Production of a hyaluronic acid gel, which comprises keeping hyaluronic acid in water at a hyaluronic acid concentration of at least 5 wt % in the presence of an acid component in an amount at least equimolar with the carboxyl groups in the hyaluronic acid.

DISCLOSURE OF THE INVENTION

Namely, the present invention provides (1) a method of producing a hyaluronic acid gel, which comprises keeping hyaluronic acid in water at a hyaluronic acid concentration of at least 5 wt % in the presence of an acid component in an amount at least equimolar with the carboxylic groups in the hyaluronic acid, (2) the method of producing a hyaluronic acid gel according to (1), which comprises keeping hyaluronic acid in water at a hyaluronic acid concentration of at least 5 wt % in the presence of an acid component in an amount at least equimolar with the carboxyl groups in the hyaluronic acid at from -10° C. to 30° C., (3) a method of producing a hyaluronic acid gel, which comprises keeping hyaluronic acid in water at a hyaluronic acid concentration of at least 5 wt % in the presence of an acid component in an amount at least equimolar with the carboxyl groups in the hyaluronic acid at from -10° C. to 30° C. to form a hyaluronic acid gel and treating the gel with a neutralizing liquid, (4) a method of producing a hyaluronic acid gel, which comprises keeping an acidic hyaluronic acid aqueous solution containing hyaluronic acid at a concentration of at least 5 wt % and an acid component in an amount at least equimolar with the carboxyl groups in the hyaluronic acid at from -10° C. to 30° C. to form a hyaluronic acid gel and treating the gel with a neutralizing liquid, (5) a method of producing a hyaluronic acid gel, which comprises keeping an acidic hyaluronic acid mixture obtained by mixing hyaluronic acid and an acidic aqueous solution containing an acid component in an amount at least equimolar with the carboxyl groups in the hyaluronic acid to a hyaluronic acid concentration of at least 5 wt % based on the acidic aqueous solution at from -10° C. to 30° C. to form a hyaluronic acid gel and treating the gel with a neutralizing liquid, (6) a method of producing a hyaluronic acid gel, which comprises keeping hyaluronic acid impregnated with an acidic aqueous solution containing an acid component in an amount at least equimolar with the carboxyl groups in the hyaluronic acid to a hyaluronic acid concentration of at least 5 wt % based on the acidic aqueous solution at from -10° C. to 30° C. to form a hyaluronic acid gel and treating the gel with a neutralizing liquid, (7) a gel made of hyaluronic acid alone which is hardly soluble in a neutral aqueous solution and has transparency, (8) the hyaluronic acid gel according to (7), which dissolves in a neutral aqueous solution at 25° C. in one day to a degree of dissolution of at most 50%, (9) the hyaluronic acid gel according to (7), which dissolves to yield solubilized hyaluronic acid having a branched structure and partly containing a molecular weight fraction with a branching degree of at least 0.5, when treated under accelerating conditions for acid hydrolysis of hyaluronic acid, (10) a biomedical material containing a gel made of hyaluronic acid alone which has transparency and dissolves in a neutral aqueous solution at 25° C. in one day to a degree of dissolution of at most 50%, (11) a biomedical material containing a gel made of hyaluronic acid alone which satisfies the following requirement and has transparency: the hyaluronic acid gel dissolves to yield solubilized hyaluronic acid having a branched structure and partly containing a molecular weight fraction with a branching degree of at least 0.5, when treated under accelerating conditions for acid hydrolysis of hyaluronic acid, (12) a biomedical material containing a hyaluronic acid gel having transparency and un-gelled hyaluronic acid, wherein the hyaluronic acid gel dissolves in a neutral aqueous solution at 25° C. in one day to a degree of dissolution of at most 50%, and dissolves to yield solubilized hyaluronic acid having a branched structure and partly containing a molecular weight fraction with a branching degree of at least 0.5, when treated under accelerating conditions for acid hydrolysis of hyaluronic acid, (13) the biomedical material according to any one of (10) to (12), wherein the hyaluronic acid gel having transparency is flaky, (14) the biomedical material according to any one of (10) to (13), which is an injection for treatment of arthrosis, (15) the biomedical material according to any one of (10) to (13), which is an embolizing material, (16) the biomedical material according to any one of (10) to (13), which is an injection for a soft tissue, and (17) the biomedical material according to any one of (10) to (13), which is an artificial vitreous body.

