Hyaluronic acid composition

Abstract

The present invention relates to a method for preparing a composition containing hyaluronic acid and / or hyaluronate and at least one bioactive molecule. The method comprises the following steps: providing a solution containing hyaluronic acid and / or hyaluronate; adding an alkali solution; adding at least one bioactive molecule; and adding an acid solution to obtain the composition. The invention also encompasses the composition prepared by the method according to the invention and the use of said composition for the aesthetics and / or treatment of humans and / or animals.

Description

Technical Field

[0001] The present invention relates to a method for preparing a composition, a composition prepared in this way and the use of said composition, wherein said composition contains hyaluronic acid and / or at least one salt of hyaluronic acid and at least one biologically active molecule. Background Art

[0002] It is known from the prior art that compositions containing hyaluronic acid are injected into human tissue for aesthetic and therapeutic purposes. Hyaluronic acid is also referred to as hyaluronan in the literature. Hyaluronic acid is a polysaccharide formed by multiple disaccharide repeating units, including D-glucuronic acid and N-acetyl-D-glucosamine in each case. It is found that hyaluronic acid is highly present in the vitreous humor of the skin, synovium and eyes. A person weighing 70 kg contains about 15 g of hyaluronic acid, half of which is present in tissues. The amount of hyaluronic acid decreases with age.

[0003] In many applications, it is known to inject compositions containing natural and / or chemically modified hyaluronic acid and / or its salt. The disadvantage is that natural hyaluronic acid and / or its salt have only a short half-life after injection, and degrade and decompose relatively quickly, so the action time of the composition is very limited. In order to increase the half-life of hyaluronic acid after the injection composition, hyaluronic acid and / or its salt are stabilized by cross-linking during the composition preparation process. However, the cross-linking agent used in this case is toxic, which impairs the safety of the composition containing cross-linked hyaluronic acid and / or its salt. Summary of the invention

[0004] The object of the present invention is therefore to eliminate the above-mentioned disadvantages of the prior art and in particular to prepare a composition which is safer to use but has the advantages of stable hyaluronic acid and / or its salts. This also applies, mutatis mutandis, to the composition and its use.

[0005] The object of the present invention is achieved by a composition containing hyaluronic acid and / or hyaluronate and at least one biologically active molecule, the method comprising the following steps:

[0006] - providing a solution containing hyaluronic acid and / or a hyaluronate,

[0007] - Add alkaline solution,

[0008] - adding said at least one biologically active molecule, and

[0009] - Adding an acid solution to obtain said composition.

[0010] The objects of the present invention are also achieved by a composition prepared by the method according to the present invention.Furthermore, the objects of the present invention are achieved by the use of a composition according to the present invention for aesthetic and / or therapeutic use in humans and / or animals.

[0011] The basic idea on which the present invention is based is that the preparation method according to the present invention eliminates the need for additives to stabilize hyaluronic acid and / or hyaluronate, for example for crosslinking. In the method according to the present invention, the stabilizing effect is achieved by the fact that the molecules of hyaluronic acid and / or hyaluronate are spatially expanded and therefore swell due to the addition of the alkaline solution. As a result, according to the present invention, at least one biologically active molecule added only after the addition of the alkaline solution can enter the gaps of the hyaluronic acid and / or hyaluronate thus produced. The subsequent addition of an acid solution not only ensures that the composition prepared according to the present invention has the desired pH value, in particular a neutral pH value, but the acid solution also changes the stereochemistry of the molecules of the hyaluronic acid and / or hyaluronate, causing it to shrink again. Therefore, the biologically active molecules are particularly enclosed in the gaps between the molecules of the hyaluronic acid and / or hyaluronate.

[0012] This results in a delayed, sustained release of the bioactive molecule after application of the composition prepared according to the invention, thereby increasing its effectiveness and in particular making it possible for at least one bioactive molecule to have a longer bioactivity. The composition prepared according to the invention thus has the same advantages as compositions with hyaluronic acid and / or hyaluronates that have been cross-linked with other additives, but without the disadvantages necessarily associated therewith.

