JP5957440B2 - An aqueous solution containing hyaluronic acid or a salt thereof - Google Patents

Description

  The present invention relates to an aqueous solution containing hyaluronic acid or a salt thereof containing a carboxylic acid or a salt thereof having a low content of divalent soluble iron and containing no amino group and thiol group.

  Hyaluronic acid is known as a polysaccharide composed of linked disaccharide units of N-acetylglucosamine and glucuronic acid. Hyaluronic acid is generally used as a raw material for pharmaceuticals, cosmetics, foods and the like. As a production method of hyaluronic acid, there are a method of producing with an extract from a chicken crown or the like, and a method of producing by a fermentation method using a microorganism.

  For example, Patent Document 1 describes that hyaluronic acid was produced by fermentation using Streptococcus tin-epidemicus. In Patent Document 1, the yield and molecular weight of hyaluronic acid are higher when ferrous sulfate is added to a concentration of 3 times during the culture (Example 3) than when not added (Example 1). It is described that it has increased.

  Patent Document 2 describes that hyaluronic acid powder was produced by fermentation using Streptococcus equi (ATCC9527). Patent Document 3 describes experimental data indicating that sodium hyaluronate crystals produced based on the method of Patent Document 2 contain a large amount of calcium, magnesium, and iron. Patent Document 3 discloses a sodium hyaluronate crystal in which the content of calcium, magnesium, and iron is reduced to less than 10 ppm by further contacting a chelate resin with the hyaluronic acid-containing liquid obtained based on the method of Patent Document 2. It is described that obtained.

JP-A-9-56394 7-2117 JP2008-280430

However, the prior art described in the above literature has room for improvement in the following points.
First, in the production method of Patent Document 1, since ferrous sulfate (iron (II) sulfate, FeSO ₄ ) is added in a large amount to the medium, iron is likely to be mixed into the purified hyaluronic acid-containing solution. Conceivable. In addition, in order to remove iron from a medium containing a large amount of ferrous sulfate, a higher cost than usual is required.

  Secondly, in the production method of Patent Document 2, the hyaluronic acid powder contained a large amount of calcium, magnesium, and iron, and the purity of sodium hyaluronate was low.

  Thirdly, in the production method of Patent Document 3, it is described that the content of calcium, magnesium, and iron in the sodium hyaluronate crystal is reduced to less than 10 ppm, but what was the specific content? Is unknown. The analysis results are in the order of ppm and lack precision. Furthermore, it is not described what specific effects can be obtained by reducing the contents of calcium, magnesium, and iron.

  The present invention has been made in view of the above circumstances, and since the content of divalent soluble iron is 5 ppb or less and includes a carboxylic acid or a salt thereof that does not contain an amino group and a thiol group, the stability is improved. An object is to provide an aqueous solution containing high hyaluronic acid or a salt thereof.

  As a result of researches on impurities contained in an aqueous solution containing hyaluronic acid or a salt thereof, the present inventors have found that the higher the content of divalent soluble iron mixed in the hyaluronate aqueous solution, the higher the hyaluronic acid molecule It was found for the first time that the stability of was reduced. In addition, the inventors surprisingly found that the stability of hyaluronic acid molecules does not decrease even when the content of trivalent soluble iron is large, and the content of divalent soluble iron is large. We have also found that the stability of hyaluronic acid molecules is reduced.

  Therefore, the present inventors reduced the content of divalent soluble iron mixed in an aqueous solution containing hyaluronic acid or a salt thereof, and improved the stability of hyaluronic acid molecules by divalent soluble iron. As a result of searching for an additive that suppresses the decrease, it is possible to suppress a decrease in the stability of hyaluronic acid molecules due to divalent soluble iron by adding a carboxylic acid or a salt thereof that does not contain an amino group and a thiol group. I found it for the first time. In addition, the inventors surprisingly added glycine, L-aspartate Na, Na bromide, Na hydrogen sulfite, sulfide Na, thioglycolate Na, glucose, purified sucrose, or ascorbic acid. Found that the decrease in the stability of hyaluronic acid molecules due to divalent soluble iron was not suppressed, and that the addition of the divalent soluble iron often resulted in a decrease in stability, and thus completed the present invention.