The present invention provides a hardly water soluble hyaluronic acid gel made of hyaluronic acid alone with transparency. The hyaluronic acid gel according to the present invention retains the structure of the biologically inherent hyaluronic acid by virtue of obviation of use of crosslinkers, and is excellently safe and biocompatible. Therefore, it is useful as a biomedical material such as an injection for treatment of arthrosis, an embolizing material, an injection for a soft tissue and an artificial vitreous body.

BEST MODE FOR CARRYING OUT THE INVENTION

Now, the present invention will be described below in detail.

In the present invention, hyaluronic acid obtained by extraction from animal tissues or by fermentation may be used without any restriction on its source.

The strain used in fermentation is preferably a hyaluronic acid-producing microorganism isolated from nature such as the genus Streptococcus or a mutant which steadily produces hyaluronic acid in high yield such as Streptococcus equi FM-100 (accession number 9027 given by National Institute of Bioscience and Human-Technology) disclosed in JP-A-63-123392 or Streptococcus equi FM-300 (accession number 2319 given by National Institute of Bioscience and Human-Technology) disclosed in JP-A-2-234689. Pure hyaluronic acid obtained from cultures of the above-mentioned mutants may be used.

The molecular weight of the hyaluronic acid to be used in the present invention is preferably within the range of from about 1×10⁵ to about 1×10⁷ Da. Hyaluronic acid having a higher molecular weight may also be used after the molecular weight is lowered into this range by treatment such as hydrolysis.

In the present invention, the concept of hyaluronic acid is used so as to include its alkali metal salts such as sodium, potassium and lithium salts, too.

In the present invention, by hyaluronic acid alone, it is meant that no chemical crosslinker or chemical modifier is used other than hyaluronic acid, that hyaluronic acid is not in the form of a complex with a cationic polymer, and that the gel is an auto-crosslinked gel.

On the other hand, substances which do not directly induce introduction of a crosslinked structure into hyaluronic acid or make hyaluronic acid insoluble or hardly soluble may be added when the hyaluronic acid gel according to the present invention is prepared.

Further, in preparation of a hyaluronic acid gel, pharmacologically or physiologically active substances may be added to give a hyaluronic acid gel containing such substances without any restriction.

The hyaluronic acid gel according to the present invention is characterized in that it is hardly soluble in a neutral aqueous solution.

In the present invention, the difficulty in dissolution is defined by the solubility in a neutral aqueous solution at 25° C. and means that the gel dissolves in a neutral aqueous solution at 25° C. in 12 hours to a degree of dissolution of at most 50%, preferably at most 30%, particularly preferably at most 10%.

The hyaluronic acid gel according to the present invention is a polymer having a three dimensional network structure or its swollen product. The three dimensional network structure is made of crosslinked hyaluronic acid.

The hyaluronic acid gel according to the present invention can be solubilized through degradation by treatment under accelerating conditions for acid hydrolysis of hyaluronic acid. When the solubilized hyaluronic acid retains the crosslinked structure, it is distinguished as branched hyaluronic acid from linear hyaluronic acid according to the theory of polymer solution.

The accelerating conditions for acid hydrolysis of hyaluronic acid according to the present invention are preferably such that the pH of the aqueous solution is 1.5 and the temperature is 60° C. It is well known that cleavage of the main chain of hyaluronic acid through hydrolysis of glycosidic bonds is remarkably accelerated in an acidic or alkaline aqueous solution as compared with that in a neutral aqueous solution. In addition, acid hydrolysis is accelerated at a higher temperature.

In the present invention, the molecular weights and branching degrees of the fractions separated by GPC according to molecular weight are measured on-line continuously by the GPC-MALLS method. In the present invention, the branching degree was measured by the elution volume method which compares the molecular weight of each fraction of the solubilized hyaluronic acid with the molecular weight of a fraction at the same elution volume of linear hyaluronic acid as a control. The branching degree is the number of branch points in one polymer chain of the solubilized hyaluronic acid and plotted against the molecular weight of the solubilized hyaluronic acid. Measurement of the branching degree by the GPC-MALLS method by the elution volume method is described in PCT/JP98/03536.