[0013] Hyaluronic acid and / or hyaluronate can be obtained from animal sources and / or by bacterial fermentation. Used hyaluronate is preferably a physiologically compatible hyaluronate, which particularly includes sodium salts, calcium salts, potassium salts, zinc salts and mixtures thereof. For physiological reasons, it is preferred to use the sodium salt of hyaluronic acid, which is also referred to as sodium hyaluronate, NaHA or is referred to as sodium hyaluronate according to international nomenclature. Sodium hyaluronate is stable, soluble and diffusible, and is therefore physiologically compatible. Preferably, after adding alkaline solution, the concentration of hyaluronic acid and / or hyaluronate can be between 5mg / ml and 150mg / ml, particularly between 30mg / ml and 130mg / ml, most preferably between 50mg / ml and 110mg / ml.

[0014] In a modification of the present invention, the alkaline solution may comprise water and at least one hydroxide component. The water of the alkaline solution may be at least one of the group consisting of purified water, ultrapure water, ultra-ultrapure water and water for injection. The hydroxide component may comprise an alkali metal hydroxide, in particular potassium hydroxide (KOH). Preferably, the hydroxide component is sodium hydroxide (NaOH), in particular at a concentration of 1N in the alkaline solution.

[0015] Embodiments of the present invention can make the concentration of the hydroxide component in the alkaline solution between 0.05N and 1.5N, in particular between 0.1N and 1N, preferably between 0.25N and 1N. After the addition of the alkaline solution, a certain time interval can pass before the addition of the biologically active molecule. Within the meaning of the present invention, this time interval is also referred to as the dissolution time and can be between 5 minutes and 120 minutes, in particular between 20 minutes and 90 minutes, preferably between 30 minutes and 60 minutes. The addition of the alkaline solution can include mixing the composition, wherein the mixing operation can be performed during and / or after the addition of the alkaline solution, which helps to speed up the homogenization of the composition. The mixing can be performed at room temperature and / or pressure and / or use ultrasonic signals to improve the homogenization of the composition.

[0016] Preferably, at least one bioactive molecule within the meaning of the present invention produces a biological effect on skin cells, in particular fibroblasts, keratinocytes, melanocytes and / or adipocytes. Preferably, the at least one bioactive molecule is selected according to the intended use of the composition according to the invention. Within the meaning of the present invention, bioactive molecules include relatively large, in particular natural molecules, such as proteins, in particular cytokines and lipids, and relatively small molecules that regulate specific signaling pathways and contribute to cell programming and / or tissue repair. Within the meaning of the present invention, the at least one bioactive molecule is preferably cell permeable, non-immunogenic and can reversibly and selectively regulate intracellular processes of the target cells of the bioactive molecule.

[0017] Preferably, the at least one bioactive molecule is selected from the group consisting of amino acids, amino esters, hydroxy esters, aldehydes, derivatives of aldehydes, ketones containing multiple hydroxy groups, derivatives of ketones containing multiple hydroxy groups, cysteine ​​and L-cysteine. Adding the at least one bioactive molecule may comprise mixing the composition, wherein the mixing may be performed during and / or after adding the at least one bioactive molecule. In this way, an improved homogenization of the components of the composition is achieved.

[0018] The acid solution may contain hydrochloric acid, in particular concentrated hydrochloric acid. Adding the acid solution may include mixing the composition, wherein the mixing of the composition may be performed during and / or after adding the acid solution. In this way, an improved homogenization of the composition components is achieved.

[0019] According to the invention, the method can provide for the addition of at least one stabilizer, wherein a stabilizer within the meaning of the present invention is preferably designed to protect the molecules of hyaluronic acid and / or hyaluronate against damaging influences.

