JPH0634814B2 - Vitreous application agent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an agent for application to the vitreous, more specifically, in the case of intractable retinal detachment in which reversion is difficult to obtain by an ordinary surgical operation from the sclera side. In contrast, a tamponad from inside the eyeball
The present invention relates to a vitreous body application agent applicable as e).

[Prior Art and its Problems] As a treatment method for intractable retinal detachment, which is difficult to reposition by a normal surgical operation from the sclera side, the intraocular period until adhesion scar formation occurs between the retina and the choroid There is a method of performing more tampon treatment. A useful material for this tampon must be highly safe and have a long retention time in the eye. At present, silicon oil, sulfur hexafluoride (SF ₆ ) and freon gas are currently attracting attention.

However, the safety of silicone oil has not been established yet, and if a gas such as sulfur hexafluoride or Freon is used, it may cause cataract. In addition, since silicone oil and gas have a low specific gravity, they are in a prone position after surgery (free dow
n position) must be strictly adhered to, and the patient's pain was not uncommon. Therefore, it is desired to develop a safer and more effective artificial vitreous body that compensates for the above drawbacks.

High-molecular-weight hyaluronic acid (hereinafter referred to as “HA”), which is a component of the vitreous body, can be said to be a more ideal material, but it deteriorates and decomposes quickly due to hyaluronidase, etc., and is therefore comparable to Freon gas, which absorbs relatively quickly. Only the effect of can be expected.

Therefore, the present inventors have a high safety, and shows resistance to tissue enzymes such as hyaluronidase, and further, when applied, for the purpose of obtaining a vitreous application agent which does not need to take a prone position. As a result of intensive studies, they have found that the object of the present invention can be achieved by using a crosslinked glycosaminoglycan (hereinafter referred to as “crosslinked GAG”) as a material, and have completed the present invention.

[Structure of the Invention] The vitreous application agent of the present invention is characterized by comprising cross-linked GAG.

As the cross-linked GAG used in the present invention, HA, chondroitin sulfate (hereinafter referred to as "ChS") (A, B, C, D, E, F, H), heparin (hereinafter referred to as "Hep"), heparan sulfate (hereinafter referred to as " HS ''
A), keratan sulfate (hereinafter referred to as “KS”) and keratan polysulfate (hereinafter referred to as “KPS”) and other glycosaminoglycans (hereinafter referred to as “GAG”) or salts thereof, preferably HA or ChS or salts thereof, Any of water-soluble substances may be used as long as they are cross-linked with a suitable cross-linking agent. Of these, high-viscosity ones, such as cross-linked HA, have a viscosity (20% ℃, shear rate 1sec ^-1 ) 1000 ~ 60,000 centipoise is preferred, in the cross-linked ChS viscosity in 10% saline solution (20 ℃, shear rate 1sec ^-1 ) 1000.
The thing of -80,000 centipoise is preferable.

Preferred cross-linking agents include, for example, polyfunctional epoxy compounds.

Here, the polyfunctional epoxy compound is a compound having at least one epoxy group, and additionally, a compound having at least one functional group suitable for crosslinking GAG, including an epoxy group.

Examples of such compounds include halomethyloxirane compounds and bisepoxy compounds. Examples of the halomethyloxirane compound include epichlorohydrin, epibromhydrin, β-methylepichlorohydrin and β-methylepibromohydrin. Bis-epoxy compounds include 1,2-bis (2,3-epoxypropoxy) ethane, 1,4-bis (2,3-epoxypropoxy) butane, 1,6-bis (2,3-epoxypropoxy)
Hexane and diglycidyl ether of bisphenol A or bisphenol F and the like can be mentioned.

Among the cross-linked GAGs used in the present invention, those using a polyfunctional epoxy compound as a cross-linking agent and a method for producing the same are described in Japanese Patent Application No.
59-88440 and 59-132885.

Usually, GAG or a salt thereof is 0.5% or more, preferably 1,0
Dissolved in an alkaline aqueous solution to a concentration of not less than%, and a water-soluble organic solvent is added so as to be not less than 30%, preferably not less than 50% of the total liquid amount. The alkaline aqueous solution preferably has a pH of 8 to 14, more preferably pH 12 to 14. Examples of the alkali include metal hydroxides such as sodium hydroxide, potassium hydroxide and calcium hydroxide, and metal carbonates such as sodium carbonate and potassium carbonate. Examples of the water-soluble organic solvent include methanol, ethanol, isopropanol, acetone, dioxane and the like, and these are used alone or as a mixture. The reaction can be effectively carried out by adding these water-soluble organic solvents, and the decomposition of GAG (decomposition into low molecular weight) due to alkali can be suppressed.

