JP2836952B2 - Crosslinked hyaluronic acid gel, its use and its production method - Google Patents

Description

The present invention relates to a crosslinked hyaluronic acid derivative which has been crosslinked by the reaction of a phosphorus-containing reagent with a phosphorus (V) acid derivative. The invention also relates to a process for the preparation of such a product, and to its use as a sustained release depot for the administration of hyaluronic acid or a medicament introduced into the gel.

Hyaluronic acid is a high molecular weight polysaccharide whose properties are extremely viscous and are composed of disaccharide repeating units of N-acetylglucosamine and glucuronic acid. It occurs naturally in human and animal organisms, for example, in synovial fluid, vitreous humor and pericardial fluid. Although the structure of hyaluronic acid is the same in all species, its molecular weight varies widely. Due to its bioabsorbability, lack of toxicity and immunological effects, hyaluronic acid can be used in medical contexts, e.g. in the treatment of joint disorders affecting the mobility of joints, and in connection with or post-operative eye surgery. It has been shown to be useful as a surgical adjunct for preventing adhesions. In such cases, hyaluronic acid has been used in the form of a viscous aqueous solution. However, in many cases, their durability is short and their mechanical stabilization is weak, and the desired therapeutic effect is not achieved.

An improvement in this regard has been achieved by chemically cross-linking hyaluronic acid to form an insoluble gel.
The preparation of such gels and their use as an alternative to vitreous humor and in the treatment of retinal detachment is described, for example, in US Pat. No. 4,716,154. First of all, PCT is a gel whose degradability can be adjusted, which is used as an anti-adhesion agent.
It is described in application WO 86/00912. A common feature of these hyaluronic acid gels is that a non-endogenous structure which is a "foreign body" to a living body is introduced into the substance during the crosslinking operation. This has undermined the effectiveness of the basic idea of using endogenous substances such as hyaluronic acid as the matrix for crosslinking. This is because this new substance actually contains a structure that is a "foreign substance" to a living body. As a result, endogenous, non-toxic, non-immunogenic hyaluronic acid has been transformed into a cross-linked substance that is recognized as "foreign" and produces an immune or inflammatory response.

The present inventors have surprisingly found a novel, biodegradable crosslinked hyaluronic acid gel derivative.
This derivative is produced by reacting hyaluronic acid with a phosphorus-containing reagent, especially a phospho (V) acid derivative,
Contains endogenous crosslinks or phosphate esters. Phosphate esters are widely present in vivo . Examples include phospholipids, DNA and RNA.

The phosphoric acid crosslinked hyaluronic acid gel of the present invention is superior in many other respects, for example, in its manufacturing process and its properties, to the manufacturing processes and properties of crosslinked hyaluronic acid materials in the prior art. The time for the crosslinking reaction is very short,
Also, since the reactive cross-linking reagent is hydrolyzed immediately, the materials of the present invention require minimal purification. The gel is degraded by the living body, and the degradation time can be varied within wide limits. Unlike conventional crosslinked hyaluronic acid gels, the gels of the present invention are capable of complete reswelling even after being completely dried.

Phosphate cross-linking of polysaccharides is a known method primarily for starch processing (eg, Koch, H. et al .: Strke, 34: 1).
6,1982). However, derivatization of starch
Treatment of insoluble materials in a heterogeneous system. The phosphoric acid cross-linking of hyaluronic acid can also be carried out heterogeneously on solid materials, for example in pyridine, but in order to obtain a highly reproducible, swellable gel substance, the cross-linking agent must The choice of the reaction to be treated is preferred. This treatment can be carried out in an organic solvent in which the hyaluronic acid is solubilized, for example, by the formation of a salt with a lipophilic cation. Surprisingly, however, we have:
It has been surprisingly found that when the reaction is carried out in an aqueous solution of a hyaluronate, a clear gel without turbidity is obtained. Performing this reaction in an aqueous solution is preferable from the viewpoint of handling and purification.

The crosslinking reagent used is a derivative of phosphorus (V) acid, in particular its halide, oxyhalide or anhydride. Examples of such crosslinking reagents include phosphorus pentachloride, phosphoryl chloride (phosphorous oxychloride) or its corresponding bromide or iodide, phosphorus pentoxide and trimetaphosphate.