  That is, according to the present invention, there is provided an aqueous solution containing hyaluronic acid or a salt thereof containing a carboxylic acid or a salt thereof not containing an amino group and a thiol group, wherein the content of divalent soluble iron is 5 ppb or less. Is done. The aqueous solution containing this hyaluronic acid or a salt thereof has been demonstrated in Examples to be described later that the intrinsic viscosity remaining rate after storage is good. Therefore, it is suitable for raw materials such as pharmaceuticals, cosmetics and foods.

  Moreover, according to this invention, the pharmaceutical composition containing the aqueous solution containing the said hyaluronic acid or its salt is provided. This pharmaceutical composition contains an aqueous solution containing hyaluronic acid or a salt thereof having a good residual intrinsic viscosity after storage. For this reason, it is difficult for a decrease in viscosity or a decrease in quality to occur over time.

  Moreover, according to this invention, the injection for treatment of arthropathy containing the aqueous solution containing the said hyaluronic acid or its salt is provided. This injection for treatment of arthropathy includes an aqueous solution containing hyaluronic acid or a salt thereof having a good residual intrinsic viscosity after storage. Therefore, good retention is obtained when administered in or around the joint.

  Moreover, according to this invention, the cosmetics composition containing the aqueous solution containing the said hyaluronic acid or its salt is provided. This cosmetic composition contains an aqueous solution containing hyaluronic acid or a salt thereof having an excellent residual viscosity after storage. For this reason, it is difficult for a decrease in viscosity or a decrease in quality to occur over time.

  Further, according to the present invention, the step of dissolving the composition containing hyaluronic acid or a salt thereof and divalent soluble iron and the carboxylic acid or a salt thereof containing no amino group and thiol group in an aqueous solution. The composition containing hyaluronic acid or a salt thereof having a divalent soluble iron content of 5 ppb or less when dissolved in an aqueous solution so that the concentration of hyaluronic acid or a salt thereof is 10 mg / mL A method for producing an aqueous solution containing the salt is provided. According to this production method, it is possible to produce hyaluronic acid or a salt thereof having an excellent residual residual viscosity after storage.

  Further, according to the present invention, the step of dissolving the composition containing hyaluronic acid or a salt thereof and divalent soluble iron and the carboxylic acid or a salt thereof containing no amino group and thiol group in an aqueous solution. The composition containing hyaluronic acid or a salt thereof having a divalent soluble iron content of 5 ppb or less when dissolved in an aqueous solution so that the concentration of hyaluronic acid or a salt thereof is 10 mg / mL A method for promoting stabilization of an aqueous solution containing the salt is provided. If the carboxylic acid or salt thereof containing no amino group or thiol group is contained in an aqueous solution containing hyaluronic acid or a salt thereof having a divalent soluble iron content of 5 ppb or less, the intrinsic viscosity remaining rate after storage It has been demonstrated in the examples described later that the improvement is significantly improved. Therefore, according to this stabilization promoting method, stabilization of an aqueous solution containing hyaluronic acid or a salt thereof can be significantly promoted.

  Further, according to the present invention, to stabilize an aqueous solution containing hyaluronic acid or a salt thereof having a divalent soluble iron content of 5 ppb or less, including a carboxylic acid or a salt thereof not containing an amino group and a thiol group. A stabilizer is provided. If the carboxylic acid or salt thereof containing no amino group or thiol group is contained in an aqueous solution containing hyaluronic acid or a salt thereof having a divalent soluble iron content of 5 ppb or less, the intrinsic viscosity remaining rate after storage It has been demonstrated in the examples described later that the improvement is significantly improved. Therefore, if this stabilizer is added to an aqueous solution containing hyaluronic acid or a salt thereof having a divalent soluble iron content of 5 ppb or less, the stabilizer can be remarkably stabilized.

  According to the present invention, since the content of divalent soluble iron is 5 ppb or less and includes a carboxylic acid or a salt thereof that does not contain an amino group and a thiol group, the highly stable hyaluronic acid or a salt thereof is included. An aqueous solution is obtained.

