KR20140050593A - Aqueous solution containing hyaluronic acid or salt thereof - Google Patents

Abstract

The present invention relates to an aqueous solution containing high stability hyaluronic acid or hyaluronic acid salt.
An aqueous solution containing hyaluronic acid or a hyaluronic acid salt containing a zinc compound having a content of divalent soluble iron of 5 ppb or less is provided. Or, it provides the aqueous solution containing hyaluronic acid or a hyaluronic acid salt whose concentration of the said hyaluronic acid or a hyaluronic acid salt is 5-15 mg / mL.

Description

Aqueous solution containing hyaluronic acid or salt

The present invention relates to an aqueous solution containing hyaluronic acid or a hyaluronic acid salt having a low content of divalent soluble iron and containing a zinc compound.

Hyaluronic acid is known to be a polysaccharide composed of N-acetylglucosamine and glucuronic acid linked with disaccharide units (disaccharide units). Hyaluronic acid is generally used as a raw material for medicines, cosmetics and food. As a production method of hyaluronic acid, there is a method which is produced by an extract from an egg of a chicken, or a method which is produced by a fermentation method using microorganisms.

For example, Patent Document 1 describes a method of producing hyaluronic acid by fermentation using Streptococcus zooepidemicus. Patent Document 1 describes that when ferrous sulfate is added at a threefold concentration during incubation (Example 3), the yield and molecular weight of hyaluronic acid are increased as compared with the case where no ferrous sulfate is added (Example 1). It is.

In addition, Patent Document 2 describes that hyaluronic acid powder is produced by a fermentation method using Streptococcus equi (ATCC9527). In Patent Literature 3, experimental data indicating that sodium hyaluronate crystal produced based on the method of Patent Literature 2 contains a large amount of calcium, magnesium, and iron is described. Further, in Patent Document 3, a hyaluronic acid crystal obtained by reducing the content of calcium, magnesium, and iron to less than 10 ppm can be obtained by further contacting a chelating resin with a hyaluronic acid-containing liquid obtained on the basis of the method of Patent Document 2. It is described.

(0001) [Patent Document 1] JP-A-9-56394 (0002) [Patent Document 2] Japanese Patent Publication No. Hei 7-2117 [0003] [Patent Document 3] Japanese Patent Application Laid-Open No. 2008-280430

However, the prior art described in the above documents has room for improvement in the following points.

First, in the production method described in Patent Literature 1, ferrous sulfate (iron sulfate (II), FeSO ₄ ) is added to the culture medium in large quantities, and therefore iron may be easily incorporated into the hyaluronic acid-containing solution after purification. In addition, in order to remove iron from the culture paper containing a large amount of ferrous sulfate, more cost than usual is required.

Second, in the production method described in Patent Document 2, the hyaluronic acid powder contains a large amount of calcium, magnesium, and iron, and the purity of sodium hyaluronate is low.

Third, in the production method described in Patent Document 3, it is described that the content of calcium, magnesium, and iron in sodium hyaluronate crystals is reduced to less than 10 ppm, but it is not clear how much the content is. The analytical results are in ppm-order and lack precision. Furthermore, it is not described what effect can be obtained specifically by reducing the content of calcium, magnesium and iron.

This invention is performed in view of the said problem, Since the content rate of bivalent soluble iron is 5 ppb or less, and contains a compound, it aims at providing the aqueous solution containing high stability hyaluronic acid or a hyaluronic acid salt.

The present inventors have conducted studies on impurities contained in an aqueous solution containing hyaluronic acid or a hyaluronic acid salt. As a result, the higher the content of the divalent soluble iron contained in the aqueous hyaluronic acid solution, the lower the stability of the hyaluronic acid molecule. I found out for the first time. In addition, the inventors found that the stability of hyaluronic acid molecules is not reduced even if the content of trivalent soluble iron is surprisingly reduced, and the stability of hyaluronic acid molecules is reduced only when the content of divalent soluble iron is large.

Here, the present inventors reduce the content of the divalent soluble iron mixed in the aqueous solution containing hyaluronic acid or the hyaluronic acid salt, and at the same time, the additive which suppresses the reduction of the stability of the hyaluronic acid molecule by the divalent soluble iron. As a result, it was found for the first time that the addition of the compound can suppress the decrease in the stability of the hyaluronic acid molecule due to the bivalent soluble iron. In addition, the inventors surprisingly, when glycine, L-aspartic acid Na, bromide Na, hydrogen sulfite Na, sulfide Na, thioglycolic acid Na, glucose, refined white sugar, or ascorbic acid is added, bivalent The present invention was completed by finding that the decrease in stability of hyaluronic acid molecules due to soluble iron is not suppressed, but that the stability is greatly reduced by addition.