The reaction is performed in an alkaline medium. Coupling and hydrolysis of the phosphoric acid derivative results in the release of a significant amount of acid. Both the phosphate ester formed and the hyaluronic acid matrix are susceptible to acid degradation. Therefore, it is very important that sufficient base is present from the very beginning of the reaction. This is because it is not possible to add anything to a viscous gelling system. At the same time, however, the pH of the initial solution must not be too high, as hyaluronic acid is susceptible to alkaline degradation. This means that in the case of crosslinking in aqueous solution, the pH must be between 9 and 14 (however, the pH value in the higher part of this range may be lower if the alkaline hyaluronate solution is prepared in situ upon cooling. Only means that the system should have sufficient buffer capacity. Bases that can be used include metal hydroxides such as sodium and potassium hydroxide. However, these hydroxides already produce high pH values at low concentrations and at the same time have low buffering capacity for acid neutralization. For this reason, other buffers with good buffering capacity should be used, for example nitrogen bases such as alkylamines, especially sterically hindered bases such as triethylamine, tributylamine and methylmorpholine. However, preference is also given to using basic metal phosphates such as trisodium phosphate or potassium phosphate for a preferred preparation by crosslinking in aqueous solution. In these gel post-treatment operations, the only by-product formed is the biologically resistant phosphate, so a further purification step to remove potentially toxic alkaline residues is required. No need to implement. If the reaction is carried out in anhydrous medium, it is also possible to use weaker bases than those mentioned above, for example pyridine.

When the cross-linking reaction is performed in a homogeneous system in a solution of hyaluronic acid, the concentration can be varied over a wide range. A practically convenient concentration range is the concentration of hyaluronic acid 2 in the reaction mixture.
~ 15% by weight. Gel formation already occurs at concentrations as low as 1%, but these gels exhibit a very liquid-like consistency and are of little value for technical purposes. There is no theoretical upper limit on the concentration of hyaluronic acid used in the crosslinking operation. However, already at moderate concentrations, high molecular weight hyaluronic acid sets a practical upper limit due to the fact that it forms a solution that is extremely difficult to treat. That is, for a hyaluronic acid having a molecular weight of, for example, about 10 ⁶ , the concentration should not exceed about 10%.

The molecular weight of the hyaluronic acid used for crosslinking can vary widely from about thousands to about millions. For example, depending on the concentration or the amount of the crosslinking agent, 20,000 to 5 × 10
It can be ⁶ . However, the preferred molecular weight range is about 10,000-4 × 10 ⁶ .

In addition to hyaluronic acid or hyaluronic acid salts such as sodium salts, other hyaluronic acid derivatives such as partially sulfated or esterified hyaluronic acid (EP
265, 116) can also be crosslinked according to the method of the invention.
This is, of course, also applicable to hyaluronic acid which has been subjected to certain other minor chemical modifications, for example as described in US Pat. No. 4,713,448.

The amount of crosslinker can also vary over a wide range depending on the molecular weight and concentration of hyaluronic acid. In a preferred embodiment using an aqueous solution of hyaluronic acid and phosphoryl chloride as the cross-linking agent, the latter amount is 10% based on hyaluronic acid.
Can vary from ~ 500% by weight.

The cross-linking reaction can also be performed at room temperature or somewhat higher. However, at these temperatures the reaction is remarkably fast, and gel formation occurs in a few minutes at room temperature. Therefore, a lower temperature, for example, a temperature in the range of 0 to 10 ° C., is selected to sufficiently control the reaction.

However, in a preferred reaction system in which hyaluronic acid is cross-linked in an aqueous solution, most of the cross-linking reagent is not completely solubilized, and these reagents and the aqueous phase form a two-phase system. It should be noted that It is therefore important that the contact area between the crosslinker phase and the aqueous phase of hyaluronic acid be as large as possible. Crosslinking depends very much on the flowability of hyaluronic acid, since the crosslinking reagents very easily form stable suspensions or emulsions. The stability of the two-phase system also increases with lower temperatures.

The gel substance can be injected as it is after swelling and pulverization in a physiologically acceptable buffer. The gel is
For example, heat stabilization can be performed by an autoclave. In addition to injectable crushed gel preparations, other preparations in the form of shaped materials, such as films, tubes, etc., can be produced.