FIG. 1 is a graph showing a change in intrinsic viscosity residual rate when sodium hyaluronate powder is added to an aqueous solution containing divalent soluble iron and Na citrate.

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

<Aqueous solution containing hyaluronic acid or a salt thereof>
One embodiment of the present invention is an aqueous solution containing hyaluronic acid or a salt thereof containing a carboxylic acid or a salt thereof containing a divalent soluble iron of 5 ppb or less and containing no amino group and thiol group. An aqueous solution containing hyaluronic acid or a salt thereof having such a composition has a high residual residual viscosity after storage and is excellent in physical stability, as demonstrated in Examples described later. In addition, the viscosity and quality are hardly lowered even after long-term storage or after a long period of time, and it is suitable for raw materials such as pharmaceuticals, cosmetics and foods.

  The carboxylic acid or salt thereof containing no amino group and thiol group is, for example, one or more compounds selected from the group consisting of citric acid, lactic acid, tartaric acid, salicylic acid, succinic acid, malic acid, and acetic acid, or salts thereof. There may be. Here, as a salt, the salt which sodium, potassium, zinc, iron, calcium, or ammonium couple | bonded can be mentioned, for example. The citrate salt may be potassium hydrogen citrate. Carboxylic acid is a compound having an R-COOH structure. The carboxylic acid may have 2, 3, 4, 5, 6, 7, 12, or 15 carbon atoms, for example. The number of carbon atoms may be greater than or equal to the value exemplified here, or may be within the range of any two values. In addition, the number of carboxyl groups in the molecule of the carboxylic acid may be 1, 2, 3, 4, 5, or 10, for example. The number of carboxyl groups may be greater than or equal to the value exemplified here, or may be in the range of any two values. The carboxylic acid may be a saturated carboxylic acid or an unsaturated carboxylic acid. The carboxylic acid may contain a hydroxyl group. The number of hydroxyl groups may be 1, 2, 3, 4, 5, or 10, for example. The number of hydroxyl groups may be greater than or equal to the value exemplified here, or may be in the range of any two values.

  The above 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 be equal to or less than the value exemplified here, or may be in the range of any two values. This content is preferably smaller from the viewpoint of improving the stability of an aqueous solution containing hyaluronic acid or a salt thereof.

  The content of the carboxylic acid or salt thereof containing no amino group and thiol group contained in the aqueous solution containing the hyaluronic acid or salt thereof of the present embodiment is, for example, 5, 10, 30, 50, 100, 150, 300. 500, 800, 1000, 1500, 2000, 5000 or 10000 μg / mL. Moreover, this content rate may be in the range of any two values exemplified here. The content is preferably 10 μg / mL or more, more preferably 500 μg / mL or more from the viewpoint of further improving the stability of the aqueous solution containing hyaluronic acid or a salt thereof. The content is preferably 2000 μg / mL or less, more preferably 1500 μg / mL or less, from the viewpoint of reducing production cost or improving operability.

  The average molecular weight of sodium hyaluronate or a salt thereof contained in the aqueous solution containing hyaluronic acid or a salt thereof of the present embodiment is, for example, 500,000, 800,000, 1,000,000, 1.5 million, 1.8 million, 2 million, 2.5 million, 300 May be 5 million, 5 million, or 8 million. This average molecular weight may be within the range of any two values exemplified here. From the viewpoint of further improving the viscosity of an aqueous solution containing hyaluronic acid or a salt thereof, it is preferably 1 million or more, more preferably 1.5 million or more. An aqueous solution containing high-viscosity hyaluronic acid or a salt thereof is excellent in retention in the affected area when used as an injection for the treatment of arthropathy. The average molecular weight of hyaluronic acid or a salt thereof contained in an aqueous solution containing hyaluronic acid or a salt thereof is calculated by measuring Laurent's formula (LAURENT et al., Biochim Biophys Acta. 1960 Aug 26; 42: 476-485 .).

  The content of hyaluronic acid or a salt thereof contained in the aqueous solution containing hyaluronic acid or a salt thereof according to the present embodiment is, for example, 0.1, 1, 5, 8, 9, 10, 11, 12, 15, or 20 mg / mL. It may be. This content may be within the range of any two values exemplified here. From the viewpoint of therapeutic effect or operability when used for injections and the like, it is preferably in the range of 5 and 15 mg / mL, more preferably in the range of 8 and 12 mg / mL.