Then, to the resulting solution, one or more of the above polyfunctional epoxy compounds is added, and 0-100 ° C, preferably 10-60 ° C,
More preferably, the reaction is carried out at 20-40 ° C. The reaction time varies depending on the reaction temperature, but is preferably 24 to 48 hours at around 20 ° C and 2 to 3 hours at around 40 ° C.

The crosslinked GAG used in the present invention can also be prepared as follows.

That is, the water-soluble organic solvent is added to the alkaline solution of GAG or a salt thereof, and the resulting water candy-like precipitate is fractionated,
When a polyfunctional epoxy compound is added to the precipitate and reacted at 50 ° C. or lower, the reaction can be performed very efficiently. In order to collect the water candy-like precipitate, the supernatant may be removed by decantation, for example. The reaction temperature is usually 10 to 50 ° C, most preferably 20 to 40 ° C. The higher the temperature, the shorter the reaction needs to be completed. Generally, about 2 hours at around 40 ℃, 24 ~ around 20 ℃
48 hours is preferred.

In the preparation of crosslinked GAG, the solubility and viscosity of the resulting crosslinked GAG or its salt can be adjusted by changing the molar ratio of GAG or its salt to the polyfunctional epoxy compound, that is, the degree of crosslinking.

In hyaluronan with a molecular weight of around 1 million, the repeating disaccharide of hyaluronan 1
The number of moles of polyfunctional epoxy compound used per mole is 1
When the amount is -10 mol, a cross-linked hyaluronan which is water-soluble and has a viscosity (20 ° C, shear rate 1 sec ^-1 ) in a 1% physiological saline solution of 1,000-60,000 centipoise can be obtained.

When injecting the cross-linked GAG into the vitreous, the cross-linked GAG is usually dissolved in physiological saline and has a viscosity capable of passing through an injection needle, that is, 50,000 centipoise (20 ° C, shear rate 1 sec ^-1 ) or less, preferably 5,000- Use at 30,000 centipoise (20 ° C, shear rate 1 sec ^-1 ). The injection is performed, for example, by the injection method (Scott method) under the binocular inversion ophthalmoscope of silicone oil.
t, JD; Trans. Ophthalmol. Soc. UK, 93, 417 (1973)).

EFFECTS OF THE INVENTION According to the present invention, a vitreous application agent that is highly safe and resistant to tissue enzymes such as hyaluronidase and does not require a prone position upon application is provided. be able to. Such a vitreous application agent of the present invention, refractory retinal detachment difficult to obtain repositioning, that is, retinal detachment with proliferative vitreoretinopathy, retinal detachment with giant lacunae, proliferative traction retinal detachment, diabetic It is useful as a therapeutic agent for tear-causing retinal detachment associated with retinopathy.

[Examples of the Invention] The present invention will be described in more detail below with reference to Preparation Examples, Test Examples and Examples, but these do not limit the scope of the present invention in any way.

Preparation Example 1 Preparation of cross-linked hyaluronan (1) Dissolve 5 g of HA sodium salt (molecular weight 730000) in 1N sodium hydroxide solution to 2%, and then add 0.22
The cells were sterilized with a μm membrane, ethanol (250 m) and epichlorohydrin (3.75 m) were added, and the mixture was reacted at 20 ° C. for 8 hours. After adjusting the pH of the reaction solution to 6.0 by adding acetic acid, ethanol 3000
m was added and precipitated to prepare crosslinked HA (hereinafter referred to as “crosslinked HA-1”).

(2) Treated in the same manner as in (1) except that the amount of epichlorohydrin, which is a cross-linking agent, is changed, and the three types of cross-links shown in Table 1 are cross-linked.
HA was prepared.

These 3 kinds of cross-linked HA and HA sodium salt used for synthesis were dissolved in 0.1M acetic acid (pH 5.0) at a concentration of 1% and measured (20 ° C, shear rate 1 sec ^-1 ), It was as follows.

Cross-linked HA-2 45,000 centipoise Cross-linked HA-3 27,000 centipoise Cross-linked HA-4 8,000 centipoise HA sodium salt 1500 centipoise Add 0.09% by weight of beef testicle hyaluronidase to these solutions and react at 50 ℃ 15, 35, 55 After 70 minutes, the viscosity was measured and the ratio to the viscosity before the reaction was calculated.

The results are shown in the figure. In the figure, □, △, ○ and ●
The marks represent the ratio of the crosslinked HA-2, 3, 4 and HA sodium salt solutions to the viscosity before the reaction at each reaction time.

From the figure, it can be seen that the crosslinked HA used in the present invention has higher resistance to hyaluronidase than HA, and the degree thereof is more remarkable as the degree of crosslinking is higher.

