JPH06107538A - Bulb of eye preserving solution for cornea graft - Google Patents

Abstract

PURPOSE:To obtain a bulb of eye preserving solution for cornea graft comprising hyaluronic acid, one polymer substance of organism, having high viscoelasticity and extremely high water retention, capable of reducing swelling properties of cornea during storage or its physiologically permissible salt. CONSTITUTION:A bulb of eye preserving solution for cornea graft comprises hyaluronic acid or its physiologically permissible salt (especially preferably one having 50,000-250,000 molecular weight). The amount of hyaluronic acid or its physiologically permissible salt is preferably 0.05-0.5wt/vol.% per preserving solution. The osmotic pressure of the bulb of eye preserving solution for cornea graft is preferably adjusted to 260-350 mOsm. An extracted whole bulb of eye or corneal piece is stored in the preserving solution. The preserving solution has low swelling properties of cornea, has low influence on corneal endothelia cell and is excellent.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an ocular preservative solution for corneal transplantation, and more particularly to a corneal ocular preservative solution containing hyaluronic acid or a physiologically acceptable salt thereof.

[0002]

2. Description of the Related Art It is desirable that whole eyeballs and scleral corneal pieces used for corneal transplantation be rapidly cryopreserved after being removed from the human body, and do not affect corneal endothelial cells and do not cause corneal swelling during storage. Is required. However, the ion pump of corneal endothelium is still 4% even if simply stored at low temperature.
It works for a long time, and when it is stored for a long period of time, the pump function is weakened, which causes corneal swelling and destruction of corneal endothelial cells. Therefore, development of a preservative solution (ocular preservative solution) for long-term preservation of whole eyeballs or scleral corneal pieces for a long period of time has been earnestly desired, and various studies have been advanced, but a good preservative solution has not been obtained.

However, when Dikstein et al. Incubated a piece of corneal cornea in a medium containing no HCO ₃ ⁻ ion, the HCO ₃ ⁻ pump of the corneal endothelium stopped and the cornea slowly swelled, but contained HCO ₃ ⁻ again. . in other liquid HCO ₃ ^- pump acts swelling was found the fact that recovery (S.Dikstein et: J.Physiol, 221, 29~
41, 1972). Therefore, cryopreservation and HCO ₃ ^- By combining dormant storage of the pump by ion-free, it becomes possible to further extend the life of the corneal pieces. From such a viewpoint, GPR (Glucose-Phosph
ate-Ringer) and other eye preservation solutions have been developed. Furthermore, an eye preservation solution (EP-II solution etc.) to which dextran which is a polysaccharide is added for the purpose of suppressing swelling of the cornea has been devised and is now widely used.

[0004]

As described above, as an eye preservation solution for corneal transplantation, a salt solution containing no NaHCO ₃ to which dextran which is a polysaccharide is added is widely used at present. However, dextran, which is a high molecular compound included in the formulation of this eye preservation solution, is already an in vitro component even though it is already used as a plasma expander, and may cause an allergic reaction. There has been pointed out (AWRichter et al: Immunol.Toda
y , 3 , 132-138, 1982). In addition, dextran is effective in blocking the swelling property of the cornea during storage, but it is taken up by cells in corneal tissue such as corneal endothelial cells as the storage period becomes longer, and then the cornea is collected in the anterior chamber fluid or inorganic salts. water taken into the corneal tissue by dextran taken and placed in a solution, can result in swelling of the cornea has become clear (DSHull et al: Invest.Ophthalmo
L. , 15 , 663-666, 1976, BEMa Carey et al .:
Invest. Ophthalmol. , 13 , pp . 165-173, 197.
4). Furthermore, it is suggested that dextran does not have the property of enhancing the preservation state of the corneal endothelium, but rather may have a harmful effect such as weakening the adhesion between the corneal endothelium and Descemet's membrane or reducing the function of corneal endothelial cells. (RHL
indstrom et al . : Br. J. Ophthalmol. , 70 , 47-54, 19
86, E. Pels et al .: Cornea , 3 , 219-227, 198.
4/1985).

