JPH0959303A - Biocompatible hyaluronic acid gel and its application - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a water-insoluble hyaluronic acid gel prepared by the condensation reaction of hyaluronic acid with a crosslinking agent such as dihydrazine, almost free from cytotoxicity, expressing appropriate elasticity, excellent in transparency, get stability and biocompatibility and suitable for vitreous body, etc. SOLUTION: This gel is prepared by the condensation reaction of hyaluronic acid with a crosslinking agent such as di(or bi)hydrazine or di(or bi)hydrazide such as succinic acid dihydrazide of formula I R is 4-8C alkylenedicarbonyl, a (substituted) bicyclic condensed-ring residue containing a six-membered heterocyclic ring group having two nitrogen atoms or formula II [X is a lower alkylene or SO2 ; (n)=0, 1]}.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel hyaluronic acid gel and its use as a biocompatible material.

[0002]

2. Description of the Related Art Hyaluronic acid has the following formula

[0003]

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It is a linear polymeric polysaccharide formed by alternately binding β-D-N-acetylglucosamine and β-D-glucuronic acid. Hyaluronic acid is distributed in large amounts in the connective tissues of mammals, and is also known to exist in the cavities of chickens, the gastric membrane of silkworms, and the capsules of streptococci. Umbilical cord, synovial fluid, vitreous body, etc. are used as extraction materials, and purified products are prepared from streptococcal cultures.

[0005] Naturally occurring hyaluronic acid is polydisperse in molecular weight, but has no species and organ specificity, and is known to show excellent biocompatibility even when transplanted or injected into a living body. Has been. Further, various chemical modifications of hyaluronic acid have been proposed because of the disadvantages associated with hyaluronic acid itself when applied to a living body, for example, the residence time in vivo is relatively short.

As a typical example of these, a highly swelling crosslinked hyaluronic acid gel obtained by using a bifunctional reagent such as divinylsulfone, bisepoxides, formaldehyde and bishalide as a crosslinking agent. (See U.S. Pat. No. 4,582,865, Japanese Patent Publication No. 6-37575, Japanese Patent Publication No. 5-37575).

[0007] Further, it is mainly intended to be used as a carrier of a drug delivery system, but it is also known that a stable acylhyaluronate urea can be produced by the reaction of a certain carbodiimide and hyaluronic acid (WO45 / 02517). See the pamphlet). By the way, it is also known that a water-insoluble biocompatible gel can be produced by reacting hyaluronic acid activated in such a reaction system with a nucleophilic reagent (for example, amines) (US Pat. No. 4, However, depending on the reaction conditions, it is said that intermolecular coupling products due to amine components are not observed (J. Kuo, et al.
Bioconjugate Chem. 1991 , 2,
232-241). The latter results show that the cross-linked product of hyaluronic acid can be obtained by using a specific carbodiimide in the absence of a nucleophile. It corresponds to the invention described in International Publication No. 93/07106 pamphlet of Kuo et al.

An example in which hyaluronic acid (oligomer) activated by carbodiimides is reacted with succinic acid dihydrazide, adipic acid dihydrazide or suberic acid dihydrazide as a nucleophilic reagent is described in T. Pouyani
Et al., Bioconjugate Chem. 1994 ,
5,339-347. More specifically, this publication discloses that an oligosaccharide of hyaluronic acid is reacted with a large excess (30 mole times) of dihydrazide in the presence of carbodiimide to attach a pendant hydrazide group to the glucuronic acid moiety. It has also been disclosed that hydrogels of hyaluronic acid, which are cross-linked with certain disulfonate cross-linkers, can be obtained utilizing the above compounds, as well as pendant hydrazide groups. However, whether or not dihydrazide itself can directly crosslink hyaluronic acid has not been published.

[0009]

DISCLOSURE OF THE INVENTION The inventors of the present invention have also examined variously the use of hyaluronic acid itself as a biocompatible material. Among them, what is interesting is the application to vitrectomy for severe retinal detachment, which has become possible due to the latest advances in microsurgical instruments and new ideas.

Originally, hyaluronic acid is contained in the vitreous humor, but due to its short residence time in the vitreous cavity, it cannot be applied to the vitreous cavity after vitreous surgery such as severe proliferative retinopathy. It did not give satisfactory results. In addition, a wide variety of polymers (natural or artificial) have been used for retinal vitreous surgery, but no sufficiently satisfactory one has been developed yet.