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

  The aqueous solution containing hyaluronic acid or a salt thereof according to the present embodiment includes, for example, a composition containing hyaluronic acid or a salt thereof and divalent soluble iron, and a carboxylic acid or a salt thereof containing no amino group and thiol group. In the aqueous solution, the content of the divalent soluble iron in the aqueous solution when the hyaluronic acid or salt thereof is dissolved in the aqueous solution so that the concentration is 10 mg / mL. It can be obtained by a method for producing an aqueous solution containing hyaluronic acid or a salt thereof that is 5 ppb or less. This production method does not necessarily require complicated steps, and is excellent in productivity or cost.

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

In the present specification, “divalent soluble iron” is iron in a soluble state and is divalent. It can also be expressed as Fe ²⁺ or divalent iron.

<Pharmaceutical composition, etc.>
The aqueous solution containing hyaluronic acid or a salt thereof according to this embodiment can be used as a raw material of a pharmaceutical composition. In this case, since the pharmaceutical composition contains an aqueous solution containing hyaluronic acid or a salt thereof excellent in stability, the viscosity and quality are hardly lowered even after long-term storage. Further, for example, when it is administered in or around the joint, it can stay in the affected area for a long time or a desired time. The dosage form of this pharmaceutical composition is not particularly limited, but an injection is preferable from the viewpoint that it can be directly administered to an affected area such as a joint. As the container for the injection, for example, a syringe, a vial, or an ampoule can be used. Upon administration, it can be administered alone, but can be mixed with one or more pharmacologically acceptable carriers or excipients and any well known in the pharmaceutical arts. It is preferably provided as a pharmaceutical preparation produced by the method. This pharmaceutical composition is prepared according to the dosage form by using a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, lidocaine hydrochloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum albumin). , Polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The pharmaceutical composition may also contain sodium hydrogen phosphate, crystalline sodium dihydrogen phosphate, and sodium chloride as additives. The adjusted pharmaceutical composition can be administered to, for example, humans and mammals (eg, rats, mice, rabbits, dogs, monkeys, sheep, pigs, cows, cats, etc.). The pharmaceutical composition includes a composition used for the purpose of prevention.

  The administration method of this pharmaceutical composition can be appropriately selected depending on the age, symptoms, affected area, etc. of the subject. When used for the treatment of osteoarthritis of the knee, for example, 2.5 mL of an adult can be administered into the knee joint cavity 5 times continuously every week. Alternatively, for the purpose of maintaining symptoms, it can be administered at intervals of 2 to 4 weeks. In addition, when used for the treatment of peri-arthritis, for example, 2.5 mL of an adult once a week for 5 consecutive shoulder joints (shoulder joint cavity, subacromial bursa, or biceps long head tendon) Can be administered within the tendon sheath). When used for the treatment of knee joint pain in rheumatoid arthritis, for example, 2.5 mL of an adult can be administered into the knee joint cavity 5 times continuously every week. It may also be administered in combination with an appropriate chemotherapeutic agent.

  Examples of the pharmacological action of this pharmaceutical composition include: a) viscoelasticity or lubrication action by hyaluronate binding to the cartilage tissue and covering the surface, b) cartilage matrix stabilization action (degeneration suppression) C) analgesic action by covering the surface of inflammatory cells and synovial cells, or by suppressing the production of analgesic substances, or d) close to arthritis with synovial and cartilage degeneration Examples thereof include inflammatory effects expressed by affecting synovial cells, chondrocytes, or inflammatory cells such as neutrophils and macrophages. By these actions, for example, reduction of pain, improvement of daily life activities and joint range of motion are expected.

  The aqueous solution containing hyaluronic acid or a salt thereof according to this embodiment can be used as a raw material of a cosmetic composition. In this case, since the cosmetic composition includes an aqueous solution containing hyaluronic acid or a salt thereof excellent in stability, the viscosity and quality are not easily lowered even after long-term storage. In addition, when used as a moisturizing cosmetic composition, the moisturizing effect tends to be sustained.