In order to eliminate such a danger caused by using dextran, sodium chondroitin sulfate, which is one of in vivo components and has a better colloid-forming ability (osmotic osmotic pressure) than dextran, is used in place of dextran. An added eye preservation solution was devised (Japanese Patent Laid-Open No. 62-198601). However, 2.5% of sodium chondroitin sulfate having an average molecular weight of 40,000
The eye preservation solution (EP-III solution) added is 3.5.
It was thought that the corneal swelling property during storage was less than that of the eye preservation solution containing 2% dextran (EP-II solution), and that it may be an excellent eye preservation solution, but there was room for further study. (Shinya Kuwayama et al .: Eki, Vol. 41, No. 1
691-1696, 1990). Furthermore, although not seen with high molecular weight sodium chondroitin sulfate, with low molecular weight sodium chondroitin sulfate, they were taken up by cells of the cornea as well as dextran as the storage period increased, resulting in remarkable swelling of the cornea after transplantation. It has been reported to cause (HE Kaufman et al .: Am.J.Ophth
almol. , 100 , 299-304, 1985),
There was also a need to remove low molecular weight sodium chondroitin sulfate having a molecular weight of 10,000 or less. As described above, although many improvements have been made to the eyeball preservation solution, none of them has been sufficiently satisfied.

Therefore, an object of the present invention is to improve the disadvantages associated with the conventionally used eye preservation solution for corneal transplantation, for example, the preservation solution having a low corneal swelling property during storage and the effect on corneal endothelial cells. An object of the present invention is to provide an excellent eye preservative solution for corneal transplantation, which has less amount of corneal transplantation.

[0007]

Means for Solving the Problems The present inventors can substitute the above-mentioned dextran and sodium chondroitin sulfate,
Moreover, as a result of studying various biopolymers capable of improving the drawbacks associated with their use, they have a high viscoelasticity and have a very high water retention property, so that they are used by being injected into the eye during ophthalmic surgery. The present invention was completed by finding that the use of hyaluronic acid at a constant concentration can unexpectedly reduce the corneal swelling property during storage and solve the above problems. That is, according to the present invention, there is provided an eye preservation solution for corneal transplant, which contains hyaluronic acid or a physiologically acceptable salt thereof.

The hyaluronic acid used in the present invention may be the free acid or its physiologically acceptable salt form. Further, it can be widely used as long as it is highly pure and does not show cytotoxicity due to impurities such as mixed proteins and endotoxin and does not cause any trouble for medical use, and its origin is not particularly limited. Specifically, hyaluronic acid obtained from umbilical cord, vitreous body, chicken comb, microorganism and the like can be used. The physiologically acceptable salt means that the salt-forming hyaluronic acid does not exhibit the above-mentioned cytotoxicity. Therefore, the type of such salt is not limited as long as it can be used for the purpose of the present invention, in particular,
Those in the form of sodium salt or potassium salt, which are compatible with other components attached to the preservation solution of the present invention, are preferable.

As such hyaluronic acid or physiologically acceptable salts thereof, those having a molecular weight in the range of 50,000 to 2,500,000 can be used, and the concentration thereof per storage solution is
It is preferable to adjust the content within the range of 0.05 to 0.5% by weight / volume before use. That is, in the present invention, the molecular weight of hyaluronic acid or a physiologically acceptable salt thereof is 50,000 or more, preferably 600,000 or more. Molecular weight is 5
If it is less than 10,000, it may be taken up by each corneal tissue like dextran or sodium chondroitin sulfate, and swelling of the cornea may be induced. If the molecular weight is less than 600,000,
In the case of a high molecular weight substance such as hyaluronic acid, its molecular weight distribution is polydisperse, so that hyaluronic acid having a low molecular weight is likely to be present, and a favorable course after corneal transplantation cannot be expected.

The concentration of hyaluronic acid or its salt in the eye preservation solution for corneal transplantation is 0.05 to 0.5% by weight / volume%, and if it is less than 0.05%, the effect of suppressing the corneal swelling is not sufficient and it is good. A good storage state cannot be obtained (see Test Example 1 below). On the other hand, if the concentration is higher than 0.5% by weight / volume, the viscosity of the preservative solution increases remarkably and becomes sticky, resulting in poor operability during eyeball handling.