That is, for use as a vitreous substitute, (1) the material is transparent, the refractive index is close to that of water, and the specific gravity is equal to or slightly larger than that of water, (2) sterile, non-antigenic. , Non-toxic, (3) Retentive enough to remain in the vitreous for a sufficient period (2 to 3 months) to place the detached retina in place, and (4) from the incision during surgery. This is because it is necessary to satisfy at least the requirement of being able to be injected or transplanted.

Materials that meet these requirements may be applicable to other biomedical applications, such as therapeutic and disposable contact lenses, artificial breasts, sliding parts of artificial joints, and artificial skin.

Therefore, it is an object of the present invention to provide a biocompatible material that meets the above requirements.

[0014]

The present inventors have investigated various chemical modifications of hyaluronic acid in order to achieve the above object. As a result, the above-mentioned J. Kuo et al., Bioco
njugate Chem. 1991 , 2,232-
241 shows that although an amide compound (condensation reaction product of a carboxyl group and an amino group of hyaluronic acid) is not obtained by the reaction of hyaluronic acid and amines in the presence of dicarbodiimide, The present inventors have found that when a specific di (or bi) hydrazine or di (or bi) hydrazide is used instead of the above, a gel of hyaluronic acid can be efficiently obtained. Furthermore, it was also confirmed that the gel thus obtained has almost the properties required for the above vitreous body substitute.

Therefore, according to the present invention, there is provided a water-insoluble hyaluronic acid gel formed by a condensation reaction of hyaluronic acid and a crosslinking agent di (or bi) hydrazine or di (or bi) hydrazide. Also provided are biocompatible compositions comprising such gels, particularly compositions that can be used in the vitreous.

[0016]

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, hyaluronic acid (hereinafter sometimes abbreviated as "HA") is a natural product of H.
If it is A, it can be used regardless of its origin,
Any molecular weight in the range of about 6 × 10 ⁴ to about 1.2 × 10 ⁷ daltons can be used. Further, as long as it has a molecular weight within the above range, from a higher molecular weight one to a low molecular weight one obtained through hydrolysis treatment or the like can be similarly used. The hyaluronic acid (or HA) referred to in the present invention is an alkali metal,
For example, it is to be understood that it is used in the concept including salts of sodium, potassium and lithium.

The cross-linking agent used for cross-linking HA may be any bifunctional di (or bi) hydrazine or di (or bi) hydrazide capable of preparing a gel for the purpose of the present invention. However, in general, it is necessary that two hydrazino groups are kept at a constant distance by three or more atoms. Specifically, in the formula _{H 2 NNH-R-NHNH 2} ( wherein, R is substituted includes a 6-membered heterocyclic group having two nitrogen atoms as the alkylene carbonyl or heteroatom C _4-8 Is a bicyclic fused ring residue which may be
When substituted, the substituent is selected from the group consisting of a lower alkyl group such as methyl and ethyl, a halogen such as fluorine and chlorine, a sulfonic acid group and a phenyl group, or

[0018]

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Here, X is a lower alkylene or sulfonyl selected from the group consisting of methylene, ethylene and propylene, n is 0 or an integer 1, and the benzene ring is divalent optionally substituted by the above substituents. Or a di (or bi) hydrazine or di (or bi) hydrazide.

More specifically, but not exclusively, succinic acid dihydrazide, adipic acid dihydrazide and suberic acid dihydrazide, and formulas

[0021]

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The di (or bi) hydrazine of can be mentioned.

Of these, in order to use it for a vitreous substitute, in addition to the properties (1) to (4) described above, it is required to have low swelling property in a physiological saline solution. Gels cross-linked with the above hydrazines which form gels which are generally less swellable are preferred.

The gel of the present invention can be produced by a dehydration condensation reaction between the carboxyl group derived from β-D-glucuronic acid of HA and the hydrazino group derived from a crosslinking agent. The gel of the present invention includes any of the reaction modes obtained by carrying out such a dehydration condensation reaction to obtain the desired gelled product. However, considering its use as a biocompatible material, it does not adversely affect the properties of HA,
It is preferable to select a reaction system that can easily separate the target gel from unreacted starting materials and byproducts.

As such a reaction system, it is preferable to react HA with a cross-linking agent in the presence of carbodiimides capable of obtaining a desired gel in an aqueous reaction solvent system. Carbodiimides that can be used include 1-ethyl-soluble in water.
3- (3-dimethylaminopropyl) carbodiimide (hereinafter referred to as "EDC"), cyclohexyl-β- (N
-Methylmorpholino) ethylene carbodiimide p-toluene sulfonate and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methiodide may be mentioned as preferred, but the most preferred is EDC.

