured that the parts are neutralized by swelling.

- Dialysis of the obtained hydrogels

In this invention, the purification process of the particles obtained is performed. Said purification process is performed by dialysis. Here, with the dialysis processes, unreacted cross-linkers, impurities and other undesirable components are effectively removed from the hydrogel. In a preferred embodiment, the dialysis process is performed in PBS using dialysis membranes. Thus, it is ensured that unreacted cross-linking agents and their byproducts are removed from a hydrogel. In this way, the safety and purity of the hydrogel product is ensured rather than directly affecting its adhesion.

In a preferred embodiment, lidocaine hydrochloride is added to the hydrogels obtained after performing dialysis processes. Said lidocaine hydrochloride is present in the mixture at a value between 0.1% to 0.8% by weight. The lidocaine hydrochloride mentioned is present in the products to act as a pain reliever.

In a preferred embodiment, the hydrogels are subjected to an autoclave process. The autoclave process takes place in syringes. In order for this procedure to take place, the syringes are at least 1 mL in volume.

In a preferred embodiment, the autoclave temperature of the hydrogels is at a temperature value between 115 °C to 125 °C.

In order to demonstrate this effect, the inventors have created Examples 1 -9 and investigated arrangements with various cross-linker/hyaluronic acid ratio values. In Table 2, the cross-linking/hyaluronic acid ratios by weight used in the production process steps characterized in the invention are given. Examples corresponding to these ratios are also indicated in Table 2.

Table 2. Cross-linker ratios present in the samples

The samples obtained with Example 1-3 behave like soft gels and have a lower elastic modulus than other gels. Since the elastic modulus is proportional to the number of cross-linking points, this result suggests that soft gels have fewer cross-linking points between molecules.

The samples obtained with Example 4-6 behave like medium hard gels and have a lower elastic modulus than hard gels and higher than soft gels. This behavior happens between soft gels and hard gels.

The samples in Example 7-9 behave as hard gels. In fact, it can be characterized by an elastic response that is almost constant with frequency and has a loss factor of about 0.1. Due to the presence of permanent chemical cross-links between molecules, under conditions of small deformation, these strong gels exhibit the typical behavior of viscoelastic solids, and elastic deformation seems to be the only way to compensate the stress. These gels have low tan 5 values, high G', high viscosity and consequently the highest cross-link density and thus the hardest internal structure.

As shown in Table 3, the rheological properties of HA hydrogels vary greatly. The rheology test has shown that dermal fillers exhibit gel behaviors of different hardness.

With regard to the HA hydrogels in this invention, example 1 -3, which is injected into the upper dermis and is an ideal filler for superficial wrinkles, is characterized by the lowest G' value and the highest tan 5 value. Example 7-9, which is injected into the medium to deep dermis and is an ideal product for deeper lines and volume increase, is characterized by the highest G' and q* value and the lowest tan 5 value. Example 4- 6, example 1-3 and example 7-9, which are designed to be injected into the middle dermis and indicated to restore or increase lip volume, are characterized by moderate G' and q* and tan 5 values.

Table 3. Rheological results of HA Hydrogels

There is no standard methodology approved as an accepted method for measuring cohesiveness compliance. "Gavard-Sundaram (GS) Compatibility Scale" is stated as the popular method in the art.

In the main embodiment of the invention, the obtained hydrogels exhibit a very compatible behavior in terms of cohesiveness scores. Cohesiveness is evaluated almost equally between the measured scores, and no statistically significant difference is detected. The gels maintain their "integrity" throughout the experiment. It is observed that the gel structure is preserved at an optimum level: No disintegration or dispersion is recorded in the gels. Based on the observed behavior, all HA hydrogels are rated as “fully cohesive” (cohesiveness score: “5”) throughout the experiment.

The cohesiveness scores of HA hydrogels obtained from the literature and market fillers are summarized in Table 4 based on the results presented by Sundaram and Gavard Molliard and which show the diversity of HA soft tissue fillers on the market. The scores in the table known in the art are indicated with reference to the articles "Evaluation of the rheologic and physicochemical properties of a novel hyaluronic acid filler range with excellent Three-Dimensional Reticulation (XTR™) technology" [1] and "Key importance of compression properties in the biophysical characteristics of hyaluronic acid soft-tissue fillers" [2],

Table 4. Cohesiveness results of HA hydrogel samples produced in the invention and fillers given as reference. According to the FT-IR results given in Figure 1 , HA hydrogels still have hydroxyl and carboxylic acid groups. This suggests that although the hydrogels have a cross-linked structure, they still resemble the natural HA structure. These groups interact with tissues. It allows the gel to adhere to the tissue. In this case, a bond occurs between the gel and the tissue. This bond is called gel-tissue integration.

