JPS6353961B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an eyeball preservation solution for corneal transplantation. More specifically, the present invention relates to an eyeball preservation solution for corneal transplantation that makes it possible to preserve the eyeballs used in corneal transplantation surgery (removed from donated human bodies after death) for a long period of time while maintaining their functions. Corneal transplant surgery in humans has a long history among organ transplant surgeries, and is a transplant surgery with an extremely high success rate, and its indications are increasingly being expanded in the ophthalmology field. Furthermore, in recent years, with the progress and spread of this surgery, there has been a strong desire to develop a method that can preserve the cornea for a long period of time without losing its function. In other words, if the eyeballs can be preserved for a long time with their functions maintained after the body is donated immediately after death, the patient undergoing surgery and the doctors and nurses performing the surgery will have more time to prepare adequately. Furthermore, it is possible to improve the quality of the eyeball that has undergone corneal transplantation. In order for corneal transplant surgery to be successful, it is strongly required that the preserved cornea maintain its vitality, but preservation solutions originally used in the field (e.g., physiological saline, glycerin solution, Hank's solution, etc.) cannot preserve the cornea for a long period of time, and in extreme cases, the cornea becomes cloudy within a few hours (3 to 5 hours), making it impossible to use it for transplant surgery. There was a problem. Therefore, research has been actively conducted in recent years to develop methods for preserving the cornea for a long period of time. Such a conventional example will be briefly explained next. For example, Mizukawa et al. reported that tissue culture medium (containing amino acids, vitamins, nucleic acid components, intermediate metabolites, supplementary growth factors, etc.), sodium bicarbonate,
Dissolve appropriate amounts of sodium chondroitin sulfate, inosine, adenosine, adenine, and antibacterial substances in distilled water, and if necessary, introduce a mixed gas of oxygen and carbon dioxide (95:5) to saturate the solution to a pH of 6.5 to 8.5.
An eye preservation solution was developed as
(See Publication No. 2036). Although the eyeball preservation solution is still widely used today, it is only effective for a short period of 2 to 3 days.
If treatment is not carried out promptly, it often happens that the function of the eyeball has already deteriorated by the time of the transplant surgery. For this reason, so-called eye banks (Eye banks), in which all eyes are placed in a preservation solution and stored at low temperatures
The preservation solution is generally used in a method similar to the Bank's preservation method. Various irrigation solutions have also been developed for preserving cut corneas. Edelhauser
et al. used rabbit corneal endothelium at a perfusion temperature of 37°C and an anterior chamber pressure of 15 mm.
Physiological saline, lactated Ringer's solution, and balanced salt solutions under Hg conditions.
salt solution (hereinafter referred to as BSS) or glutathione bicarbonate Ringer's (Glutathion bicarbonate ringer)
When perfused for 4 hours using a Bicarbonate Ringer (hereinafter referred to as GBR), the corneal swelling rates were 98 μm/hr, 39 μm/hr, 24 μm/hr, and 0 to 4 μm, respectively.
m/hr, suggesting the usefulness of GBR in corneal preservation (Edelhauser et al.
HF, Van Horn DL, Hyndiuk NAand
Schulz RO, Amer. J. Ophthalmol., 93 , 643
(1975)). In addition, McEnerney et al. conducted a similar experiment using GBR with its components glutathione and/or adenosine removed and measured the corneal swelling rate, and reported that no damage to the corneal endothelium was observed. (McEnerney JKand
Peyman GA, Invest.Opthalmol., 16 , 657
(1977)). However, when the above-mentioned irrigating solution is applied to corneal transplant surgery, it is satisfactory if it can maintain the function of the cornea for at most several hours. However, when these irrigation solutions are used as eye preservation solutions, the bicarbonate ions (HCO ^- ₃ ) contained in these formulations activate the pumping action of the corneal endothelium (KRMayes, MV Graham
and S.Hodson, Exp.Eye Research, 26 , 555~
560 (1978)), and as a result, the cornea quickly swells and becomes cloudy, rendering it useless as an eye preservation solution. In order to avoid the pump effect, some solutions have been proposed that can be applied to the eye without added bicarbonate ions. For example, Alcon in the United States uses bicarbonate ion-free BSS.
However, it does not contain glucose, which serves as an energy source and has an anti-swelling effect, and has a K ⁺ content of approximately 2 compared to normal aqueous humor components.
Contains about 3 times as much Ca ²⁺ ,
Furthermore, very large amounts of acetate and citrate are used for buffering, which makes them unsuitable for biocomposition preparations. Glucose Phosphate Ringer, which has an ideal composition as a bicarbonate ion-free product, is
Bicarbonate free medium (hereinafter referred to as GPR)
There is. However, GPR is prescribed with Ca ²⁺ and Mg ²⁺ , which are also prescribed at the same time.
In practice, formulation is difficult because Na ₂ HPO ₄ forms an insoluble salt (cloudy precipitate), and white precipitate also occurs during heat sterilization. The present invention was developed in view of the current state of the art, and it halts the corneal pump effect, suppresses corneal swelling and opacity for a long period of time, and improves stability during formulation and storage stability after formulation. Furthermore, another object of the present invention is to provide an eyeball preservation solution for corneal transplantation that enables long-term preservation of the eyeball. As a result of intensive research in line with this objective, the present inventors have found that (a) constant concentrations of electrolytes such as sodium ions, potassium ions, calcium ions, magnesium ions, and chloride ions, which are essential components of aqueous humor; containing, buffered with a phosphate buffer, and using one or more dextrans (preferably with a molecular weight of 40,000 to 150,000) at 1 to 10% (W/V) as a corneal swelling inhibitor. (b) By adding glucose, preferably 0.5 to 15mM, cell activation is achieved when the cornea is restored to the body temperature immediately before surgery. (c) Furthermore, by adding citrate ion and/or acetate ion, preferably 0.5 to 5mM, the effect of preventing corneal swelling and the formation of white precipitate due to calcium ions and magnesium ions is evaluated. (d) When the pumping effect was stopped by not adding hydrogen carbonate ions, the inventors discovered that the above object could be achieved and completed the present invention. That is, the present invention uses sodium ion (Na ⁺ ), potassium ion (K ⁺ ), calcium ion (Ca ²⁺ ), and magnesium ion (Mg ²⁺ ) as cations, and chloride ion (Cl ^- ), phosphate as anions. An eye preservation solution containing ions (PO ₄ ^3- ), sulfate ions (SO ₄ ^2- ), citrate ions and/or acetate ions, but not bicarbonate ions, to which dextran and glucose are added. This invention relates to an eyeball preservation solution for corneal transplantation, characterized by: The dextran used in the eye preservation solution of the present invention preferably has a molecular weight of 40,000 to 150,000, and commercially available dextrans have a molecular weight of 40,000 to 150,000, for example.
Examples include Dextran 70 (manufactured by Meito Sangyo Co., Ltd.) with a molecular weight of 70,000 and 40 (manufactured by Pharmacia Fine Chemicals, Inc., USA). Preferred concentration ranges and particularly preferred concentration ranges of each component used in the eye preservation solution of the present invention are summarized in Table 1 below.