Hereinafter, in this preferred reaction system, adipic acid dihydrazide (hereinafter sometimes abbreviated as "AAD") or 1,4-dihydrazinophthalazine represented by the above formula (a) [dihydralazine (hereinafter referred to as "DHZ" May be abbreviated as ") as a cross-linking agent, and about 2 x as HA
The present invention will be described in more detail with reference to the case of using 10 ⁶ daltons. The reaction behavior is HA
Is hardly affected by the change in the molecular weight of.

According to the above reaction system, the HA concentration is 0.5.
It is convenient to set the amount to ˜5.0% by weight in terms of treatment of the reaction solution, but it is preferably set to 0.8 to 1.5% by weight. Taking the case of setting the HA sodium salt concentration to about 1.2% by weight / weight, the β-D-
When converted into disaccharide units of N-acetylglucosamine and β-D-glucuronic acid, this disaccharide unit is about 0.03mo.
Equivalent to 1 / l. The amounts of the carbodiimide EDC and the cross-linking agents AAD and DHZ can be appropriately selected on the basis of this converted value, but the EDC is usually about 0.13 to 0.1 with respect to HA depending on the intended cross-linking ratio. It is advisable to use them so as to be 67 molar equivalents. Similarly, the cross-linking agent is also preferably selected so as to have a molar equivalent of 0.13 to 0.67 with respect to HA. Needless to say, the crosslinking rate depends on the reaction time and the reaction temperature, but for the sake of convenience, the explanation will be given according to the reaction time and the reaction temperature at which the reaction proceeds until the reaction is almost completed.

In addition, since the progress of the reaction is influenced by the pH of the reaction solution, it is usually preferable to adjust the pH of the reaction solution to 3 to 4 for di (or bi) hydrazide.

On the other hand, with respect to di (or bi) hydrazines, the pH of the reaction solution can be adjusted to pH 3 to 8 and the condensation reaction can be carried out, so that there is usually no need to adjust the pH. The reaction temperature is conveniently room temperature of 20-25 ° C.,
In this case, using AAD completes in about 40-60 minutes and using DHZ completes in about 120-140 minutes.

With respect to such reaction conditions, various absorbances of the reaction solution, such as an ultraviolet absorption spectrum or a fluorescence spectrum, may be measured with time or, if more certain is desired, by measuring an infrared absorption spectrum (IR). It can be appropriately selected by determining whether or not the degree of crosslinking can be achieved. Fourier transform infrared absorption spectrum (FT-I
In the case of R), for example, HA may be selected as a control, and then the next change in absorption may be observed. For example, when DHZ is used, 1413 cm ⁻¹ : C—O of carboxyl (—COO—)
Absorption, and 1377 cm ^-1 by: paying attention to the absorption of deformation vibration of C-H, the change in (absorbance / 1413Cm absorbance ^-1 1377 cm ^-1) in the art will reflect the progress of cross-linking,
Also, 1650 cm ⁻¹ : absorption of amide (—CO—NH—) by C—O, and 1614 cm ⁻¹ : carboxy (—COO—) of C—O
Focusing on the absorption due to (absorbance at 1650 cm ^-1 /
Changes in the absorbance at 1614 cm ^-1 ) will also reflect the progress of crosslinking.

Thus, a gel having a desired degree of crosslinking can be prepared.

Generally, the crosslinking ratio can be increased as the carbodiimide and the crosslinking agent are increased with respect to the number of moles of the above disaccharide unit of HA. The cross-linking rate is approximately 29% to about 29% when the amount of cross-linking agent used as a unit involved in cross-linking is used as a unit involved in cross-linking and the amount of unreacted cross-linking agent is taken into consideration in consideration of the change in absorbance with the progress of the above reaction. The 85% one forms a transparent gel, exhibits appropriate viscoelasticity, and the change (increase) in swelling degree due to physiological saline solution is hardly observed,
In addition, it shows almost no toxicity (suppression of cell growth) in a cytotoxicity test using cultured cells.

Therefore, the crosslinked HA gel of the present invention is a biocompatible material, for example, an artificial vitreous body (vitreous body substitute), an artificial crystalline lens (an artificial crystalline lens having accommodative power), a therapeutic contact lens, a disposable contact lens, It could be used for artificial breasts, sliding parts of artificial joints, artificial skin, etc.