The hydrogels obtained in the invention have high cohesiveness. The hydrogels obtained according to this having higher cohesiveness than other products in the art can be explained by the effects of dissolution of HA used during the cross-linking reaction, homogenization of the cross-linker, removal of bubbles and purification stage. The protection scope of the invention is specified in the appended claims and cannot be limited to what is described for illustrative purposes in this detailed description. It is clear that a person skilled in the art can produce similar embodiments in the light of what is explained above, without deviating from the main theme of the invention.

Claims

1. A method for the producing hyaluronic acid-containing hydrogel, which can be applied to the upper, middle or deep dermis and used as a dermal filler in the technical field of dermatology, characterized in that it comprises the following process steps enabling the production of hyaluronic acid-containing hydrogel with the highest cohesiveness score values, even though it has variable rheological properties depending on the upper, middle or deep dermis:

- adding sodium hyaluronate to sodium hydroxide solution and holding it at a temperature between 2 to 8 °C for at least 1 hour,

- after being held for at least 1 hour, performing the mixing process by means of a mixer under vacuum and obtaining the homogeneous first mixture,

- adding at least one cross-linker to the obtained first mixture in the determined ratios by weight and mixing for at least 1 minute to obtain a homogeneous second mixture in which the cross-linking reaction is achieved, wherein the amount of the cross-linker in said second mixture, in order to obtain hydrogels with a G' value at a value between 20 to 150 Pa and a q* value between 3 to 45, has a m_Cross-iinker/m_hyaiuronic acid value between 0.02 to 0.13, wherein the amount of the cross-linker in said second mixture, in order to obtain hydrogels with a G' value at a value between 150 to 400 Pa and a q* value between 25 to 80, has a m_Cross-iinker/m_hyaiuronic add value between 0.04 to 0.20, wherein in order to obtain hydrogels with 400 to 1200 Pa and a q* value between 50 to 250, m_Cross-iinker/m_hyaiuronicacid is at a value between 0.08 to 0.35,

- removing air bubbles formed in the obtained second mixture by vacuum at a pressure value of at least 1 .0 bar,

- adding the particles obtained by cutting the parts obtained as a result of crosslinking to a buffer solution and performing washing processes with at least one acid solution in order to neutralize them, wherein the holding time of the gel particles in the buffer solution continues until the swelling amount of the hydrogels reaches between 2 to 10 times of value, particulating the obtained hydrogels and dialyzing with a buffer solution at a temperature at a value between 2 to 8 °C.

2. A method according to Claim 1 , characterized in that the amount of sodium hyaluronate compound in the first mixture is at a value in the range of 5% to 15% by weight.

3. A method according to Claim 1 or Claim 2, characterized in that in the first mixture, sodium hydroxide is at a concentration value of 0.25 M.

4. A method according to one of the preceding claims, characterized in that at least one of a group of materials such as 1 ,4-butanediol diglycidyl ether, divinyl sulfone, glutaraldehyde, 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) is used as a cross-linker in said second mixture.

5. A method according to Claim 4, characterized in that the cross-linker is 1 ,4- butanediol diglycidyl ether.

6. A method according to one of the preceding claims, characterized in that said cross-linking processes are performed at a temperature at a value between 35 to 50 °C.

7. A method according to Claim 6, characterized in that cross-linking processes are performed for a period of 150 to 200 minutes.

8. A method according to one of the preceding claims, characterized in that said buffer solutions are phosphate buffer solutions.

9. A method according to one of the preceding claims, characterized in that the acid solution for neutralization processes is at least one of hydrochloric acid, acetic acid, phosphoric acid, carbonic acid, formic acid.

10. A method according to one of the preceding claims, characterized in that lidocaine hydrochloride is further added to the hydrogels obtained.

11. A method according to claim 10, characterized in that the amount of said lidocaine hydrochloride added is at a value between 0.1% to 0.8% by weight.