Preparation Example 2 Preparation of cross-linked ChS 3.1 g of ChS-C sodium salt (molecular weight 53000) was dissolved in 0.75N aqueous sodium hydroxide solution to 12.5%, and 1 volume of ethanol was added with stirring to produce a candy-like precipitate. Was collected. 0.18 m of epichlorohydrin was added to this candy-like precipitate.
Was added and kneaded thoroughly, and the mixture was allowed to stand at 20 ° C. for 24 hours.
30 m of water was added to the reaction solution to dissolve it, pH was adjusted to 6.0 with acetic acid, and ethanol precipitation was performed. It was again dissolved in water, precipitated with ethanol, and dried under reduced pressure to prepare crosslinked ChS-C (hereinafter referred to as "crosslinked ChS").

Test Example 1 Relationship between Crosslinking Degree of Crosslinked HA and Viscosity 100 mg of HA sodium salts having molecular weights of 370000 and 730,000 were dissolved in 5.0 m of 1N sodium hydroxide, and 5 m of ethanol and epichlorohydrin, 25, 50 and 100, respectively. , 200 μm, and reacted at 40 ° C. for 2 hours. After the reaction, post-treatment was carried out according to Preparation Example 1 (1).

In addition, 75 mg of HA sodium salt having a molecular weight of 1,700,000 was dissolved in 7.5 ml of 1N sodium hydroxide to prepare a solution of 7.5 m of ethanol.
And epichlorohydrin 40μ or 80μ are added, 40 ℃
And reacted for 2 hours. Further, at the same time under the same conditions as in the reaction subjected to reaction with [2- ¹⁴ C] epichlorohydrin (available from Amersham Japan Co.) was calculated degree of crosslinking from the radiation activity of the labeled compound. Table 2 shows the relationship between the degree of crosslinking and the viscosity.

It can be seen from Table 2 that the crosslinked HA and the viscosity have a proportional relationship in the crosslinked HA.

Test Example 2 Acute toxicity test 4-week-old male ddY mice were preliminarily bred for 1 week. The weight of the mice used in the experiment at the start of the test was 21 to 27 g.

The mice were divided into 15 groups each, and 0.5m saline solution of cross-linked HA-1 in 1m / 10g body weight (cross-linked H-1
A-1 500 mg / Kg), the same 1% physiological saline solution 1 m / 10 g body weight (crosslinked HA-1 1000 mg / Kg) or physiological saline 1/10 g body weight were intraperitoneally administered.

General symptoms were observed daily at the same time according to the Irwin method. Five mice in each group were sacrificed 7 days after the administration, and the remaining surviving mice were sacrificed on the 14th day, and macroscopic observation of the administration site and major organs was performed. The results are shown below.

(i) There were no deaths in each group.

(ii) There was no difference in general symptoms, body weight change, intake, and water intake between the 500 mg / Kg administration group, the 1000 mg / Kg administration group, and the control group.

(iii) There was no difference between the control group and the 500 mg / Kg administration group in the autopsy on the 7th and 14th days, but only in the 1000 mg / Kg administration group.
It was estimated that the amount of uronic acid in the intraperitoneal residual fluid was higher than that in the control group on both the 14th and 14th days, and 3.6 to 3.8 mg / m of cross-linked HA-1 remained on the 14th day.

(iv) Each group had almost no effect on organs.

Example 1 Substitution of vitreous body with cross-linked HA and measurement of reversion rate The following experiment was conducted using a 1% physiological saline solution of cross-linked HA-1.

10 rabbits (3 kg body weight) that had been experimentally retinally detached by pulling the cortical vitreous body with tweezers (ophthalmic forceps) created an outlet hole in the sclera of the right eye to remove subretinal fluid and remove hair. A single incision was made on the pars plana, and the crosslinked HA-1 was slowly injected by advancing the crosslinked HA injection needle to the back of the lens under observation with a binocular indirect ophthalmoscope. When the subretinal fluid was eliminated and the retina was restored, the injection needle and drainage needle were removed, the needles were removed from the opposite holes, injection was repeated, and the entire vitreous cavity was replaced with crosslinked HA-1 as much as possible. .

On the 10th day, the reversion position was observed with a fundus camera. As a result, it was observed that the retinal detachment was completely cured in 7 birds (reversion rate 70%).

Example 2 Substitution of vitreous body with crosslinked ChS and measurement of reversion rate Instead of 1% physiological saline solution of crosslinked HA-1, 1 of crosslinked ChS was used.
The same experiment as in Example 1 was conducted except that 0% physiological saline solution was used. As a result, it was observed that the retinal detachment was completely cured in 7 out of 10 rabbits (reversion rate 70%).

[Brief description of drawings]

The figure is a diagram showing the relationship between the decrease in viscosity and the time when various crosslinked HAs and HAs were treated with hyaluronidase.

Claims (1)

[Claims] 1. A vitreous application agent comprising a crosslinked glycosaminoglycan.