Solubilized hyaluronic acid was diluted with the GPC eluent for concentration adjustment and filtered through a membrane filter of 0.2 μm before measurement.

If the hyaluronic acid gel according to the present invention has a crosslinked structure which is stable under accelerating conditions for acid hydrolysis of hyaluronic acid, a branched structure is recognized in the solubilized hyaluronic acid according to the theory of polymer solution. The hyaluronic acid gel according to the present invention has a branching degree of at least 0.5.

In the present invention, transparency means that the visible light transmittance of the hyaluronic acid gel of the present invention in a spectrometric cuvette of 10 mm thick measured at 340 nm to 800 nm is at least 50%, preferably 70%, particularly preferably 90%, based on the transmittance of water.

The transparent hyaluronic acid gel of the present invention is obtained by keeping hyaluronic acid in water at a hyaluronic acid concentration of at least 5 wt % in the presence of an acid component in an amount at least equimolar with the carboxyl groups in the hyaluronic acid, without freezing.

The amount of the acid component to be used for acidification in the present invention is usually preferred to be at least equimolar with the carboxyl groups in hyaluronic acid, although it is set depending on various factors such as the type of the counterion in the hyaluronic acid salt, the molecular weight of hyaluronic acid, the concentration of hyaluronic acid and the properties of the resulting gel such as strength.

As the acid component, any acid that is stronger than hyaluronic acid in acidity may be used. Preferably, a strong acid such as hydrochloric acid, nitric acid and sulfuric acid is used to decrease the amount of an acid.

When the hyaluronic acid concentration is below 5 wt %, a hyaluronic acid gel is not obtained, whether or not a sufficient proportion of the carboxyl groups in the hyaluronic acid undergo protonation.

In the present invention, it is necessary to put hyaluronic acid, water and an acid component in an amount at least equimolar with the carboxyl groups in the hyaluronic acid together so that the hyaluronic acid concentration is 5 wt %, and then keep their coexistence so that gellation proceeds, for example, by letting them stand until a certain period of time passes.

Further, though the keeping temperature and time are set depending on various factors such as the type of the counterion in the hyaluronic acid salt, the molecular weight of hyaluronic acid, the concentration of hyaluronic acid and the properties of the resulting gel such as strength, the temperature is preferably from -10° C. to 30° C. to prevent water from freezing and prevent acid decomposition of hyaluronic acid.

If the acidic hyaluronic acid aqueous solution freezes, an opaque hyaluronic acid gel is obtained.

The acidic hyaluronic acid aqueous solution with a hyaluronic acid concentration of at least 5 wt % to be used in the present invention may be prepared by any methods, for example, by mixing hyaluronic acid with an acidic aqueous solution, by impregnating hyaluronic acid with an acidic aqueous solution, by concentrating a low concentration acidic hyaluronic acid aqueous solution to a predetermined concentration or by adding an acid component to a dense hyaluronic acid aqueous solution.

In the present invention, hyaluronic acid may be used in any form, for example, in powdery form, in the form of a molded block obtained from hyaluronic acid powder by compacting or in the form of a cast film or a sponge obtained from an aqueous solution of hyaluronic acid in distilled water by air-drying or freeze-drying. The mixing of hyaluronic acid with an acidic aqueous solution may be accomplished by addition of the acidic aqueous solution to hyaluronic acid and subsequent kneading.

The impregnation of hyaluronic acid with an acidic aqueous solution may be conducted so as to attain a predetermined hyaluronic acid concentration.

In the case where a dilute acidic hyaluronic acid aqueous solution is concentrated to a predetermined concentration, the low concentration acidic hyaluronic acid aqueous solution is prepared firstly by adding an acid component to hyaluronic acid dissolved in distilled water or dissolving hyaluronic acid directly in an acidic aqueous solution and may be prepared from any form of hyaluronic acid. The low concentration is intended to be lower than the hyaluronic acid concentration of the intended hyaluronic acid gel and is preferably lower than 5 wt % for easy handling. The concentration may be accomplished by ultracentrifugation, air-drying, vacuum drying or freeze-drying.