[0034]

Now, the present invention will be described in further detail with reference to specific examples. In the following description, percentage means (weight / weight)% unless otherwise specified.

Examples 1 to 16: Preparation of gels using DHZ 1.2% HA aqueous solution 1 in a test tube at room temperature (around 25 ° C)
Add mL and add 0.05 to 0.2 mol / L of EDC.
After adding 1 mL and stirring for 3 minutes, 0.05 to 0.2 mol
0.1 mL of DHZ / L was added and stirred for 3 minutes. 120 mL of physiological saline was added to 1.2 mL of the obtained gel and dialyzed for 19 hours, and then the state of the gel in the test tube was observed. The results are summarized in FIG. The gels thus obtained are all insoluble in physiological saline and have an inherent swelling property, which indicates that hydrogels are formed. Further, DHZ has an absorption maximum around a wavelength of 310 nm, but it has been confirmed that unreacted DHZ can be easily separated from the gel by the above dialysis.

The example numbers of the respective combinations in the drawing are Examples 1 to 16 from left to right, and from the top column to the bottom column. Of these examples, the use concentration of EDC is 0.2 mol / L and the use concentration of DHZ is 0.1 mol.
The gel according to Example 8, which is / L, shows a theoretical crosslinking ratio of about 56.4% according to the above calculation formula of the crosslinking ratio.

Examples 17 to 20: Change in weight of produced gel by dialysis with physiological saline (average of two trials) The above Example 4 (concentration used: DHZ = 0.05 mol / L, E
DC = 0.2 mol / L), Example 8 (concentration used: DHZ =
0.1 mol / L, EDC = 0.2 mol / L), Example 12
(Use concentration: DHZ = 0.15 mol / L, EDC = 0.
2 mol / L) and Example 16 (concentration used: DHZ = 0.2)
(3 mol / L, EDC = 0.2 mol / L), 3 ml of the gel obtained was dialyzed with 300 ml of physiological saline at room temperature (25 ° C.). The dialysis was treated with saline and the gel was collected. The results of measuring the weight of the obtained gel over time are shown in FIG.

As can be seen from the figure, DHZ is 0.05
The mol / L (Example 4) gel swelled on dialysis for 21 days, but the DHZ was 0.12 and 0.2 mol / L (respectively,
The gels of Examples 12 and 16) separated and condensed. The gel having a DHZ of 0.1 mol / L (Example 8) did not change.

Considering the use of these gels in the eye, it is preferable that the change due to dialysis is small.
When setting the DC concentration to 0.2 mol / L, DH
A Z concentration of 0.1 mol / L is suitable.

Examples 21 to 25: Preparation of gel using DHZ under various pH 2 mL of 1% HA aqueous solution having pH adjusted beforehand by dialysis method into a test tube at room temperature (around 25 ° C.).
H1, pH 2 in Example 22, pH 4 in Example 23, pH 6 in Example 24, pH 8 in Example 25) and 0.04.
1 mL of mol / L EDC was added and stirred for 3 minutes, and then 1 mL of 0.04 mol / L DHZ was added and stirred for further 3 minutes. The change in absorbance of the reaction solution with time at a wavelength of 400 nm was measured to trace the reaction behavior. The results are shown in FIG.

From the figure, the reaction is completed in about 2-3 hours,
Further, it can be seen that the reaction does not occur at pH 1. Example 21
As in Examples 1 to 16, the gels obtained in -25 were dialyzed by adding physiological saline, and then the gels were visually observed. All of them were transparent gels, but became whiter as the pH increased. Was confirmed.

Examples 26 to 29: Preparation of gel using AAD At room temperature (around 25 ° C.), 1 mL of 1.2% HA aqueous solution was put into each test tube, and 0.04 mol / L of EDC 0.1 was added.
Add mL and stir for 3 minutes, then add 1N HCl to pH
To 3.1 (Example 26), 3.3 (Example 27), 3.5 (Example 2)
8), or, without adjustment (Example 29), 0.1 mL of 0.06 mol / L AAD was added and stirred for 3 minutes.

In the case of Example 29, a white gel was produced, but in all the other examples a transparent gel was produced. Examples 17-20
FIG. 4 shows the change in weight of the gel over time when the obtained gel was dialyzed against a physiological saline solution according to the method of 1. These results are average values of four trials.

From the figure, when AAD is used as the crosslinking agent, D
It is observed that the gel swells as compared to the case of using HZ.