[0020] Stabilizer can include at least one antioxidant, particularly antioxidant vitamins and / or antioxidant vitamin derivatives, to protect the hyaluronic acid and / or its salt present in the composition from free radical degradation. For example, stabilizer includes at least one carboxylic acid selected from the group of antioxidant vitamins and / or its amides. Stabilizer preferably includes nicotinic acid, also referred to as vitamin B3, pyridine-3-carboxylic acid or nicotinic acid, and / or its amides, nicotinamide (nicotinamide), also referred to as nicotinamide (niacinamide), nicotinamide (nicotinic acid amide) or pyridine-3-carboxamide. At least one stabilizer, particularly at least one antioxidant concentration can be between 0.01mg / ml and 60mg / ml, preferably between 1mg / ml and 50mg / ml, most preferably between 5mg / ml and 30mg / ml.

[0021] In order to simplify the method according to the invention, at least one stabilizer can be added together with the acid solution. Preferably, the at least one stabilizer is dissolved in the acid solution and has in particular a concentration between 0.05N and 1.5N, in particular between 0.1N and 1N, most preferably between 0.25N and 1N. Preferably, the acid solution, in particular in which the at least one stabilizer is dissolved, is added at a temperature lower than ambient temperature, in particular between 1°C and 12°C, preferably between 4°C and 8°C. It has been found that an acid solution lower than ambient temperature improves the effect on the molecules of hyaluronic acid and / or its salts.

[0022] Preferably, provision is made for sterilizing the composition, in particular using heat, such as moist heat. In particular, autoclaving of the composition can be provided for this purpose. The composition can be sterilized at a temperature above 100° C., preferably above 110° C., in particular above 120° C. In addition, the duration of the sterilization can be between 1 second and 30 minutes. The sterilization can have at least one sterilization cycle, wherein the sterilization cycle can be, for example, sterilization at 121° C. for 20 minutes, at 126° C. for 7 minutes, or at 130° C. for 3 minutes.

[0023] The molecular weight of the hyaluronic acid and / or hyaluronate of the composition according to the invention may be between 0.01 Da and 6 MDa, in particular between 0.1 Da and 3.5 MDa. Furthermore, the composition of the invention may be in the form of a hydrogel.

[0024] The concentration of hyaluronic acid and / or its salts according to the composition of the present invention may be between 0.01 mg / ml and 60 mg / ml, particularly between 5 mg / ml and 35 mg / ml, preferably between 10 mg / ml and 25 mg / ml. In addition, the concentration of at least one bioactive molecule according to the composition of the present invention may be between 0.01 mg / ml and 50 mg / ml, particularly between 1 mg / ml and 30 mg / ml, preferably between 1 mg / ml and 20 mg / ml. The concentration of at least one stabilizer according to the composition of the present invention may be between 1 mg / ml and 60 mg / ml, particularly between 5 mg / ml and 50 mg / ml, preferably between 10 mg / ml and 30 mg / ml. The stabilizer may be particularly diluted in an acid solution and may particularly have a concentration between 0.05 N and 1.5 N, particularly between 0.1 N and 1 N, preferably between 0.25 N and 1 N. The composition according to the present invention may be injected, particularly contained in a syringe and / or vial.

[0025] According to the present invention, the composition can be used for stimulating and / or promoting tissue, particularly the regeneration of skin tissue, and / or for moisturizing tissue and / or for protecting tissue and / or for stimulating hair growth. In addition, it can be used by intradermal and / or subcutaneous injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Further advantages and features of the invention can be found in the claims and in the following description, in which exemplary embodiments of the invention are explained in detail.

[0027] Figure 1 Table showing the concentration of compositions G1 and REF1 over time. DETAILED DESCRIPTION

[0028] In the following, a method for preparing composition G1 according to the invention and a method for preparing reference composition REF1 are described.