<Stabilization method, etc.>
Another embodiment includes dissolving a composition containing hyaluronic acid or a salt thereof and divalent soluble iron and a carboxylic acid or a salt thereof containing no amino group and thiol group in an aqueous solution. The composition is hyaluronic acid or a salt thereof, wherein the content of divalent soluble iron in the aqueous solution is 5 ppb or less when dissolved in the aqueous solution so that the concentration of hyaluronic acid or a salt thereof is 10 mg / mL Is a method for promoting the stabilization of an aqueous solution containing. According to this method, an aqueous solution containing hyaluronic acid or a salt thereof having high physical stability can be obtained. Further, this method does not necessarily require a complicated process and is excellent in convenience.

  Moreover, the carboxylic acid or its salt which does not contain the said amino group and thiol group can be used as a raw material of the stabilizer of the aqueous solution containing a hyaluronic acid or its salt. If this stabilizer is used, the physical stability of an aqueous solution containing hyaluronic acid or a salt thereof having a divalent soluble iron content of 5 ppb or less can be improved. This stabilizer may be in powder or liquid form. In the case of liquid, a buffer may be included.

  As mentioned above, although embodiment of this invention was described, these are illustrations of this invention and various structures other than the above are also employable. Moreover, it is also possible to adopt a combination of the configurations described in the above embodiments.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these.

<Example 1>
A sodium hyaluronate aqueous solution was prepared by the following procedure. First, 1 liter of medium consisting of 5% glucose, 0.2% monopotassium phosphate, 1.0% polypeptone, and 0.5% yeast extract was heat sterilized, and then Streptococcus ex FM-100 (Mikken Kenjo) No. 9027). The culture was carried out for 20 hours at 200 rpm with stirring, at a temperature of 33 ° C., and at pH 8.5 (control by automatic dropping of 20% sodium hydroxide) while aeration of air at 1 vvm. This solution was filtered with a cell filtration device and dialyzed against water for 12 hours to recover the hyaluronic acid solution in the dialysis membrane. The collection container used was an inner surface made of glass in order to prevent iron contamination. Sodium chloride was added to 2.5% here, and ethanol was added twice as much as the hyaluronic acid solution to precipitate sodium hyaluronate. The precipitate was washed 5 times with ethanol, and the impurities were thoroughly washed out and then air-dried at 40 ° C. for 10 hours to obtain Na hyaluronic acid powder.

  Next, the obtained sodium hyaluronate powder was dissolved in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride, and sodium hyaluronate containing 5, 8, 10, 12, 15 mg / mL of hyaluronic acid Na, respectively. An aqueous solution was obtained. Furthermore, the intrinsic viscosity was measured, and the molecular weight of Na hyaluronate was calculated using Laurent's formula.

<Comparative Example 1>
Streptococcus equi FM-100 was cultured according to the procedure described in Example 1, and a sodium hyaluronate aqueous solution was purified by the same procedure as that described in Example 1 of Patent Document 3 (Japanese Patent Laid-Open No. 2008-280430). Specifically, first, 1 liter of a medium consisting of 5% glucose, 0.2% potassium phosphate, 1.0% polypeptone, and 0.5% yeast extract is heat sterilized, and then Streptococcus ex FM-100 is added. Vaccinated. The culture was carried out for 20 hours at 200 rpm with stirring, at a temperature of 33 ° C., and at pH 8.5 (control by automatic dropping of 20% sodium hydroxide) while aeration of air at 1 vvm. The culture solution is diluted 10 times with ion-exchanged water, and 5 g of activated carbon (Shirakaba RW50-T manufactured by Takeda Pharmaceutical Co., Ltd.) and pearlite (LocaHelp # 409, Mitsui Mining & Smelting Co., Ltd.) are added to the 2.5 L aqueous solution. 30 g was added, treated for 1 hour, and filtered using Nutsche. This operation was repeated twice to remove organic components in the medium and prepare a sodium hyaluronate-containing solution. Next, 68 ml of Mitsubishi Chemical's Diaion CR11 was packed in 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 above-mentioned sodium hyaluronate solution was passed through this chromatographic column at SV = 18 (1200 ml / hr). 2 g of sodium chloride was dissolved in 1 L of the chromatography column passing solution, adjusted to pH 7, precipitated with 2-propanol, and vacuum dried at 40 ° C. to obtain Na hyaluronic acid powder.