[Table] Sodium citrate and sodium acetate shown in Table 1 are added as appropriate to prevent white precipitate formation if it is observed during the production of the eyeball preservation solution of the present invention. In addition, Dextran 40 and Dextran 70 contain either one or both of them in an appropriate amount (preferably 1 to 10%).
(W/V)) Blend. The concentration ranges of Na ⁺ components shown in Table 1 are sodium citrate, sodium acetate, Na ₂ HPO ₄ ,
Added as NaH ₂ PO ₄ as well as common salt (NaCl)
Adjust by adding appropriate amount. Further, it is preferable that K ⁺ is added as potassium chloride (KCl), Ca ²⁺ as calcium chloride (CaCl ₂ ), and Mg ²⁺ as magnesium sulfate (MgSO ₄ ). The eye preservation solution of the present invention can be manufactured (formulated) by methods conventional in the art and has many advantages. One embodiment of such a manufacturing method will be briefly described below. First, NaCl, KCl, CaCl ₂ (・2H ₂ O), MgSO ₄
(・7H ₂ O) is dissolved in sterile purified water at room temperature or below 60℃. Next, glucose, sodium citrate, dextran, NaH ₂ PO ₄ (・H ₂ O),
Add and dissolve Na ₂ HPO ₄ in that order, and finally sterilize. The eye preservation solution obtained in this way is
It usually has a liquid property with a pH of 6.5 to 7.8, an osmotic pressure of 270 to 350 mOsM, and a relative viscosity of about 2 to 3 cPs. Further, for sterilization, it is possible to employ a heat sterilization method using pressure. The following (i) to (vi) are the advantages of the eyeball preservation solution of the present invention when manufacturing or storing it compared to conventional eyeball preservation solutions. (i) A mixed gas of oxygen and carbon dioxide (95:
5) There is no need for bubbling. Also, ice cooling during preparation is not necessary. (ii) Using NaOH aqueous solution or HCl aqueous solution, etc.
No need to adjust PH. (iii) Sterilization, for example, when autoclaved at 115°C for 10 minutes, it becomes a clear liquid after cooling. (iv) As shown in (i) to (iii), since the problems often encountered in the past during manufacturing have been resolved, the time required for manufacturing is considerably shortened. (v) Environmental changes during storage (e.g. temperature
The appearance of the liquid does not change depending on the temperature (40 to 60℃). (vi) It can be sterilized by pressure and heat, and since it does not contain tissue culture fluid, there is little risk of bacterial growth, and as long as it is not opened, it can be stored stably for a long period of time until it is ready for use. Next, the eyeball preservation solution for corneal transplantation of the present invention will be explained in more detail with reference to prescription examples, but the present invention is not limited to these prescription examples. Prescription example 1 7.5g of NaCl, 0.41g of KCl in 2 flasks,
0.172 g of CaCl _{2.2H 2} O and MgSO _{4.7H 2} O
0.217g was taken, sterilized purified water was added, and the mixture was stirred at room temperature to about 60°C or lower to dissolve it. Next, 0.404 g of glucose was added and dissolved, followed by 0.4 g of sodium citrate dihydrate, 10.0 g of dextran 40,
0.2 g of NaH ₂ PO ₄ H ₂ O and 1.0 g of Na ₂ HPO ₄
were added and dissolved in that order to bring the total amount to 1. This product was dispensed and sealed into 100-500 ml ampoules or vials, and then sterilized by heating at 115°C for 10 minutes. The obtained eyeball preservation solution is a colorless to slightly yellow clear liquid, and the physical properties of the liquid are as follows: PH7.09, osmotic pressure
It had a relative viscosity of 295 mOsM and 1.94 cPs. Formulation Examples 2 to 7 Experiments were conducted in the same manner as Formulation Example 1, except that the ingredients and/or their amounts used in the formulation were changed as shown in Table 2. The physical properties of the obtained eyeball preservation solution are also listed in Table 2.