In other words, according to the present invention, an aqueous solution containing hyaluronic acid or a hyaluronic acid salt containing a zinc compound having a content of divalent soluble iron of 5 ppb or less and containing a zinc compound is provided. The aqueous solution containing hyaluronic acid or a hyaluronic acid salt is demonstrated by the Example mentioned later that the intrinsic viscosity residual ratio after storage is favorable. Therefore, it is suitable as a raw material for medicine, cosmetics, food and the like.

In addition, according to the present invention, there is provided a pharmaceutical composition comprising an aqueous solution containing the hyaluronic acid or hyaluronic acid salt. The pharmaceutical composition contains an aqueous solution containing hyaluronic acid or hyaluronic acid salt having excellent intrinsic viscosity residual ratio after storage. Therefore, the fall of the viscosity or the fall of the quality which generate | occur | produce over time hardly arise.

Moreover, according to this invention, the injection for arthrosis treatment containing the aqueous solution containing the said hyaluronic acid or a hyaluronic acid salt is provided. The injection for treating arthrosis includes an aqueous solution containing hyaluronic acid or hyaluronic acid salt having excellent intrinsic viscosity residual ratio after storage. Therefore, when administered in or around the joint, good retention can be obtained.

Moreover, according to this invention, the cosmetic composition containing the said aqueous solution containing hyaluronic acid or a hyaluronic acid salt is provided. The cosmetic composition contains an aqueous solution containing hyaluronic acid or hyaluronic acid salt having good intrinsic viscosity residual ratio after storage. Therefore, the fall of the viscosity or the fall of the quality which generate | occur | produce over time hardly arise.

According to the present invention, there is also provided a composition containing a hyaluronic acid or a hyaluronic acid salt and a divalent soluble iron, and a step of dissolving the zinc compound in an aqueous solution, wherein the composition has a concentration of 10 mg of hyaluronic acid or a hyaluronic acid salt. Provided is a method for producing an aqueous solution containing hyaluronic acid or a hyaluronic acid salt having a content of divalent soluble iron in an aqueous solution of 5 ppb or less when dissolved in an aqueous solution to be / mL. According to the above production method, hyaluronic acid or hyaluronic acid salt having excellent intrinsic viscosity residual ratio after storage can be produced.

According to the present invention, there is also provided a composition containing a hyaluronic acid or a hyaluronic acid salt and a divalent soluble iron, and a step of dissolving the zinc compound in an aqueous solution, wherein the composition has a concentration of 10 mg of hyaluronic acid or a hyaluronic acid salt. Provided is a method for promoting stabilization of an aqueous solution containing hyaluronic acid or a hyaluronic acid having a content of divalent soluble iron in an aqueous solution of 5 ppb or less when dissolved in an aqueous solution at / mL. When the zinc compound is contained in an aqueous solution containing hyaluronic acid or hyaluronic acid having a content of divalent soluble iron of 5 ppb or less, it is demonstrated in the examples described below that the intrinsic viscosity residual ratio after storage is remarkably improved. Therefore, according to the said stabilization promotion method, stabilization of the aqueous solution containing hyaluronic acid or a hyaluronic acid salt can be promoted remarkably.

Moreover, according to this invention, the stabilizer for stabilizing the aqueous solution containing the hyaluronic acid or the hyaluronic acid containing a zinc compound and whose content rate of bivalent soluble iron is 5 ppb or less is provided. When the zinc compound is contained in an aqueous solution containing hyaluronic acid or hyaluronic acid having a content of divalent soluble iron of 5 ppb or less, it is demonstrated in Examples described later that the intrinsic viscosity residual ratio after storage is remarkably improved. Therefore, when the said stabilizer is added to the aqueous solution containing hyaluronic acid or a hyaluronic acid whose content rate of bivalent soluble iron is 5 ppb or less, it can remarkably stabilize.

According to this invention, since the content rate of bivalent soluble iron is 5 ppb or less and contains a zinc compound, the aqueous solution containing high stability hyaluronic acid or a hyaluronic acid salt can be obtained.

1 is a graph showing a change in intrinsic viscosity residual ratio when sodium hyaluronate powder is added to an aqueous solution containing divalent soluble iron and zinc chloride.

Hereinafter, embodiments of the present invention will be described in detail. In addition, description is abbreviate | omitted suitably about the same content, in order to avoid repetitive complicated description.