  Next, the obtained sodium hyaluronate powder was dissolved in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride, and sodium hyaluronate containing 5, 8, 10, 12, 15 mg / mL of hyaluronic acid Na, respectively. An aqueous solution was obtained. Furthermore, the intrinsic viscosity was measured, and the molecular weight of Na hyaluronate was determined using the Laurent equation.

<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 the hyaluronic acid Na was dissolved in 5, 8 An aqueous sodium hyaluronate solution containing 10, 12, or 15 mg / mL was prepared (No. 1 to 10), respectively. Next, for each aqueous solution, the amount of iron with a bivalent iron content, the amount of bivalent and trivalent soluble iron, and the total amount of iron are measured by the following procedures (1-1) to (1-3). did. The total iron amount includes soluble and insoluble iron amounts. The detection limit of the bivalent soluble iron amount, the bivalent and trivalent soluble iron amount, and the total iron amount was 5 ppb.

(1-1) Method for Analyzing Divalent Soluble Iron a) 2.75 g of an aqueous sodium hyaluronate solution is dispensed into 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) After dilution to about 10 mL, leave for about 10 minutes.
d) After adding 5 mL of chloroform, seal tightly. Shake for 5 minutes and extract.
e) After standing, 4 mL of chloroform phase is collected and transferred to another sample bottle.
f) After evaporation to dryness on a hot plate, 0.5 mL of concentrated nitric acid is added to cause acid decomposition.
g) Add 2 mL of dilute nitric acid (1 + 100) and dissolve the residue.
h) Quantify Fe by ICP emission analysis.

(1-2) Analysis method of divalent and trivalent soluble iron a) 2.75 g of hyaluronic acid Na aqueous solution is dispensed into a sample bottle.
b) Add 0.2 mL of hydroxylamine hydrochloride solution.
c) Add 0.2 mL of 1,10-phenanthroline solution and 0.5 mL of potassium thiocyanate solution.
d) After dilution to about 10 mL, leave for about 10 minutes.
e) After adding 5 mL of 4-methyl-2-pentanone, seal tightly. Shake for 1 minute and extract.
f) After standing, 4 mL of 4-methyl-2-pentanone phase is collected and transferred to another sample bottle.
g) After evaporation to dryness on a hot plate, 0.5 mL of concentrated nitric acid is added to cause acid decomposition.
h) Add 2 mL of dilute nitric acid (1 + 100) and dissolve the residue.
i) Quantify Fe by ICP emission analysis.

(1-3) Analytical method of total iron a) Dispense 2.75 g of sodium hyaluronate aqueous solution into a sample bottle.
b) Add 0.1 mL of HCl (1 + 1) and heat on a hot plate.
c) Analyzing Method of Divalent and Trivalent Soluble Iron (1-2) Analysis of Soluble Iron b) The following operations are performed.

(1-4) Analysis result of iron content Table 1 shows the analysis results of the above (1-1) to (1-3).

(2) Intrinsic Viscosity Measurement With respect to an 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 has a sodium hyaluronate concentration of 10 mg / mL. It melt | dissolved so that it might become (No. 11-12). Furthermore, after storing at 80 ° C. for 24 hours, the results of measuring intrinsic viscosity and intrinsic viscosity residual ratio (%) are shown in Table 2. The intrinsic viscosity was measured in accordance with the description of the viscosity section of the 15th revision Japanese Pharmacopoeia second supplement “Purified Sodium Hyaluronate”. The results are shown in Table 2.

As shown in Table 3, iron (II) chloride (FeCl ₂ ) was dissolved in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride (No. 13 to 19). Next, the sodium hyaluronate powder prepared in Example 1 was dissolved in each aqueous solution so that the sodium hyaluronate concentration was 10 mg / mL. Furthermore, after storing at 80 ° C. for 24 hours, the intrinsic viscosity and the intrinsic viscosity residual ratio (%) were measured. The results are shown in Table 3.