【table】

[Table] In order to compare the differences between the conventional solution described above (M-K medium will be described later) and the eyeball preservation solution of the present invention, the main compositions of each are summarized in Table 3.

[Table] Next, an example of an eyeball preservation test in which the usefulness of the eyeball preservation solution of the present invention was investigated will be described. The preservation solutions used in the following test examples were those obtained in Formulation Example 3 or 5 above, and GBR having the composition shown in Table 4.
An eye preservation solution containing GPR and sodium chondroitin sulfate (manufactured by Kaken Yakuhikako Co., Ltd.) and "Culture of human cancer cells" (written by Shoichi Ohno and Haruo Kanno, published by Asakura Shoten, pp. 20-21) M-K medium (McCarey-Kaufman medium) obtained by the composition and preparation method.

[Table] *3 Manufactured by Kaken Yakuh Kako Co., Ltd., trade name: Eyeball Preservation Solution (Test Method) A piece of sclerocornea removed from a white rabbit weighing approximately 3 kg was used. First, pieces of sclerocornea cut from the entire enucleated eyeball were stored in each of the above-mentioned eyeball preservation solutions at a temperature of 4°C for a certain period of time (up to 14 days). Next, the sclerocorneal piece was mounted on a plastic chamber bar, and GBR containing bicarbonate ions was placed in the endothelial chamber to apply an intraocular pressure of 15 cm of water column.Then, the entire chamber was placed in a beaker containing GBR. Then, the beaker was immersed in a constant temperature bath at 37°C for 1 hour. In other words, the cryopreserved scleroderma was subjected to temperature reversal for 1 hour in GBR. Thereafter, the structure of the corneal endothelium was observed using a specular microscope and an electron microscope. (Test Results) Table 5 briefly shows the results of observing the endothelial structure using a specular microscope when the scleroderma pieces were stored in each solution for 5, 9, or 12 days. In the observation, a piece of scleroderma (hereinafter referred to as normal) from a freshly removed eyeball was used as a control example. The eyeball preservation solution of the present invention can be satisfactorily used not only for scleroderma but also for the entire eyeball.