Example 30: Measurement of infrared absorption spectrum (1) Gel using DHZ Example 4 (EDC 0.2 mol / L, DHZ 0.05 mo)
1 / L), Example 8 (EDC 0.2 mol / L, DHZ
0.1 mol / L), Example 12 (EDC 0.2 mol / L)
L, DHZ 0.15 mol / L), and Example 16 (ED
The gel obtained according to C 0.2 mol / L, DHZ 0.2 mol / L) was dried and powdered, and each was made into a KBr tablet, and FT-IR was measured. As a control, HA FT-
The IR was measured.

The instrument used was a Magna IR ™ spectrometer 550 (Nicore
t) was used.

FT-of HA and the gel obtained according to Example 8
IR spectra are shown in Figures 5 and 6, respectively.

Compared to HA, HA-gel (DHZ) has 1
C- of carboxyl (-COO-) near 413 cm ^-1
C- in the vicinity of 1377 cm ^-1 relative to the intensity of absorption by O
The ratio of the absorption strength due to the H bending vibration becomes large (that is, the reduction of free carboxyl groups), and 1614 cm ^-1
C-of amide (-CO-NH-) around 1650 cm ^-1 with respect to the absorption intensity of carboxyl by C-O in the vicinity
The ratio of the intensity of absorption by O is increased (ie, reduction of free carboxyl groups and formation of amide bond are observed).

1650 cm ^-1 / 1614 for the gels prepared according to the control, Example 4, Example 8, Example 12 and Example 16.
cm ^-1 and a plot of the absorbance ratio of 1377cm ^-1 / 1413cm ^-1 shown in Fig.

From this figure, it can be seen that as the concentration of the cross-linking agent DHZ is increased, the free carboxyl groups are relatively decreased and the cross-linking is further promoted.

(2) Gel using AAD 0.04 mol / L EDC and 0.03, 0.06 and 0.03.
Gels were prepared in the same manner as in Examples 26 to 29, except that 12 mol / L AAD was used and the reaction was performed at pH.
FT- of the gel obtained with 0.06 mol / L AAD
The IR spectrum is shown in FIG. -C as in (1) above
Ratio of absorbance derived from O-NH- / absorbance derived from -COO-, and absorbance derived from -CH / -COO-
The graph plotted against the concentration of AAD using the ratio of absorbance derived from is shown in FIG. From these figures, −
It is observed that gelation due to the formation of CO-NH-bonds proceeds depending on the concentration of AAD.

Example 31: Measurement of light transmittance (light scattering) of gel The following experiment was conducted in order to examine the light transmittance of HA-gel after injection into the vitreous body. 2.5 mL syringe (made by Terumo)
The tip of the is cut off with a cutter, and the tip of a plastic tip for pipette (C-5000 / GILSON) is cut there (inner diameter of cut end: 7 mm, 2 mm, 1.2 m
m) were prepared and the HA-gel was put into those applicators. HA-gel was injected into the disposable cuvette (1 cm x 1 cm x 4.5 cm) filled with 1 mL of physiological saline in advance from those applicators, and the light transmittance of each was measured by a spectrophotometer at a wavelength of 550 nm. As a control, a 1.2% HA physiological saline solution was also measured. The gel used was that prepared according to Example 8 (0.2 mol / L EDC, 0.1 mol / L DHZ). Control (H
Graphs in which the average values of the measured values of A) and the gel [HA-gel (DHZ)] are plotted over time are shown in (a) and (b) of FIG. 10, respectively.

According to the figure, the larger the opening diameter of the pipe, the higher the H
Both A and HA-gel have high transmittance, and H is higher than HA.
The A-gel had a slightly lower transmittance. 90 after the second day
A high transmittance of not less than% was obtained. Therefore, it is considered that HA-gel does not seriously damage the visual acuity even after being injected into the vitreous cavity.

Example 32: Determination of elasticity Example 4 (0.2 mol / L EDC, 0.05 mol / L DHZ), Example 8 (0.2 mol / L EDC, 0.1 mo).
1 / L DHZ), Example 12 (0.2 mol / L ED)
C, 0.15 mol / L DHZ) and Example 16 (0.2 m
gel prepared according to ol / L EDC, 0.2 mol / L DHZ, as well as control (1.2% HA, 0.2mo).
The dynamic storage modulus (G ′) of 1 / L EDC, 0 mol / L DHZ) and HA was determined.

The measurement was carried out using a MR-101 type rheometer (Rheology) measuring mode: parallel plate with a diameter of 30 mm, automatic strain control, gap 2 mm. The results are shown in Fig. 11.