<Aqueous solution containing hyaluronic acid or hyaluronic acid salt>

One embodiment of the present invention is an aqueous solution containing hyaluronic acid or a hyaluronic acid salt containing a zinc compound having a content of divalent soluble iron of 5 ppb or less. The aqueous solution containing the hyaluronic acid or the hyaluronic acid salt having such a composition has a high intrinsic viscosity residual ratio after storage and excellent physical stability, as demonstrated in Examples described later. Moreover, even after long-term storage or after a long time elapses, a decrease in viscosity or quality hardly occurs, and is suitable as a raw material such as pharmaceuticals, cosmetics and foods.

Examples of the zinc compound include zinc salicylate, zinc phosphate, zinc sulfate, zinc nitrate, zinc iodide, zinc fluoride, zinc chloride, zinc bromide, zinc carbonate, pt-butyl benzoate and zinc thiocyanate. Zinc lactate, zinc acetate, or zinc oxide. Moreover, it can use also as a complex like a salt of zinc acid ion. When using as a raw material of a pharmaceutical composition, zinc acetate, zinc sulfate, zinc chloride, or zinc oxide is preferable. When used as a raw material for injection, zinc acetate or zinc chloride is preferable. In addition, preferably, an endotoxin concentration is reduced.

The 5 ppb or less may be, for example, 0.001, 0.01, 0.1, 0.5, 1, 2, 4, or 5 ppb. The content may also be within the range of values shown here or in any two values. The content ratio is preferably as small as possible from the viewpoint of improving the stability of the aqueous solution containing hyaluronic acid or hyaluronate.

The content rate of the said zinc compound contained in the aqueous solution containing the hyaluronic acid or the hyaluronic acid salt of this embodiment is 0.005, 0.01, 0.1, 0.5, 0.8, 1, 1.5, 2, 5, 10, 50, Or 100 microgram / mL may be sufficient. The content may also be in the range of any two values exemplified herein. From the viewpoint of further improving the stability of the aqueous solution containing hyaluronic acid or hyaluronic acid salt, the content rate is preferably 0.01 µg / mL or more, and more preferably 0.5 µg / mL or more. Moreover, 10 micrograms / mL or less is preferable and, as for the said content rate from a viewpoint of production cost reduction or operability improvement, 2 microgram / mL or less is more preferable.

The average molecular weight of sodium hyaluronate or hyaluronic acid contained in the aqueous solution containing hyaluronic acid or hyaluronic acid salt of this embodiment is 500,000, 800,000, 1 million, 1.5 million, 1.8 million, 2 million, 250, for example. 3 million, 5 million, or 8 million may be sufficient. The average molecular weight may be in the range of any two values exemplified herein. From the viewpoint of further improving the viscosity of the aqueous solution containing hyaluronic acid or hyaluronate, it is preferably at least 1 million, more preferably at least 1.5 million. An aqueous solution containing highly viscous hyaluronic acid or hyaluronic acid salt has good retention in affected areas when used as an injection for treating arthrosis. The average molecular weight of the hyaluronic acid or the hyaluronic acid salt contained in the aqueous solution containing the hyaluronic acid or the hyaluronic acid salt was determined by measuring the intrinsic viscosity, followed by the Laurent equation (LAURENT et al., Biochim Biophys Acta 1960 1960 26; 42: 476-485.).

The content of hyaluronic acid or hyaluronic acid contained in the aqueous solution containing hyaluronic acid or hyaluronic acid salt of the present embodiment is, for example, 0.1, 1, 5, 8, 9, 10, 11, 12, 15, or 20 mg. / mL may be sufficient. The said content rate may be in the range of any two values illustrated here. From the viewpoint of therapeutic effect, or operability when used in injections and the like, a range of values of 5 and 15 mg / mL is preferable, and a range of values of 8 and 12 mg / mL is more preferable.

PH of the aqueous solution containing the hyaluronic acid or the hyaluronic acid salt of this embodiment may be 5.5, 6, 6.5, 6.8, 7, 7.8, 8, 8.5, or 9, for example. The pH may be in the range of any two values exemplified herein. From the viewpoint of stability, the pH is preferably in the range of values of 6.5 and 8, and more preferably in the range of values of 6.8 and 7.8.

The aqueous solution containing the hyaluronic acid or the hyaluronic acid salt of the present embodiment includes, for example, a composition containing hyaluronic acid or a hyaluronic acid salt and a bivalent soluble iron, and a step of dissolving the zinc compound in the aqueous solution, The composition is a method for producing an aqueous solution containing hyaluronic acid or a hyaluronic acid having a content of divalent soluble iron in an aqueous solution of 5 ppb or less when dissolved in an aqueous solution such that the concentration of hyaluronic acid or hyaluronic acid salt becomes 10 mg / mL. Can be obtained by Since the production method does not necessarily require a complicated process, it is excellent in productivity or cost.