【expressed.
Figure 1 shows a normal specular microscope photograph (magnification: approximately 30x, the same applies below).
The cells are uniformly bright and the mosaic pattern is clearly visible. Figures 2 and 3 show GPR (no dextran added) or 5% dextran 70.
Specular after being stored in GPR for 5 days.
A microscope photo is shown. It can be seen that when dextran is not added, swelling of the endothelial cells is observed, whereas when dextran is added, there is no swelling of the endothelial cells and the boundaries between cells are clear. 4 to 20 show specular microscope photographs corresponding to the observation results in Table 5. As can be seen from Figures 4 to 7, the sclerocorneal endothelium preserved for 3 to 12 days in the eyeball preservation solution of the present invention (obtained in Prescription Example 3) had no abnormalities and was well preserved. . In addition, when we investigated the addition of dextran, which is a feature of the present invention, to other solutions, we found that it had some effect, especially with M-K medium and GPR, which is free of hydrogen carbonate ions. (See Figures 8-18).
However, with the sodium chondroitin sulfate eye preservation solution that has been commonly used as an eye preservation solution, as can be seen in Figure 20, cell deformation, separation, and shedding due to cell swelling and rupture of the juncture complex was observed. It was hot. Figure 21 is a normal electron micrograph (magnification:
30,000 times, the same applies hereafter), Figure 22 shows dextran 70
Figure 23 is an electron micrograph of corneal endothelium stored for 5 days in sodium chondroitin sulfate ocular preservation solution containing 5% dextran 70. Figure 24 is an electron micrograph of the corneal endothelium preserved in the eyeball preservation solution obtained in Formulation Example 5 for 8 days. As can be seen by comparing Figure 21 with Figures 22 to 24, the corneal endothelium preserved in sodium chondroitin sulfate eyeball preservation solution for 5 days showed intracellular vacuolization and fragments of mitochondria and rough endoplasmic reticulum. was observed, indicating that endothelial cells were severely damaged. On the other hand, GPR containing 5% dextran 70
In the corneal endothelium stored for 8 days in the eyeball preservation solution obtained in Prescription Example 5, the nucleus and nuclear membrane are well preserved, and chromatin can be seen within it. In addition, the intercellular space is well preserved,
Furthermore, the rough endoplasmic reticulum and mitochondria were also well preserved. Furthermore, the adhesion between endothelial cells and Descemet's membrane was also good. Therefore, it can be said that these preservation solutions hardly damage endothelial cells. As can be seen from the above test results, the eye preservation solution of the present invention contains GPR and M
- It had an eye preservation effect equal to or better than that of K. Medium. Furthermore, GPR has the aforementioned problems during formulation, and M-K Medium has a complex composition consisting of more than 60 types of ingredients, making it expensive and containing many unstable ingredients such as glutathione. Since it cannot be sterilized by heating and has poor stability, it must be prepared each time it is needed, which is not practical. Furthermore, since M-K Medium is a tissue culture solution, there is a risk that bacteria may proliferate while the eyeball is stored for a long period of time, for example, 10 days. In contrast, the eyeball preservation solution of the present invention is easy to formulate and is stable, so it can be said to be extremely excellent as an eyeball preservation solution.

[Brief explanation of drawings]

Figure 1 is a normal specular microscope photograph, Figures 2 to 20 are 3 to 12
Specular microscopic photographs of sclerocorneal endothelium stored for 5 days, Figure 21 is a normal electron microscope photograph, and Figures 22 to 24 are each stored in a sodium chondroitin sulfate ocular preservation solution containing 5% dextran 70. This is an electron micrograph of corneal endothelium stored in GPR for 8 days and in the eyeball preservation solution obtained in Prescription Example 5 for 8 days.

Claims (1)

[Scope of Claims] 1. An eye preservation solution containing sodium ions, potassium ions, calcium ions, and magnesium ions as cations and chloride ions, phosphate ions, and sulfate ions as anions and not containing hydrogen carbonate ions, and glucose. 2. The eyeball preservation solution according to claim 1, to which citrate ions and/or acetate ions are added. 3. The eyeball preservation solution according to claim 1 or 2, wherein a phosphate buffer is added to adjust the pH to 6.5 to 7.8. 4. The eyeball preservation solution according to claim 1, 2, or 3, wherein the dextran has a molecular weight of 40,000 to 150,000 and a concentration of 1 to 10% (weight/volume). 5. The eyeball preservation solution according to claim 2, 3, or 4, wherein the concentration of the citrate ion and/or acetate ion is 0.5 to 5 mmol/. 6. The eyeball preservation solution according to claim 1, 2, 3, 4, or 5, which has an osmotic pressure of 270 to 350 mOsM.