The eye preservation solution for corneal transplantation of the present invention is
Other components for adjusting osmotic pressure, pH and the like are added as necessary. Other components to be used are usually sodium ions, potassium ions, calcium ions and magnesium ions as cations, and chloride ions, phosphate ions, sulfate ions and citrate ions as anions. Glucose can be included.

Each of the above components is formulated in an appropriate concentration so that the pH is 6.5 to 7.8 and the osmotic pressure is 260 to 3
When adjusted to the range of 50 mOsm, the eye preservation solution for corneal transplant of the present invention is provided. In addition, each component is preferably added as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, sodium citrate, monosodium phosphate, and disodium phosphate.

As a method for producing the eyeball preservation solution for corneal transplantation of the present invention, for example, first, NaCl, KCl, CaCl ₂
2H ₂ O and MgSO ₄ .7H ₂ O dissolved in water for injection at room temperature, then glucose, sodium citrate dihydrate, sodium hyaluronate, NaH ₂ PO ₄ , Na
Dissolve ₂ HPO ₄ .12 H ₂ O respectively and combine, and finally sterilize. If necessary, an indicator such as phenol red can be added before sterilization. The preservative thus obtained usually has a pH of 6.5 to 7.8 and an osmotic pressure of 260 to 350 mOsm. It should be noted that it is also possible to provide it as a combination of a series of reagents (components) in which each component is individually packaged so that the preservation solution can be prepared on site immediately before use.

[0014]

EXAMPLES Next, the present invention will be described in more detail with reference to specific formulation examples, test examples and examples, but the present invention is not limited to these formulation examples and examples. Formulation Example 1 5.39 g NaCl, KCl 0.
41 g, CaCl ₂ .2H ₂ O 0.172 g and M
0.218 g of gSO ₄ .7H ₂ O was taken, water for injection was added, and the mixture was stirred and dissolved at room temperature. Next, 0.782 g of glucose was added and dissolved, and further 0.9 g of sodium citrate dihydrate, 0.5 g of purified sodium hyaluronate (molecular weight 1.8 million) of microbial origin, NaH ₂ PO
₄ 0.25 g, and Na ₂ HPO ₄ · 12H ₂ O
Add 3.6 g each in that order and dissolve to make the total amount 1
It was set to L. After sterilizing this by filtration, 100-500
The mixture was dispensed and sealed in an ampule or a vial of ml volume. This product has a pH of 7.34 and an osmotic pressure of 275 mOsm.
Met. Formulation Example 2 5.39 g NaCl, KCl 0.
41 g, CaCl ₂ .2H ₂ O 0.172 g and M
0.218 g of gSO ₄ .7H ₂ O was taken, water for injection was added, and the mixture was stirred and dissolved at room temperature. Next, 0.782 g of glucose was added and dissolved, and further 0.9 g of sodium citrate dihydrate, 5.0 g of purified sodium hyaluronate (molecular weight 2,000,000) of microbial origin, NaH ₂ PO
₄ 0.25 g, and Na ₂ HPO ₄ · 12H ₂ O
Add 3.6 g each in that order and dissolve to make the total amount 1
It was set to L. After sterilizing this by filtration, 100-500
The mixture was dispensed and sealed in an ampule or a vial of ml volume. This product has a pH of 7.20 and an osmotic pressure of 280 mOsm.
Met. Formulation Example 3 NaCl 7.8 g, KCl 0.4 in a 2 L flask.
1 g, CaCl ₂ .2H ₂ O 0.172 g and Mg
0.218 g of SO ₄ .7H ₂ O was taken, water for injection was added, and the mixture was dissolved by stirring at room temperature. Next, glucose 0.
782 g was added and dissolved, and then 0.9 g of sodium citrate dihydrate, 1.0 g of purified sodium hyaluronate (molecular weight 800,000) of chicken cob origin, NaH ₂ PO ₄ .0.
25 g and Na ₂ HPO ₄ .12H ₂ O (3.6 g) were added in that order and dissolved to make the total amount 1 L.
The pH of this product is 7.21, and the osmotic pressure is 314 mOs.
It was m.