In this specification, "hyaluronic acid salt" may be sodium hyaluronate, potassium hyaluronate, zinc hyaluronate, calcium hyaluronate, magnesium hyaluronate, or ammonium hyaluronate, for example. Among them, sodium hyaluronate is preferable from the viewpoint that a desired effect of viscosity or arthropathy can be expected. The chemical name of sodium hyaluronate is, for example, [→ 3) -2-acetamino-2-deoxy-β-D-glucopyranosyl- (1 → 4) -β-D-sodium glucopyranosyluronate- (1 →) n ([→ 3) -2-acetamido-2-deoxy-β-D-glucopyranosyl- (1 → 4) -β-D-sodium glucopyranosyluronate- (1 →] n) (IUPAC) have.

In the present specification, &quot; bifunctional soluble iron &quot; means iron in a soluble state and bivalent iron. It may be expressed as Fe ^{2 +} Na 2 gacheol.

<Medicinal composition, etc.>

The aqueous solution containing hyaluronic acid or hyaluronic acid salt of this embodiment can be used as a raw material of a pharmaceutical composition. In this case, since the said pharmaceutical composition contains the aqueous solution containing hyaluronic acid or hyaluronic acid salt which is excellent in stability, it is hard to produce the fall of a viscosity and a quality even after long-term storage. In addition, when administered, for example, in or around the joint, it can stay in the affected area for a long time or for a desired period of time. Although the formulation of the said pharmaceutical composition is not specifically limited, Injectable agent is preferable from a viewpoint that it can administer directly to a affected part, such as a joint. As an injection container, a syringe, a vial, or an ampule can be used, for example. In the case of administration, it may be administered alone or mixed with one or more pharmacologically acceptable carriers or excipients, and any method well known in the art of pharmaceuticals. It is preferable to provide in the pharmaceutical preparation manufactured by the. Depending on the dosage form, the pharmaceutical composition may be formulated with buffers (e.g., phosphate buffers, sodium acetate buffers), analgesic agents (e.g., lidocaine hydrochloride, procaine hydrochloride, etc.), stabilizers (e.g. For example, human serum albumin, polyethylene glycol, etc.), a preservative (for example, benzyl alcohol, phenol, etc.), antioxidant, etc. may be combined. The pharmaceutical composition may also contain sodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride as additives. The adjusted pharmaceutical composition can be administered, for example, to humans or mammals (eg rats, mice, rabbits, dogs, monkeys, sheep, pigs, cattle, cats, etc.). The pharmaceutical composition also includes a composition used for the purpose of prevention.

) 내에 투여할 수 있다. 또한 관절 류머티즘에 있어서, 무릎 관절통(關節痛)의 치료에 사용될 경우에는, 예를 들면, 성인은 1회당 2.5mL을 매주 연속 5회 무릎 관절강 안에 투여할 수 있다. 또한, 적절한 화학요법약과 병용하여 투여해도 좋다. The administration method of the said pharmaceutical composition can be suitably selected according to the age, a symptom, an affected part, etc. of a subject. When used in the treatment of deformed knee arthropathy, for example, an adult may administer 2.5 mL per one time in a consecutive 5 times a week in the knee joint cavity (关节 腔). Alternatively, for the purpose of maintaining the symptoms, it may be administered every 2 to 4 weeks. In addition, when used for the treatment of shoulder periarthritis, for example, an adult may take 2.5 mL per dose five times per week for five consecutive shoulder joints (shoulder joint cavity, acromioclavicular vacuoles, or upper arm). Biceps curls

). &Lt; / RTI &gt; Also, in the case of joint rheumatism, when used in the treatment of knee arthralgia (关节痛), for example, an adult can administer 2.5 mL per one time in the knee joint cavity five times in a week. It may also be administered in combination with an appropriate chemotherapeutic drug.

As the pharmacological action of the pharmaceutical composition, for example, a) hyaluronic acid is bound to the cartilage tissue and coats the surface, so that viscoelastic or lubricating action, b) articular cartilage protection action (degeneration) by stabilizing the cartilage matrix Inhibitory action, restorative action, etc.), c) analgesic action by coating the surface of inflammatory cells and synovial cells, or by inhibiting the production of pain-inducing substance, d) synovial cell and cartilage degeneration. Inflammatory effects are found by affecting synovial cells, chondrocytes, or inflammatory cells such as neutrophils or macrophage that are closely related to arthritis. These actions are expected to reduce pain, for example, or improve daily activities and joint movement areas.