[0029] In order to prepare the composition G1 according to the invention, 22 g of powdered sodium hyaluronate (NaHA) corresponding to the sodium salt of hyaluronic acid, with a molecular weight of about 2.1 MDa and a water content of 9.7% were first weighed and prepared. Then 132 g of an aqueous solution of sodium hydroxide (NaOH) with a concentration of 0.25 N was added thereto as an alkaline solution. The powder was dissolved within 1 hour under mechanical homogenization. Then 320 g of a phosphate solution containing L-cysteine ​​was added, L-cysteine ​​being the naturally occurring enantiomer of cysteine, wherein L-cysteine ​​was diluted in a ratio of 1:15. The composition was then mechanically homogenized for 30 minutes. A phosphate buffer solution cooled to a temperature of 4°C to 8°C was then added so that the pH value of the composition was 7.2, and then mechanical homogenization was performed. A phosphate solution containing 30 g of nicotinamide, corresponding to the amide of nicotinic acid, was added to the composition, and then mechanical homogenization was performed for a duration of 2 hours. The resulting gel-like composition according to the invention was named G1. The composition G1 according to the invention has a hyaluronic acid concentration of 10 mg / g, an L-cysteine ​​concentration of 10 mg / g and a nicotinamide concentration of 15 mg / g, respectively, relative to the total composition. The composition G1 according to the invention is placed in a glass vial made of borosilicate glass and sterilized by autoclave at a temperature of 121° C. for 20 minutes.

[0030] To prepare the reference composition REF1, 22 g of sodium hyaluronate (NaHA) powder with a molecular weight of about 2.1 MDa and a water content of 9.7% was weighed and prepared. 20 g of L-cysteine ​​and 30 g of nicotinamide were added thereto. A phosphate buffer solution was then added and the composition was then mechanically homogenized for a duration of 3.5 hours. The gel-like reference composition then obtained was named REF1, which had a hyaluronic acid concentration of 10 mg / g, an L-cysteine ​​concentration of 10 mg / g, and a nicotinamide concentration of 15 mg / g, respectively, relative to the total composition. The reference composition REF1 was placed in a borosilicate glass vial and autoclaved at a temperature of 121° C. for 20 minutes.

[0031] The concentrations of hyaluronic acid, L-cysteine ​​and nicotinamide of the composition G1 prepared according to the invention correspond to the corresponding concentrations of the reference composition REF1, wherein the composition G1 according to the invention differs from the reference composition REF1 in the method of preparation.

[0032] In the following, according to Figure 1 The release kinetics of L-cysteine ​​and nicotinamide were studied, on the one hand, for the composition G1 prepared according to the invention and on the other hand for the reference composition REF 1. As shown in the figure, the release kinetics of the two compositions G1 and REF 1 are different, which is mainly due to the different preparation methods.

[0033] 5 g of the composition G1 prepared according to the present invention and 5 g of the reference composition REF1 were placed in small dialysis bags containing 20 g of physiological saline or NaCl solution, and dialyzed at different time intervals. Figure 1 After the duration indicated by "t" in the first row, the dialysis was stopped, the compositions G1 and REF1 were homogenized with a spatula, and the concentrations of L-cysteine ​​and nicotinamide were determined by UV spectroscopy. The concentrations of the compositions G1 and REF1 thus determined were Figure 1 The concentrations are shown in the rows of Figure 1 The remaining proportion of the component mentioned in relative to the original concentration of the component before the start of dialysis. Therefore, Figure 1 The concentration data in the table should be understood as relative concentrations. Each concentration is determined as weight percent (w / w).

[0034] Figure 1 The first row shows the different times at which the dialysis was performed. The second row shows the concentration of L-cysteine ​​("L-cysteine") of the composition G1 prepared by the method according to the invention determined at the times mentioned in the first row, while the third row shows the concentration of L-cysteine ​​of the reference composition REF 1. Figure 1 The fourth row of shows the concentration of nicotinamide ("niacinamide") at the time mentioned for composition G1 prepared according to the invention and the fifth row shows the concentration of niacinamide for reference composition REF 1.

[0035] It should be noted that for compositions G1 and REF1, L-cysteine ​​and nicotinamide are almost completely released after 8 hours at the latest. However, there are significant differences between the release kinetics of compositions G1 and REF1, which is related to the significant advantages of composition G1 prepared according to the invention in clinical applications.