Epoxy cross-linked hyaluronate gel (Laurent, TC et al .: A
cta Chem. Scand., 18 : 274-275,1964)
The gels of the invention can be completely re-swelled even after drying, for example by freeze-drying. This is very advantageous from a manufacturing point of view. This is because storing the dried and stable intermediate product avoids degradation that occurs when the gel is stored in a swollen state in a buffer solution having a pH of 7 or more. It also facilitates changing the concentration and swelling medium of the final gel composition.

The phosphorus content in the dried gel can vary from the order of 1/100% to as low as 1%. For gels cross-linked in a homogeneous aqueous solution, the solid content of the gel completely swollen in the aqueous solution is 0.1-10%. On the other hand, the degree of swelling of a gel crosslinked in a heterogeneous system using hyaluronic acid that is not completely dissolved is much lower. Usually, the solids content of the film thus produced is 30%.

The gels of the present invention show maximum stability at pH 5.75, but degrade at physiological pH (7.3). However, compared to the carboxylate cross-linked hyaluronic acid gel, which is also degradable and described in patent application WO 86/00912, the phosphoric acid cross-linked gel has much higher stability. In other words, the phosphoric acid crosslinking has made it possible to obtain a hyaluronate gel in which the decomposition time is significantly longer than that of the conventional type gel and the decomposition time can be further varied. Gels with degradation times of about 1 day to 2 months can be produced. Patent application EP 272,300 also describes a method of extending the degradation time of hyaluronate gels by a combination of a carboxylic acid ester and an ether bridge, but these gels cannot be manufactured in an injectable form. Can not.

According to one aspect of the present invention, a gel that also contains other components, such as a drug, can be produced. This is achieved by adding the desired components to the gel before or after crosslinking, for example, to achieve a sustained release effect.

In another aspect of the invention, the gel is used for sustained release of soluble hyaluronate in vivo . The effectiveness of topical administration of hyaluronic acid, eg, into a joint, is well known in the literature. One of the previous problems in this case is that despite its high viscosity, hyaluronic acid quickly disappears from the administration area by diffusion. As described above, the decomposition products of the gel of the present invention are hyaluronic acid and harmless phosphate, and when these gels are implanted, they become excellent sustained-release depots of hyaluronic acid.

The present invention therefore relates to a method for cross-linking hyaluronic acid by subjecting a solution of hyaluronic acid or a derivative thereof to treatment with a phosphorus-containing reagent, in particular a phosphoric acid (V) acid derivative. In this case, first, an aqueous solution of hyaluronic acid or a derivative thereof is treated under alkaline conditions, preferably at pH 9-1.
4. The method according to 4, wherein the reaction is carried out with a phosphorus (V) acid derivative. Preferred phosphorus (V) acid derivatives currently include halides, oxyhalides or anhydrides, such as phosphorus pentachloride, phosphoryl chloride (phosphorous oxychloride) or its corresponding bromide or iodide, phosphorus pentoxide, and trimetaphosphoric acid Can be considered.

The present invention further includes hyaluronic acid gels containing phosphate ester crosslinks prepared as described above, and the use of the gels as pharmaceutical dosage forms and sustained release depots for hyaluronic acid administration.

Many examples are set forth below to illustrate the invention, but they are not intended to limit the invention in any way.

Unless otherwise specified, high molecular weight sodium hyaluronate (MW 3 × 10 ⁶ ) was used for gel production.

The gel was made with isotonic Srensen buffer pH 5.75 [100 ml Na
₂ HPO ₄ (9.470g /), 900ml Na ₂ PO ₄・ H ₂ O (9.208g /)
And 5200 mg NaCl / solution], washed and autoclaved.

Gel degradation time was 37 ° C in phosphate buffered saline solution pH 7.3
The gel was solubilized and checked for complete solubilization.

Example 1 500 mg of sodium hyaluronate was weighed into a glass centrifuge tube. 8.3m saturated sodium phosphate (Na ₃ PO ₄ ) solution
l was added. The polysaccharide was swelled in a phosphate solution in a refrigerator overnight.