The aqueous solution containing the hyaluronic acid or the hyaluronic acid salt of this embodiment can be used as a raw material of a cosmetic composition. In this case, since the said cosmetic composition contains the aqueous solution containing hyaluronic acid or hyaluronic acid salt which is excellent in stability, it is hard to produce the fall of a viscosity or quality even after long-term storage. Moreover, when used as a cosmetic composition for moisturizing, a moisturizing effect becomes easy to persist.

<Stabilization method, etc.>

Another embodiment includes a composition containing hyaluronic acid or hyaluronic acid salt and divalent soluble iron, and a step of dissolving the zinc compound in an aqueous solution, wherein the composition has a concentration of 10 mg / mL of hyaluronic acid or hyaluronic acid salt. It is a stabilization acceleration | stimulation method of the aqueous solution containing hyaluronic acid or a hyaluronic acid whose content rate of the bivalent soluble iron in aqueous solution when it melt | dissolves in an aqueous solution is 5 ppb or less. According to the said method, the aqueous solution with high physical stability and containing hyaluronic acid or a hyaluronic acid salt can be obtained. Moreover, since the said method does not necessarily require a complicated process, it is excellent in convenience.

In addition, the zinc compound may be used as a stabilizer raw material of an aqueous solution containing hyaluronic acid or hyaluronic acid salt. When the stabilizer is used, the physical stability of the aqueous solution containing hyaluronic acid or hyaluronic acid having a content of divalent soluble iron of 5 ppb or less can be improved. The stabilizer may be in a powder state or a liquid state. In the liquid state, a buffer may be included.

As mentioned above, although embodiment of this invention was described, these are the illustrations about this invention, Various structures of that excepting the above are also employable. Moreover, it can also employ | adopt combining the structure as described in the said embodiment.

[Example]

Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited thereto.

&Lt; Example 1 >

The aqueous hyaluronic acid Na solution was adjusted in the following procedure. First, 1 liter of culture medium consisting of 5% of glucose, 0.2% of potassium phosphate, 1.0% of polypeptone and 0.5% of yeast extract was heated and sterilized. Streptococcus equi FM-100 No. 9027, Ministry of Agriculture, Forestry and Fisheries). The mixture was stirred at 200 revolutions / minute while passing air at 1 vvm, and cultured for 20 hours at a temperature of 33 ° C and a pH of 8.5 (controlled by automatic dropwise addition of 20% sodium hydroxide). The liquid was filtered with a bacterial filter and dialyzed against water for 12 hours to recover the hyaluronic acid solution in the dialysis membrane. In order to prevent the iron from being mixed with the recovery container, a glass container whose inner surface was made of glass was used. Sodium chloride was added to 2.5%, and ethanol was added twice as much as the hyaluronic acid solution to precipitate Na hyaluronic acid. The precipitate was washed 5 times with ethanol, and the impurities were washed sufficiently, and then dried at 40 ° C. for 10 hours to obtain a powder of hyaluronic acid Na.

Next, the obtained hyaluronic acid Na powder is dissolved in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride, and the hyaluronic acid Na aqueous solution containing 5, 8, 10, 12, and 15 mg / mL of hyaluronic acid Na, respectively. Got. In addition, when the intrinsic viscosity was measured and the molecular weight of hyaluronic acid Na was calculated using the Laurent equation, it was 2.75 million.

&Lt; Comparative Example 1 &

Streptococcus equi FM-100 was cultured in the order described in Example 1 and the aqueous hyaluronic acid Na solution was purified in the same procedure as described in Example 1 of Patent Document 3 (Japanese Patent Laid-Open No. 2008-280430). Specifically, first, 1 liter of a culture medium consisting of glucose 5%, potassium phosphate 0.2%, polypeptone 1.0% and yeast extract 0.5% was heated and sterilized, and Streptococcus equi FM-100 . The mixture was stirred at 200 revolutions / minute while passing air at 1 vvm, and cultured for 20 hours at a temperature of 33 DEG C and pH 8.5 (control by automatic dropwise addition of 20% sodium hydroxide). The culture solution was diluted 10 times with ion exchange water and 5 g of activated charcoal (Shirasagawa RW50-T manufactured by Takeda Pharmaceutical Co., Ltd.) was added to 2.5 L of the aqueous solution, and perlite (manufactured by Mitsui Mining & 30 g of ROKAHELP # 409 (manufactured by Mitsui Chemicals, Inc.) was added, and the mixture was treated for 1 hour and filtered using Nutsche. The above operation was repeated twice to remove organic components in the culture medium, and the hyaluronic acid Na-containing liquid was adjusted. Next, 68 ml of diaion CR11 made by Mitsubishi Chemical Corporation was charged (filled) into a chromatographic column having an inner diameter of 15 mm and a height of 300 mm to regenerate the resin. 2.5 L of the Na-containing hyaluronic acid solution was passed through the chromatograph column at a SV = 18 (1200 ml / hr). 2 g of sodium chloride was dissolved in 1 L of a chromatographic column, adjusted to pH 7, precipitated with 2-propanol, and vacuum-dried at 40 캜 to obtain a hyaluronic acid Na powder.