From the figure, in the control and HA, G ′ was about 150 (dyn / cm ² ) to about 1, as the angular velocity (ω) increased.
While it rises to 200 (dyn / cm ² ), HA-
In the gel (DHZ), it is recognized that the increase in G ′ with the increase in ω is slow. From these results, it is understood that all HA-gels are soft elastic bodies and are suitable for application to a living body.

Example 33: Measurement of cytotoxicity Example 8 (0.2 mol / L EDC, 0.1 mol / L D)
HZ) and Example 27 (0.04 mol / L EDC, 0.0).
1% HA according to 6 mol / L AAD, pH 4.0)
Examine the cytotoxicity of gels prepared aseptically using an aqueous solution.

HA-gel (from Example 8 and from Example 27)
0.2 mL of each was placed in the bottom of a 24-well plate or in an intercup, and dialyzed with physiological saline in the wells. 2.5 × 10 ⁴ human skin-derived fibroblasts were seeded in the well and cultured (medium RITC 80
-7, temperature 37 ° C., under 5% CO ₂ ). After 3, 7, and 10 days, the state of cell proliferation was observed with an inverted microscope.

After 10 days of culture, all the cells grew well. When HA-gel (from Example 8 or from Example 27) was placed in an intercup and cultured without contact with cells, the cell proliferation was almost the same as that of the control group, or a tendency of slightly suppressing was observed.

[0060]

EFFECTS OF THE INVENTION According to the present invention, there is provided a gel which is transparent in a certain range of cross-linking degree, has little cytotoxicity, exhibits appropriate elasticity, and is stable. This gel can be used as a biocompatible material.

[Brief description of drawings]

FIG. 1 is a schematic view showing the appearance of the gel of the present invention in response to changes in the concentration of the crosslinking agent used and changes in the concentration of the carbodiimide used.

FIG. 2 is a graph showing swelling characteristics over time when DHZ is used as a cross-linking agent and the gel obtained at each cross-linking agent concentration is dialyzed against a physiological saline solution.

FIG. 3 is a graph showing the influence of pH change during gel preparation on gel formation when AAD is used as a crosslinking agent.

FIG. 4 is a graph showing swelling characteristics over time when AAD is used as a cross-linking agent and a gel obtained at various pHs is dialyzed against a physiological saline solution.

FIG. 5 is a diagram showing an FT-IR spectrum of HA.

FIG. 6: FT of gel obtained using DHZ as crosslinker
It is a figure showing an -IR spectrum.

FIG. 7 is a graph in which the absorbance ratio at a specific wavelength of the FT-IR spectrum of the gel obtained by using various concentrations of the cross-linking agent (DHZ) used is plotted against the concentration change.

FIG. 8: FT of gel obtained using AAD as crosslinker
It is a figure showing an -IR spectrum.

FIG. 9 is a graph in which the absorbance ratio at a specific wavelength of the FT-IR spectrum of a gel obtained by using various crosslinking agent (AAD) concentrations is plotted against the concentration change.

FIG. 10 is a graph showing the transmittance of a gel obtained by using HA (control) and DHZ as a cross-linking agent [(a): control, (b): gel].

FIG. 11 is a graph showing the elastic moduli of gels obtained with various crosslinking agent (DHZ) concentrations.

Claims (5)

[Claims] 1. Hyaluronic acid and a crosslinking agent di (or bi)
A water-insoluble hyaluronic acid gel formed by a condensation reaction with hydrazine or di (or bi) hydrazide. 2. The cross-linking agent is represented by the formula H ₂ NNH—R—NHNH ₂ (wherein R is a C _4-8 alkylenedicarbonyl or a 6-membered heterocyclic group having two nitrogen atoms as hetero atoms). An optionally substituted bicyclic fused ring residue containing
Alternatively, the formula: Here, X is lower alkylene or —SO ₂ —, n is 0 or integer 1, and the benzene ring is a divalent group or a single bond which may be substituted). The hyaluronic acid gel according to claim 1. 3. The crosslinking agent is succinic acid dihydrazide, adipic acid dihydrazide, and suberic acid dihydrazide, and a compound represented by the formula: The hyaluronic acid gel according to claim 1, which is a compound selected from the group consisting of di (or bi) hydrazines represented by or a salt thereof. 4. A biocompatible composition comprising the hyaluronic acid gel according to claim 1. 5. A biocompatible composition according to claim 4 for use in the vitreous.