In order to examine the usefulness of the ocular preservation solution of the present invention, the one obtained in Formulation Example 1, the one in which the concentration of sodium hyaluronate was adjusted to 0.01% in Formulation Example 1 and sodium chondroitin sulfate were contained. The whole eye preservation effect was tested using a formulation according to Matsuyama et al.'S eye preservation solution for corneal transplantation (see JP-A-62-198601). Test Example 1 After removing the whole eyeball of a Japanese white male rabbit (body weight 2-3 kg), the concentration of sodium hyaluronate was 0.0 in Formulation Example 1 and Formulation Example 1 containing sodium hyaluronate.
It was immersed in a preservative solution for cornea transplantation containing 2.5% of sodium chondroitin sulfate in place of sodium hyaluronate, and stored at 4 ° C.
The eye preservation solution was replaced with a new one every 24 hours. The thickness of the central portion of the cornea was measured with a Pachymeter manufactured by Silco Co. every 24 hours after total enucleation and after being placed in an eye preservation solution. In addition, this test was carried out with 7 eyes for each storage solution.

FIG. 1 is a graph showing the measurement results of the corneal thickness when all the eyeballs were stored for 7 days. In FIG. 1, black circles are the prescription example 1 containing 0.05% of sodium hyaluronate, black triangles are those obtained by adjusting the concentration of sodium hyaluronate to 0.01% in the prescription example 1, and white circles are sodium chondroitin sulfate. Fig. 2 shows an eyeball preservation solution for corneal transplant containing 2.5%. In Formulation Example 1 containing 0.05% sodium hyaluronate, the rate of increase in corneal thickness was 2 days or more after all eye preservation compared to the eye preservation solution for corneal transplant containing 2.5% sodium chondroitin sulfate. It was significantly suppressed. On the other hand, prescription example 1
In the case where the concentration of sodium hyaluronate was set to 0.01%, there was no difference in the rate of increase in corneal thickness as compared with the eye preservation solution for corneal transplant containing sodium chondroitin sulfate. Test Example 2 In this test, the effect on corneal endothelial cells was compared between EP-II solution, which is an eye preservation solution containing dextran, and Formulation Example 1 containing 0.5% sodium hyaluronate.

A piece of a corneal cornea obtained from a Japanese white male rabbit (body weight: 2 to 3 kg) was added to EP-II solution or prescription example 1 at a sodium hyaluronate concentration of 0.5% at 4 ° C. Stored for 9 days. Immediately after storage, or after leaving it at room temperature for 1 hour, scleral corneal pieces were treated with 25 mM NaHCO 3.
_It was transferred to a stock solution containing ₃ and incubated at 37 ° C. for 1 hour (temperature reversal was performed).
After the incubation, the corneal pieces were fixed with 2.5% glutaraldehyde, and the corneal endothelial cells were subjected to scanning electron microscope observation according to a conventional method.

FIG. 2 shows a scanning electron micrograph of normal corneal endothelial cells. The mosaic pattern of the cells is clearly visible, and the fine structure of the corneal endothelial cell surface, that is, the fine projections is clearly observed. 3 to 6 show scanning electron micrographs of Test Example 2. As can be seen from FIGS. 3 and 4, the corneal endothelium was subjected to temperature reversal immediately after storage in the corneal endothelium in EP-II solution and in Formulation Example 1 in which the sodium hyaluronate concentration was set to 0.5%. All cells were well preserved without any abnormalities. However, as shown in FIG. 5, in the scleral corneal piece stored at 4 ° C. and left at room temperature for 1 hour, E
In the case of the P-II solution, the fine projections on the corneal endothelial cell surface were deformed or atrophied. On the other hand, in Formulation Example 1 in which the concentration of sodium hyaluronate was set to 0.5%, as shown in FIG. 6, no deformation or atrophy was observed in the fine projections, and the morphology was well preserved.