Next, the resulting hyaluronic acid Na powder was dissolved in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride to obtain an aqueous hyaluronic acid Na solution containing 5, 8, 10, 12 and 15 mg / mL of hyaluronic acid Na, respectively. In addition, the intrinsic viscosity was measured and the molecular weight of hyaluronic acid Na was calculated using Laurent formula, and it was 2.4 million.

<Evaluation Example 1>

(1) Analysis of soluble iron

The hyaluronic acid Na powder prepared in Example 1 and Comparative Example 1 was dissolved in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride and hyaluronic acid containing 5, 8, 10, 12, or 15 mg / mL hyaluronic acid Na Aqueous solutions of sodium nitrite were prepared (Nos. 1 to 10). Next, for each of the aqueous solutions, the soluble iron amount of divalent iron, the soluble iron amount of divalent and trivalent iron, and the total iron amount were measured in the following order (1-1) to (1-3). Total iron content includes soluble and insoluble iron content. The detection limit of soluble iron, divalent and trivalent soluble iron, and total iron was 2 ppb and 5 ppb, respectively.

(1-1) Analysis method of soluble iron 2

 a) 2.75 g of an aqueous solution of sodium hyaluronate is sampled in a sample bottle.

 b) 0.1 ml of potassium thiocyanate solution and 0.05 ml of 1, 10-phenanthroline solution are added and mixed.

 c) Dilute to about 10 mL, and allow to stand for about 10 minutes.

 d) Add 5 mL of chloroform and tightly stop. Shake for 5 minutes, and then extracted (extracted).

 e) After standing, 4 mL of the chloroform phase is dispensed and transferred to another sample bottle.

 f) After evaporating to dryness on a hot plate, 0.5 mL of concentrated nitric acid is added to decompose the acid (acid decomposition).

 g) 2 mL of dilute acid (1 + 100) is added to dissolve the remainder.

h) Quantification of Fe by ICP emission analysis.

(1-2) Analytical method of bivalent and trivalent soluble iron

 a) 2.75 g of an aqueous solution of sodium hyaluronate is collected into a sample bottle.

 b) 0.2 ml of a hydrochloric acid hydroxylamine solution is added.

 c) 0.2 ml of a 1, 10-phenanthroline solution and 0.5 ml of potassium thiocyanate solution are added.

 d) Dilute to about 10 mL and leave for about 10 minutes.

 e) Add 5 mL of 4-methyl-2-pentanone, and then tighten. Mix by shaking for 1 minute and extract.

 f) After standing, 4 mL of the 4-methyl-2-pentanone phase is collected and transferred to another sample bottle.

 g) After evaporating to dryness on a hot plate, 0.5 ml of nitric acid is added to effect acid decomposition.

 h) 2 mL of dilute acid (1 + 100) is added to dissolve the remainder.

i) Determine Fe by ICP luminescence analysis.

 (1-3) Analysis method of total iron

 a) 2.75 g of an aqueous solution of sodium hyaluronate is collected into a sample bottle.

 b) Add 0.1 mL of HCl (1 + 1) and heat with a hot plate.

c) Analysis method of said (1-2) divalent and trivalent soluble iron It performs by operation after b) of analysis of soluble iron.

(1-4) Analysis of iron content

Table 1 shows the analysis results of the above (1-1) to (1-3).

(2) Measurement of intrinsic viscosity

For the aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride, the hyaluronic acid Na powder prepared in Example 1 or Comparative Example 1 was dissolved to a concentration of 10 mg / mL of hyaluronic acid Na (No. 11 to 12) . In addition, Table 2 shows the intrinsic viscosity and the intrinsic viscosity remaining ratio (%) after storage at 80 ° C for 24 hours. On the other hand, the intrinsic viscosity was measured according to the contents described in the item "Viscosity of purified hyaluronate sodium" of the 15th Revised Japanese Pharmacopoeia Control 2 (Fifteenth revised edition, Nippon Pharmaceutical Co., Ltd.). The results are shown in Table 2.

Iron (II) chloride (FeCl ₂ ) shown in Table 3 was dissolved in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride (Nos. 13 to 19). Next, the hyaluronic acid Na powder prepared in Example 1 was dissolved in each aqueous solution so that the concentration of hyaluronic acid Na became 10 mg / mL. In addition, after preservation at 80 ° C for 24 hours, the intrinsic viscosity and the intrinsic viscosity residual ratio (%) were measured. The results are shown in Table 3.