The sample was stirred with a glass rod until the hyaluronic acid was completely dissolved. The pH of the 6% hyaluronate solution thus prepared is about 12.8.

The solution was cooled to about + 1 ° C in an ice bath. With vigorous stirring, 167μ phosphoryl chloride (POCl ₃ ) was added.
Upon stirring for a few minutes, the solution gelled. The sample was stirred for a total of 5 minutes and then centrifuged until a uniform clear gel was obtained.

After a further 10 minutes of reaction, the resulting gel with a neutral pH was cut into thin slices. This slice
In Srensen buffer. The gel was washed with shaking for 2 days, during which time the buffer was changed three times. The yield of swollen gel was 35 ml.

The gel was crushed by passing it through a fine mesh steel wire net, filled into a syringe, and then autoclaved. The soft, slightly sticky gel can be easily injected through a fine needle.

The degradation time of the gel without autoclaving was 5 weeks, but it degraded to gel-claved gel within 3-4 weeks. The dialyzed and dried gel had a phosphorus content of 0.081%. The solids content of the swollen gel was 1.4%.

Example 2 300 mg of sodium hyaluronate was taken in 15 ml of water and placed in a refrigerator to swell overnight. Upon stirring, the hyaluronate was completely dissolved to form a 2% solution. To this, 3.7 g of solid sodium phosphate (Na ₃ PO ₄ .12H ₂ O) was added. After the salts have dissolved, the solution is cooled in an ice bath and then
Crosslinking was carried out in the same manner as in Example 1 using 400 μl of POCl ₃ .

The resulting gel was dialyzed against distilled water for 2 days, then the product was crushed and freeze-dried. The phosphorus content was 0.10%. The dried gel was swollen in Srensen buffer for 3 days and then autoclaved. The gel thus obtained was sticky, uniform, completely transparent and showed considerable mobility. The degradation time of the autoclaved gel was 4-5 days.

Example 3 300 mg of hyaluronate in 10 ml of water and 2.5 g of N as base
The same experiment as in Example 2 was repeated except that a 3% solution was prepared using a ₃ PO ₄ .12H ₂ O. POCl ₃ 2 as a crosslinking agent
50μ was used.

In this case, the solids content of the swollen gel was 3.6%.
The decomposition time of the autoclaved gel was 10 days, and the phosphorus content of the dried gel was 0.085%.

Example 4 400 mg of sodium hyaluronate was dissolved in 3.3 ml of water,
A very viscous 12% solution was formed. About +
Cool to 10 ° C, then triethylamine (this is + 18 ° C
(Which is water-soluble below) was added with stirring. Solution pH
Was about 13.5. The sample was further cooled to + 1 ° C and crosslinked with 183μ of POCl ₃ . A hard gel with a phosphorus content of 0.12% was obtained. The gel decomposition time in the autoclaved state was 4 weeks.

Example 5 5 ml of a 6% hyaluronate solution was mixed with 1 ml of 4-methoxymorpholine. The pH of the solution was about 11.2. This solution was crosslinked with 200 μl of POCl ₃ as described above.

A slightly liquefied gel was obtained. Gel with pH 5.0 Sren
After swelling with sen buffer, the mixture was autoclaved.
The degradation time of the non-autoclaved gel was 15-19 days, but after autoclaving the degradation time was 10 days.

Example 6 In this example, a cross-linking test was performed using various concentrations of sodium hydroxide as a base in an aqueous solution.

Various amounts of sodium hydroxide solution were added to 2.75 g of the cold 10% hyaluronate solution, and then a predetermined amount of the crosslinking agent, 100 μl
Crosslinking was carried out with POCl ₃ .

a) After addition of 100 μM 5M NaOH: POCl ₃ , the solution rapidly became very acidic (pH about 2). Only a very small amount of water-insoluble gel was obtained.

b) 250μ of 5M NaOH; again, the formation of a very acidic reaction solution occurred rapidly. The resulting gel had a very liquid consistency and was degraded by autoclaving.

c) 5 μM NaOH 500μ: After crosslinking, a gel was obtained which showed considerably higher consistency. The hyaluronate content in its fully swollen state was 0.6%. However, autoclaving resulted in significant degradation of the gel. The decomposition time of the autoclaved sample was 4 days.

d) 5 μM NaOH 1000μ: Hyaluronate was rapidly decomposed by the alkaline solution to produce a low viscosity solution. No gel formation occurred with the addition of the crosslinking agent.