As shown in Table 4, iron (III) chloride (FeCl ₃ ) was dissolved in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride (No. 20 to 22). Next, the sodium hyaluronate powder prepared in Example 1 was dissolved in each aqueous solution so that the sodium hyaluronate concentration was 10 mg / mL. Furthermore, after storing 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) that the method described in Example 1 can be used, it is possible to prepare an aqueous solution of sodium hyaluronate having a mixed amount of divalent soluble iron of 5 ppb or less, and 2) hyaluronic acid can be obtained by divalent soluble iron. It was found that the intrinsic viscosity after storage of the aqueous acid Na solution was lowered, and 3) trivalent soluble iron did not affect the intrinsic viscosity after storage of the aqueous hyaluronate solution.

<Evaluation Example 2>
Various additives were dissolved in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride as shown in Table 5 (No. 23 to 36). Next, the sodium hyaluronate powder prepared in Example 1 was dissolved in each aqueous solution so as to have a concentration of 10 mg / mL. Furthermore, after storing at 80 ° C. for 24 hours, the intrinsic viscosity and the intrinsic viscosity residual ratio (%) were measured. Additives include: Na citrate, Na lactate, Na tartrate, Na salicylate, glycine, Na-L-aspartate, Na bromide, Na hydrogen sulfite, Na sulfide, Na thioglycolate, glucose, purified white sugar, and ascorbic acid It was used.

Additive: Na citrate: Wako Pure Chemical Industries, 194-13245
Lactic acid Na: Wako Pure Chemical Industries, 195-05965
Na tartrate: Wako Pure Chemical Industries, 190-03455
Na salicylate: Wako Pure Chemical Industries, 191-03142
Glycine: Wako Pure Chemical Industries, 036435
L-aspartate Na: Wako Pure Chemical Industries, 193-01262
Na bromide: Wako Pure Chemical Industries, 193-01505
Sodium hydrogen sulfite: Wako Pure Chemical Industries, 190-01375
Sodium sulfide: Wako Pure Chemical Industries, 197-03362
Sodium thioglycolate: Wako Pure Chemical Industries, 590-11762
Glucose: Wako Pure Chemical Industries, 076-05705
Purified white sugar (sucrose): Wako Pure Chemical Industries, 196-13705
Ascorbic acid Na: Wako Pure Chemical Industries, 196-01252

  As a result, when the aqueous solution in which the powder of sodium hyaluronate prepared in Example 1 is dissolved contains sodium citrate, sodium lactate, sodium tartrate, or sodium salicylate, the stability of the aqueous sodium hyaluronate solution is improved. I understood it. These are carboxylates that do not contain amino and thiol groups. Further, when Na bromide, Na hydrogen sulfite, Na sulfide, Na thioglycolate, glucose, or ascorbic acid was added, the stability was rather lowered.

<Evaluation Example 3>
Various additives were dissolved in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride as shown in Table 6 (Nos. 37 to 45). Further, 11.3 ppb (5 ppb as divalent soluble iron) was added to each aqueous solution. Next, the sodium hyaluronate powder prepared in Example 1 was dissolved in each aqueous solution so as to have a concentration of 10 mg / mL. Then, after storing for 24 hours at 80 ° C., the intrinsic viscosity and the intrinsic viscosity residual ratio (%) were measured.

  As a result, it was shown that the aqueous solution in which the sodium hyaluronate powder prepared in Example 1 was dissolved contained 5 ppb of divalent soluble iron, and the stability of the aqueous hyaluronate solution was lowered. Furthermore, it was found that the decrease in stability was significantly suppressed by Na citrate, Na lactate, Na tartrate, or Na salicylate.

<Evaluation Example 4>
Na citrate was dissolved at a concentration shown in Table 7 in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride (No. 46 to 52). Next, the sodium hyaluronate powder prepared in Example 1 was dissolved in each aqueous solution so as to have a concentration of 10 mg / mL. Furthermore, after storing 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 sodium hyaluronate powder prepared in Example 1 was dissolved contained sodium citrate at 10 μg / mL or more, and the stability of the aqueous hyaluronate solution was improved. In addition, the stability improvement was moderated when Na citrate was contained at 1000 μg / mL.