Iron (III) chloride (FeCl ₃ ) shown in Table 4 was dissolved in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride (Nos. 20 to 22). Next, the hyaluronic acid Na powder prepared in Example 1 was dissolved in each aqueous solution so that the concentration of hyaluronic acid Na became 10 mg / mL. In addition, after preservation at 80 ° C for 24 hours, the intrinsic viscosity and the intrinsic viscosity residual ratio (%) were measured. The results are shown in Table 4.

From the above results, 1) by employing the method described in Example 1, the aqueous solution of hyaluronic acid Na containing 5 ppb or less of divalent soluble iron can be adjusted, and 2) the aqueous solution of hyaluronic acid Na is preserved by divalent soluble iron. It was found that the intrinsic viscosity after the lowering and 3) trivalent soluble iron did not affect the intrinsic viscosity after storage of the aqueous solution of hyaluronic acid Na.

<Evaluation Example 2>

Various additives shown in Table 5 were dissolved in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride (Nos. 23 to 36). Next, the hyaluronic acid Na powder prepared in Example 1 was dissolved in each aqueous solution to a concentration of 10 mg / mL. In addition, after preservation at 80 ° C for 24 hours, the intrinsic viscosity and the intrinsic viscosity residual ratio (%) were measured. As the additive, zinc chloride, zinc salicylate, zinc iodide, zinc acetate, glycine, L-aspartic acid Na, bromide Na, hydrogen sulfite Na, sulfide Na, thioglycolic acid Na, glucose, refined white sugar, and ascorbic acid were used.

*additive

Zinc chloride: Wako Pure Chemical Industries, 263-00271

Zinc Salicylate: Wako Pure Chemical Industries, 269-00692

Zinc iodide: Wako Pure Chemical Industries, 268-01061

Zinc acetate: Wako Pure Chemical Industries, 263-00232

Glycine: Wako Pure Chemical Industries, 036435

L-aspartic acid Na: Wako Joon Pharmaceutical Co., Ltd., 193-01262

Na bromide: Wako Jun Pharmacist, 193-01505

Hydrogen sulfite Na: Wako Jun Pharmacist, 190-01375

Na sulfide: Wako Jun Pharmacist, 197-03362

Thioglycolic acid Na: Wako Pure Chemical Industries, 590-11762

Glucose: Wako Jun Pharm, 076-05705

Purified white sugar (sucrose): Wako Joon Pharmaceutical Co., Ltd., 196-13705

Ascorbic acid Na: Wako Pure Chemical Industries, 196-01252

As a result, it was found that the aqueous solution in which the hyaluronic acid Na powder prepared in Example 1 was dissolved contains zinc chloride, zinc salicylate, zinc iodide, or zinc acetate, thereby improving the stability of the aqueous hyaluronic acid Na solution. Moreover, when Na bromide, hydrogen sulfite Na, sulfide Na, thioglycolic acid Na, glucose, or ascorbic acid were added, stability rather fell.

&Lt; Evaluation Example 3 &

Various additives shown in Table 6 were dissolved in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride (Nos. 37 to 43). In addition, 11.3 ppb of iron (II) chloride (5 ppb as bivalent solubility iron) was added to each aqueous solution. Next, the hyaluronic acid Na powder prepared in Example 1 was dissolved in each aqueous solution to a concentration of 10 mg / mL. Thereafter, after preservation at 80 DEG C for 24 hours, the intrinsic viscosity and the intrinsic viscosity remaining ratio (%) were measured.

From the above results, it was found that when the aqueous solution containing the powder of hyaluronic acid Na prepared in Example 1 contained 5 ppb of divalent soluble iron, the stability of the aqueous hyaluronic acid Na solution was lowered. Moreover, it turned out that the fall of the stability is remarkably suppressed by zinc chloride or zinc acetate.

&Lt; Evaluation Example 4 &

Zinc chloride was dissolved in the concentration shown in Table 7 in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride (No. 44 to 50). Next, the hyaluronic acid Na powder prepared in Example 1 was dissolved in each aqueous solution to a concentration of 10 mg / mL. In addition, after preservation at 80 ° C for 24 hours, the intrinsic viscosity and the intrinsic viscosity residual ratio (%) were measured.

From the above results, it was found that the aqueous solution in which the hyaluronic acid Na powder prepared in Example 1 was dissolved contains more than 0.01 µg / mL of zinc chloride, thereby improving the stability of the aqueous hyaluronic acid Na solution. Moreover, the improvement of the stability became slow in the vicinity containing 1 microgram / mL of zinc chloride.