These examples clearly show that the crosslinking system requires proper buffer control.

Example 7 3000 mg of sodium hyaluronate was added to 3 ml of a saturated Na ₃ PO ₄ solution.
In POCl ₃ 25 to form a 10% solution.
Crosslinked with μ. A hard gel is obtained with a degradation time of 14
It was a day.

Example 8 80 mg of acid-decomposed sodium hyaluronate having a molecular weight of 100,000
Was dissolved in 1 ml of a saturated Na ₃ PO ₄ solution. To this was added an additional 0.2 g of solid Na ₃ PO ₄ .12H ₂ O. This solution was crosslinked with 75μ of POCl ₃ .

A milky, friable gel was obtained with a degradation time of 2 weeks.

Example 9 300 mg of sodium hyaluronate was dissolved in 5 ml of a saturated Na ₃ PO ₄ solution. The solution is cooled and phosphorus pentachloride (PCl ₅ ) 200m
g was added in small portions with vigorous stirring. Phosphorus pentachloride reacted faster and more vigorously than phorphoryl chloride. A slightly milky gel was obtained with a degradation time of 2 weeks.

Example 10 200 mg of tetrabutylammonium hyaluronate was dissolved in 2 ml of dimethylformamide. The dissolution was mixed with 1 ml of triethylamine, then the solution was cooled and POCl ₃ 100
μ was added. Gel formation occurred almost instantaneously. The resulting gel consisted of white flakes and showed little swelling in water. The gel had a hyaluronate content of 30%.

Example 11 100 mg of tetrabutylammonium hyaluronate was dissolved in 2 ml of dimethylformamide. Triethylamine 200
Add μ, cool the solution and add phosphorus pentoxide (P ₂ O ₅ ) 200mg
Mix with. Almost immediately, a white gel precipitate formed. This gel exhibited the same properties as the gel prepared in Example 10.

Example 12 100 mg of sodium hyaluronate was co-evaporated with 3 × 10 ml of dry pyridine. This material was suspended in 10 ml of pyridine, then the suspension was cooled and 400 μl of POCl ₃ was added. This solution was shaken for 15 minutes. The hyaluronate thus treated is insoluble in water, is prone to irregular swelling, and produces a mixture of a hard white portion and a clear gel portion. The hardest crosslinked white gel part does not decompose at physiological pH, but dissolves when subjected to alkali treatment. The phosphorus content in the dialysis dried gel was 0.77%.

Example 13 Dialyzed, salt-free sodium hyaluronate was dried in a Petri dish to form a clear, flat film. The film was swollen for a few seconds with a cold mixture of 1 part water and 9 parts triethylamine. The swollen film was immersed in phosphoryl chloride for a few seconds or kept in the vapor of phosphoryl chloride for about 1 minute to perform phosphoryl chloride treatment. In each case, a clear, water-insoluble, crosslinked film was obtained with a solids content in the swollen state of about 30%.

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C08B 37/08 A61K 9/00, 31/725

Claims (7)

(57) [Claims] 1. A method for producing a gel of crosslinked hyaluronic acid or a derivative thereof, comprising reacting a solution of hyaluronic acid or a derivative thereof with a phosphorus-containing reagent to crosslink the solution. 2. The method according to claim 1, wherein the reagent is a derivative of phosphorus (V) acid. 3. The method according to claim 2, wherein the phosphorus (V) acid derivative is a halide, oxyhalide or anhydride. 4. The method according to claim 3, wherein the phosphorus (V) acid derivative is phosphorus pentachloride, phosphoric chloride or phosphorus pentoxide. 5. A gel of cross-linked hyaluronic acid or a derivative thereof produced by reacting hyaluronic acid or a derivative thereof with a phosphorus (V) acid derivative. 6. A gel of cross-linked hyaluronic acid or a derivative thereof, wherein the cross-link is a phosphate ester bridge. 7. A sustained-release depot for administration of soluble hyaluronic acid or a medicament comprising a gel of cross-linked hyaluronic acid or a derivative thereof.