<Evaluation Example 5>
Citrate Na was dissolved in an aqueous solution containing 2 mM phosphate buffer and 0.9% sodium chloride so that the concentration would be 1000 μg / mL. Furthermore, iron chloride (II) having a concentration shown in Table 8 was added to each aqueous solution (No. 53 to 58). Next, the sodium hyaluronate powder prepared in Example 1 was dissolved in each aqueous solution so as to have a concentration of 10 mg / mL. Then, after storing for 24 hours at 80 ° C., the intrinsic viscosity and the intrinsic viscosity residual ratio (%) were measured.

  The results of Table 8 and Table 3 are graphed as shown in FIG. From FIG. 1, it can be seen that the stability of the aqueous solution of sodium hyaluronate is improved by adding 1000 μg / mL of sodium citrate to the hyaluronic solution containing a low concentration of divalent soluble iron. This stabilizing effect was particularly remarkable when the divalent soluble iron concentration was 5 ppb or less.

  As described above, the aqueous solution of sodium hyaluronate having a low content of divalent soluble iron had an improved residual intrinsic viscosity after storage. Furthermore, when the sodium hyaluronate aqueous solution contained Na citrate, the intrinsic viscosity remaining rate was further improved. This aqueous sodium hyaluronate solution has high stability and is less likely to cause a decrease in viscosity or quality even after long-term storage, and thus is excellent as a raw material for pharmaceutical compositions, for example. Moreover, since it is suitable for long-term storage, the cost can be reduced.

  In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

Claims (11)

An aqueous solution containing hyaluronic acid or a salt thereof, wherein the content of divalent soluble iron is 5 ppb or less and contains at least one compound selected from the group consisting of citric acid, lactic acid, tartaric acid, and salicylic acid, or a salt thereof.   The aqueous solution containing hyaluronic acid or a salt thereof according to claim 1, wherein the concentration of the hyaluronic acid or a salt thereof is 5 to 15 mg / mL. The aqueous solution containing hyaluronic acid or a salt thereof according to claim 1 or 2 , wherein the content of the compound or a salt thereof is 10 to 10,000 µg / mL. The aqueous solution containing hyaluronic acid or a salt thereof according to any one of claims 1 to 3 , wherein the hyaluronic acid or a salt thereof has an average molecular weight of 1,000,000 or more. The aqueous solution containing the hyaluronic acid or a salt thereof according to any one of claims 1 to 4 , wherein the salt of the hyaluronic acid is sodium hyaluronate. A pharmaceutical composition comprising an aqueous solution containing the hyaluronic acid or a salt thereof according to any one of claims 1 to 5 . An injection for treating arthritis, comprising an aqueous solution containing the hyaluronic acid or a salt thereof according to any one of claims 1 to 5 . Cosmetic composition containing the aqueous solution containing the hyaluronic acid in any one of Claims 1-5, or its salt. A composition containing hyaluronic acid or a salt thereof and divalent soluble iron;
One or more compounds selected from the group consisting of citric acid, lactic acid, tartaric acid and salicylic acid, or salts thereof;
A step of dissolving in an aqueous solution,
The composition has a content of divalent soluble iron in the aqueous solution of 5 ppb or less when dissolved in the aqueous solution so that the concentration of hyaluronic acid or a salt thereof is 10 mg / mL.
A method for producing an aqueous solution containing hyaluronic acid or a salt thereof. A composition containing hyaluronic acid or a salt thereof and divalent soluble iron;
One or more compounds selected from the group consisting of citric acid, lactic acid, tartaric acid and salicylic acid, or salts thereof;
A step of dissolving in an aqueous solution,
The composition has a content of divalent soluble iron in the aqueous solution of 5 ppb or less when dissolved in the aqueous solution so that the concentration of hyaluronic acid or a salt thereof is 10 mg / mL.
A method for promoting stabilization of an aqueous solution containing hyaluronic acid or a salt thereof. Including one or more compounds selected from the group consisting of citric acid, lactic acid, tartaric acid and salicylic acid, or salts thereof,
A stabilizer for stabilizing an aqueous solution containing hyaluronic acid or a salt thereof having a content of divalent soluble iron of 5 ppb or less.