[0036] If Figure 1Comparing the second row with the third row in the graph, it can be seen that after 1.5 hours, the L-cysteine ​​concentration of composition G1 is still 0.63, i.e. 63%, while the L-cysteine ​​concentration of reference composition REF 1 at the same time has dropped to 0.25, i.e., it has dropped by more than half compared to composition G1. After 4 hours, the L-cysteine ​​concentration of composition G1 is 0.38, which is 4 times higher than the L-cysteine ​​concentration of 0.08 of reference composition REF 1 at the same time point. Therefore, in composition G1 prepared according to the present invention, the release of L-cysteine ​​as a biologically active molecule is significantly delayed compared to reference composition REF 1. One explanation for this is that the alkaline solution initially causes the hyaluronic acid chain to extend, and then the cooled hydrochloric acid added after the addition of L-cysteine ​​as a biologically active molecule to neutralize the composition causes steric hindrance of the hyaluronic acid molecules, causing them to be shrunk or compressed by the hydrochloric acid, thereby enclosing the L-cysteine ​​molecules between them for a longer time. As a result, a sustained release of L-cysteine ​​is caused.

[0037] About Niacinamide, Figure 1 The concentration gradient in shows that after 4 hours, the niacinamide concentration in both composition G1 and REF 1 has dropped to almost 0; at this point, almost all of the niacinamide has been released. In contrast, it is noteworthy that after 1.5 hours, the niacinamide concentration of composition G1 has dropped faster, with a concentration value of 0.17, while the niacinamide concentration of reference composition REF 1 after 1.5 hours is 0.23. For niacinamide, steric hindrance may not be significant or only significant to a small extent, because niacinamide is added only at the end of the method for preparing composition G1, in particular, after the shrinkage of the hyaluronic acid molecules, and therefore the niacinamide molecules are not affected by any delayed release kinetics.

Claims

1. A method for preparing a composition containing hyaluronic acid and / or hyaluronate and at least one biologically active molecule, wherein the method comprises the following steps: - providing a solution containing hyaluronic acid and / or a hyaluronate, - Add alkaline solution, - adding said at least one biologically active molecule, and - Adding an acid solution to obtain said composition.

2. The method according to claim 1, characterized in that: The alkaline solution comprises water and a hydroxide component, in particular an alkali metal hydroxide.

3. The method according to claim 1 or 2, characterized in that: The at least one biologically active molecule produces a biological effect on skin cells, in particular fibroblasts, keratinocytes, melanocytes and / or adipocytes.

4. The method according to any one of claims 1 to 3, characterized in that The at least one biologically active molecule is selected from the group consisting of amino acids, amino esters, hydroxy esters, aldehydes, derivatives of aldehydes, ketones containing multiple hydroxy groups, derivatives of ketones containing multiple hydroxy groups, cysteine ​​and L-cysteine.

5. The method according to any one of claims 1 to 4, characterized in that The acid solution contains concentrated hydrochloric acid.

6. The method according to any one of claims 1 to 5, characterized in that The method includes adding at least one stabilizer.

7. The method according to claim 6, characterized in that The at least one stabilizer comprises at least one antioxidant, in particular an antioxidant vitamin.

8. The method according to claim 6 or 7, characterized in that: The at least one stabilizer is added together with the acid solution.

9. The method according to claim 8, characterized in that The at least one stabilizer is dissolved in the acid solution and has in particular a concentration of between 0.05N and 1.5N, in particular between 0.1N and 1N, preferably between 0.25N and 1N.

10. The method according to any one of claims 1 to 9, characterized in that The composition is sterilized.

11. A composition prepared by the method of claims 1 to 10.

12. Use of a composition according to claim 11 for the aesthetic and / or therapeutic use of humans and / or animals.

13. The use according to claim 12, characterized in that The use is for stimulating and / or promoting the regeneration of tissue, in particular skin tissue, and / or for moisturizing tissue and / or for protecting tissue and / or for stimulating hair growth.

14. The use according to claim 12 or 13, characterized in that The use is carried out by intradermal and / or subcutaneous injection.

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