&Lt; Evaluation Example 5 &

For an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride, zinc chloride was dissolved to a concentration of 1 μg / mL. In addition, iron (II) chloride at the concentrations shown in Table 8 were added to each aqueous solution (Nos. 51 to 56). Next, the hyaluronic acid Na powder prepared in Example 1 was dissolved in each aqueous solution to a concentration of 10 mg / mL. Thereafter, after preservation at 80 DEG C for 24 hours, the intrinsic viscosity and the intrinsic viscosity remaining ratio (%) were measured.

When the result of Table 8 and the result of Table 3 are shown graphically, it is as showing in FIG. From Fig. 1, it was found that when 1 µg / mL of zinc chloride was contained in the hyaluronic solution containing a low concentration of divalent soluble iron, the stability of the aqueous solution of hyaluronic acid Na was improved. The stabilizing effect was particularly remarkable when the divalent soluble iron concentration was 5 ppb or less.

As mentioned above, the intrinsic viscosity residual ratio after the hyaluronic acid Na aqueous solution with low content of bivalent soluble iron was preserve | saved improved. In addition, when zinc chloride was contained in the aqueous solution of hyaluronic acid Na, the intrinsic viscosity residual ratio further improved. The aqueous solution of hyaluronic acid Na is high in stability and hardly deteriorates in viscosity and quality even after long-term storage, and thus is excellent as a raw material such as a pharmaceutical composition. Moreover, since it is suitable for long term storage, cost can be held down.

The present invention has been described above based on the examples. It is to be understood by those skilled in the art that the foregoing embodiments are merely illustrative and that various modifications are possible and that these modifications are also within the scope of the present invention.

Claims (12)

The content rate of divalent soluble iron is 5 ppb or less,
An aqueous solution containing hyaluronic acid or a hyaluronic acid salt containing a zinc compound. The method of claim 1,
An aqueous solution containing hyaluronic acid or hyaluronic acid salt having a concentration of 5 to 15 mg / mL of the hyaluronic acid or hyaluronic acid salt. 3. The method according to claim 1 or 2,
The zinc compound is zinc salicylate, zinc phosphate, zinc sulfate, zinc nitrate, zinc iodide, zinc fluoride, zinc chloride, zinc bromide, zinc carbonate, pt-butyl benzoate, zinc thiocyanate, zinc lactate, zinc acetate, and An aqueous solution containing hyaluronic acid or a hyaluronic acid salt, which is at least one zinc compound selected from the group consisting of zinc oxide. 4. The method according to any one of claims 1 to 3,
Aqueous solution containing hyaluronic acid or a hyaluronic acid whose content of the said zinc compound is 0.01-100 microgram / mL. 5. The method according to any one of claims 1 to 4,
Aqueous solution containing hyaluronic acid or a hyaluronic acid salt whose average molecular weight of the hyaluronic acid or a hyaluronic acid salt is 1 million or more. The method according to any one of claims 1 to 5,
An aqueous solution containing hyaluronic acid or hyaluronic acid salt, wherein the hyaluronic acid salt is sodium hyaluronate. A pharmaceutical composition comprising an aqueous solution containing the hyaluronic acid or the hyaluronic acid salt according to any one of claims 1 to 6. An injection for treating arthrosis, comprising an aqueous solution containing hyaluronic acid or a hyaluronic acid salt according to any one of claims 1 to 6. Cosmetic composition containing the aqueous solution containing the hyaluronic acid or hyaluronic acid salt in any one of Claims 1-6. A composition containing hyaluronic acid or a hyaluronic acid salt and a bivalent soluble iron, and a step of dissolving the zinc compound in an aqueous solution,
The composition is a method for producing an aqueous solution containing hyaluronic acid or a hyaluronic acid salt having a divalent soluble iron content in the aqueous solution of 5 ppb or less when dissolved in an aqueous solution such that the concentration of hyaluronic acid or hyaluronic acid salt is 10 mg / mL. A composition containing hyaluronic acid or a hyaluronic acid salt and a bivalent soluble iron, and a step of dissolving the zinc compound in an aqueous solution,
The composition is a method for promoting stabilization of an aqueous solution containing hyaluronic acid or a hyaluronic acid having a content of divalent soluble iron in an aqueous solution of 5 ppb or less when dissolved in an aqueous solution such that the concentration of hyaluronic acid or a hyaluronic acid salt is 10 mg / mL. . Containing zinc compound,
Stabilizer for stabilizing the aqueous solution containing hyaluronic acid or a hyaluronic acid whose content rate of bivalent soluble iron is 5 ppb or less.

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