As is clear from the above test results, the eye preservation solution for corneal transplantation of the present invention, which is one of biological components and contains sodium hyaluronate which is a constituent in human aqueous humor, is dextran or chondroitin. It had an excellent corneal preservation effect equivalent to or higher than that of an eye preservation solution containing sodium sulfate. During corneal transplant surgery, temperature reversal is not intentionally performed, and is performed by contacting the anterior chamber fluid of the corneal recipient with the transplanted cornea after the transplant operation is completed. That is, in daily clinical practice,
The cornea for transplantation may be exposed to room temperature in an HCO ₃ ⁻ ion-free environment for a while before the ature reversal is performed. Even in consideration of such matters, the eyeball preservative solution for corneal transplant containing sodium hyaluronate is extremely excellent as can be seen from its corneal endothelial cell preserving effect. In vivo test Whole eyeballs were extracted from two cynomolgus monkeys and immersed in Formulation Example 3 containing 0.1% sodium hyaluronate at 4 ° C. After soaking for 24 hours, the soaked cornea was transplanted to one eye of another 4 cynomolgus monkeys. That is, a small amount of an ophthalmic surgery auxiliary agent (Healon: registered trademark) [Kabi Pharmacia] was injected into the anterior chamber of all eyes, and then the cornea was punched out with a trepan with a diameter of 7 mm and cut with a corneal scissor. The monkey receiving the cornea was anesthetized by intramuscular injection of ketamine hydrochloride (Ketalal 50: registered trademark) [Sankyo] 45 mg / kg and xylazine hydrochloride (Seractal: registered trademark) [Bayer] 1.8 mg / kg. The cornea of one eye was punched out with a trepan with a diameter of 7 mm, and the cornea for transplantation was placed thereon. 10-0
After suturing 16 needles with nylon thread, physiological saline was injected into the anterior chamber to form the anterior chamber. 0.3 after surgery
% Gentamicin (gentasin: registered trademark) [Essex Japan] was instilled 5 times a day. The handling and operability of the eyeball during the surgery were good, and no opacity of the cornea or inflammatory reaction in the anterior chamber was observed in the examination with a slit lamp 7 days after the operation. Further, although it was observed up to 6 months, the thickness of the cornea was normal, and the observation result by the specular microscope showed that the reduction rate of corneal endothelial cells was as low as 6.3% on average.

[0020]

EFFECTS OF THE INVENTION According to the present invention, there is provided an excellent ocular preservation solution for corneal transplantation, which has a low corneal swelling property during storage and a small influence on corneal endothelial cells. Such an effect is achieved by including hyaluronic acid or a physiologically acceptable salt thereof in the preservation solution, and thus is equivalent to a conventionally used eye preservation solution for corneal transplantation to which dextran or sodium chondroitin sulfate is added. Alternatively, it becomes possible to provide an eyeball preservation solution that exhibits a corneal preservation effect higher than that.

[Brief description of drawings]

FIG. 1 is a characteristic graph showing changes in corneal thickness of whole rabbit eyeballs in Test Example 1.

FIG. 2 is a scanning electron micrograph as an alternative to an enlarged view of corneal endothelial cells (normal) of a piece of a corneal cornea immediately after being removed from the living body.

[Fig. 3] After excision from the living body, te immediately after storage in EP-II solution
It is a scanning electron micrograph which replaces the enlarged view of the said cell which performed mperature reversal.

FIG. 4 is a scanning electron micrograph as an alternative to an enlarged view of the cells when treated in the same manner as in FIG. 3 by using the preservative solution of Formulation Example 1 in place of the EP-II solution in FIG.

FIG. 5 is a scanning electron micrograph as an alternative to an enlarged view of the cells subjected to temperature reversal after being removed from a living body, stored in an EP-II solution, and further left at room temperature for 1 hour.

FIG. 6 is a scanning electron micrograph as an alternative to an enlarged view of the cells in the case where the stock solution of Formulation Example 1 was used instead of the EP-II solution of FIG. 5 and treated in the same manner as in FIG. (Note that Figure 2
All the micrographs of ~ 6 have a magnification of 4000 times. )

Claims (4)

[Claims] 1. An eye preservation solution for corneal transplantation, which comprises hyaluronic acid or a physiologically acceptable salt thereof. 2. The eye preservative solution for corneal transplantation according to claim 1, wherein the hyaluronic acid has a molecular weight of 50,000 to 2,500,000 and a concentration thereof is 0.05 to 0.5% by weight / volume based on the preservative solution. 3. The eyeball preservation solution for corneal transplantation according to claim 1, wherein the osmotic pressure is adjusted to 260 to 350 mOsm. 4. The ocular preservative solution for corneal transplant according to claim 1, which can be used for preservation of isolated whole eyeball or a piece of scleral cornea.