In the case where an acid component is added to a dense hyaluronic acid aqueous solution, the dense hyaluronic acid aqueous solution is prepared firstly by mixing hyaluronic acid with distilled water or by concentrating a dilute hyaluronic acid aqueous solution and may be prepared from any form of hyaluronic acid. The addition of an acid component to the dense hyaluronic acid aqueous solution may be accomplished by exposure to an atmosphere of a gaseous acid such as hydrogen chloride or by immersion in a solution of an acid in a solvent with little solvency for hyaluronic acid such as an ethanol-hydrochloric acid solution.

The hyaluronic acid gel obtained in accordance with the present invention has to be subjected to neutralization of hyaluronic acid in the acid form in which the carboxyl groups have protonated and the acid component used for acidification in order to avoid acid hydrolysis of hyaluronic acid. For neutralization, an aqueous solvent such as phosphate buffer or aqueous sodium hydroxide is usually used, though there is no restriction on the aqueous solvent so long as it does not functionally impair the hyaluronic acid gel.

Herein, such a solvent is generically referred to as a neutralizing liquid.

Although there is no particular restriction on the treatment with a neutralizing liquid (the neutralization method), a batch method, a filtration method or a method in which a solvent is passed through a loaded column is usually used. The neutralization conditions, inclusive of the volume of the neutralization liquid and the number of times of neutralization, may be selected appropriately considering the shape and the use of the hyaluronic acid gel so that hyaluronic acid in the acid form and the acid component used for acidification are neutralized.

The hyaluronic acid gel treated with a neutralizing liquid is used in an immersed state with a solvent, in a wet state with a solvent or in a dry state after air-drying, vacuum drying or freeze drying depending on the use.

With a view to shaping the hyaluronic acid gel, by selecting hyaluronic acid, the vessel for the acidic hyaluronic acid aqueous solution and the procedure, a hyaluronic acid gel of desired shape such as a sheet-like, filmy, flaky, spongy, massive or tubular shape can be obtained. For example, from compacted hyaluronic acid power, a gel in the form of a block or a sheet is obtained. Preparation of a hyaluronic acid gel may be followed by post-treatment such as mechanical fragmentation, freezing-thawing, rolling or spinning to make the gel into fine flakes, a sponge, a film or the like.

The hyaluronic acid gel of the present invention is obtainable endotoxin-free and aseptically if care is taken over the reagents, water and the vessels.

The hyaluronic acid gel thus prepared is transparent in itself and retains transparency even after crushed and suspended. It may be filled into a syringe or a bag before use. If pharmaceutically or physiologically active substances are added at the time of gelation, the resulting hyaluronic acid gel contains these substances in it.

For example, addition of thrombin which coagulates blood by catalyzing conversion of fibrinogen into fibrin in the blood coagulation cascade with a view to accelerating embolization and addition of various antitumor agents with a view to obstructing tumor arteries may be mentioned without any restriction.

The hyaluronic acid gel of the present invention shows great improvement in in vivo residency and persistency over hyaluronic acid solution and excellently safe and biocompatible by virtue of the absence of crosslinkers. Therefore, it can be used as a biomedical material such as an injection for treatment of arthrosis, an embolizing material, an injection for a soft tissue and an artificial vitreous body.
 

Claim 1 of 21 Claims

1. A biomedical material which contains a transparent gel consisting of hyaluronic acid or a salt thereof; a second acid or a salt thereof; and water;

wherein the transparent gel does not contain any cross-linkers, and

wherein at least one of the following requirements (a), (b) and (c) is satisfied:

(a) the hyaluronic acid gel dissolves in a neutral aqueous solution at 25° C. in one day to a degree of dissolution of at most 50%,

(b) the hyaluronic acid gel dissolves to yield a solubilized hyaluronic acid having a branched structure and partly containing a molecular weight fraction with a branching degree of at least 0.5, when treated under accelerating conditions for acid hydrolysis of hyaluronic acid, or

(c) the hyaluronic acid gel satisfies the above requirements (a) and (b), and the biomedical material also contains an un-gelled hyaluronic acid.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.